THE INTRODUCTION

TO MICROBIOLOGY

Marisa Cases
Manchester Community College
A remix of OpenStax: Microbiology
by PARKER, SCHNEEGURT, THI TU, FORESTER and LISTER

For Manchester Community College

edited by Marisa Cases
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TABLE OF CONTENTS
Licensing
Front Matter from OpenStax Microbiology

1: Depth and Breadth of Microbiology

1.1: What Our Ancestors Knew
1.2: Spontaneous Generation
1.3: Foundations of Modern Cell Theory
1.4: A Systematic Approach
1.5: Types of Microorganisms
1.6: Tools and Media Used for Bacterial Growth
Chapter 1 Exercises

2: Chemistry and Biochemistry

2.1: Atoms, Isotopes, Ions, and Molecules - The Building Blocks
2.2: Water
2.3: Carbon and Organic Molecules
2.4: Carbohydrates
2.5: Lipids
2.6: Proteins
2.7: Nucleic Acids
Chapter 2 Exercises

3: Microscope and the Cell

3.1: How Microscopes Work
3.2: Staining Microscopic Specimens and Descriptions
3.3: Cells as Living Things
Chapter 3 Exercises

4: Prokaryotic Diversity
4.1: Unique Characteristics of Prokaryotic Cells
4.2: Classifying Prokaryotes and Examples
Chapter 4 Exercises

5: The Eukaryotes of Microbiology

5.1: Characteristics of Eukaryotic Cells
5.2: Classifying Eukaryotic Microbes and Examples
Chapter 5 Exercises

6: Mechanisms of Microbial Genetics

6.1: Using Microbiology to Discover the Secrets of Life
6.2: Structure and Replication of DNA
6.3: Structure and Transcription of RNA
6.4: Protein Synthesis (Translation)
6.5: Mutations

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 6.6: How Asexual Prokaryotes Achieve Genetic Diversity
6.7: Gene Regulation and Operon Theory
Chapter 6 Exercises

7: Microbial Metabolism
7.1: Energy, Matter, and Enzymes
7.2: Catabolism of Carbohydrates
7.3: Alternate Forms of Catabolism: Fermentation, Lipids and Proteins
7.4: Photosynthesis and the Importance of Light
Chapter 7 Exercises

8: Microbial Growth
8.1: How Microbes Grow
8.2: Oxygen Requirements for Microbial Growth
8.3: The Effects of pH and Temperature on Microbial Growth
8.4: Other Environmental Conditions that Affect Growth
8.5: Microbial Relationships
Chapter 8 Exercises

9: Acellular Pathogens
9.1: Viruses
9.2: The Viral Cycles
9.3: Isolation, Culture, and Identification of Viruses
9.4: Viroids, Virusoids, and Prions
Chapter 9 Exercises

10: Modern Applications of Microbial Genetics

10.1: Microbes and the Tools of Genetic Engineering
10.2: Visualizing and Characterizing DNA
10.3: Whole Genome Methods and Industrial Applications
10.4: Genetic Engineering - Risks, Benefits, and Perceptions
Chapter 10 Exercises

11: Control of Microbial Growth

11.1: Controlling Microbial Growth
11.2: Using Physical Methods to Control Microorganisms
11.3: Using Chemicals to Control Microorganisms
11.4: Discovering Antimicrobial Drugs
11.5: Drug Targets on Prokaryote Microorganisms
11.6: Drugs for Non-prokaryote Microbes
11.7: Mechanisms for Resistance
11.8: Testing the Effectiveness of Antimicrobial Chemicals and Drugs
Chapter 11 Exercises

12: Microbial Interactions Flora, Pathogenicity and Epidemiology

12.1: Normal Microbiota of the Body
12.2: Characteristics and Steps of Infectious Diseases
12.3: Virulence Factors in Infection
12.4: How Diseases Spread

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 12.5: The Language of Epidemiologists
12.6: Tracking Infectious Diseases
Chapter 12 Exercises

13: Innate Nonspecific Host Defenses

13.1: First Line defense- Physical, Mechanical and Chemical Defenses
13.2: Second Line Defenses: Cells and Fluids
13.3: Pathogen Recognition and Phagocytosis
13.4: Inflammation and Fever
Chapter 13 Exercises

14: Specific Adaptive Host Defenses

14.1: Architecture of the Immune System
14.2: T Lymphocytes and Cellular Immunity
14.3: B Lymphocytes and Humoral Immunity
14.4: Vaccines
14.5: Practical Applications of Monoclonal and Polyclonal Antibodies
Chapter 14 Exercises
Index

Index
Glossary

Detailed Licensing

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Licensing
A detailed breakdown of this resource's licensing can be found in Back Matter/Detailed Licensing.

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Front Matter from OpenStax Microbiology
SENIOR CONTRIBUTING AUTHORS
NINA PARKER, SHENANDOAH UNIVERSITY
MARK SCHNEEGURT, WICHITA STATE UNIVERSITY
ANH-HUE THI TU, GEORGIA SOUTHWESTERN STATE UNIVERSITY BRIAN M. FORSTER, SAINT JOSEPH'S
UNIVERSITY
PHILIP LISTER, CENTRAL NEW MEXICO COMMUNITY COLLEGE
OpenStax
Rice University
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Revision Number MB-2016-002(05/18)-BB

Original Publication Year 2016

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Preface
Welcome to Microbiology, an OpenStax resource. This textbook was written to increase student access to high-quality learning
materials, maintaining highest standards of academic rigor at little to no cost.
About OpenStax
OpenStax is a nonprofit based at Rice University, and it's our mission to improve student access to education. Our first openly
licensed college textbook was published in 2012, and our library has since scaled to over 20 books for college and AP® Courses
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About OpenStax Resources

Customization
Microbiology is licensed under a Creative Commons Attribution 4.0 International (CC BY) license, which means that you can
distribute, remix, and build upon the content, as long as you provide attribution to OpenStax and its content contributors.
Because our books are openly licensed, you are free to use the entire book or pick and choose the sections that are most relevant to
the needs of your course. Feel free to remix the content by assigning your students certain chapters and sections in your syllabus, in
the order that you prefer. You can even provide a direct link in your syllabus to the sections in the web view of your book.

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Instructors also have the option of creating a customized version of their OpenStax book. The custom version can be made
available to students in low-cost print or digital form through their campus bookstore. Visit your book page on openstax.org for
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Errata
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Format
You can access this textbook for free in web view or PDF through openstax.org, and for a low cost in print.
About Microbiology
Microbiology is designed to cover the scope and sequence requirements for the single-semester Microbiology course for non-
majors. The book presents the core concepts of microbiology with a focus on applications for careers in allied health. The
pedagogical features of Microbiology make the material interesting and accessible to students while maintaining the career-
application focus and scientific rigor inherent in the subject matter.
Coverage and Scope
The scope and sequence of Microbiology has been developed and vetted with input from numerous instructors at institutions across
the US. It is designed to meet the needs of most microbiology courses for non-majors and allied health students. In addition, we
have also considered the needs of institutions that offer microbiology to a mixed audience of science majors and non-majors by
frequently integrating topics that may not have obvious clinical
relevance, such as environmental and applied microbiology and the history of science.
With these objectives in mind, the content of this textbook has been arranged in a logical progression from fundamental to more
advanced concepts. The opening chapters present an overview of the discipline, with individual chapters focusing on microscopy
and cellular biology as well as each of the classifications of microorganisms. Students then explore the foundations of microbial
biochemistry, metabolism, and genetics, topics that provide a basis for understanding the various means by which we can control
and combat microbial growth. Beginning with Chapter 15, the focus turns to microbial pathogenicity, emphasizing how interactions
between microbes and the human immune system contribute to human health and disease. The last several chapters of the text
provide a survey of medical microbiology, presenting the characteristics of microbial diseases organized by body system.
While we have made every effort to align the Table of Contents with the needs of our audience, we recognize that some instructors
may prefer to teach topics in a different order. A particular strength of Microbiology is that instructors can customize the book,
adapting it to the approach that works best in their classroom.

American Society of Microbiology (ASM) Partnership

Microbiology is produced through a collaborative publishing agreement between OpenStax and the American Society for
Microbiology Press. The book has been developed to align to the curriculum guidelines of the American Society for Microbiology.
About ASM
The American Society for Microbiology is the largest single life science society, composed of over 47,000 scientists and health
professionals. ASM's mission is to promote and advance the microbial sciences.
ASM advances the microbial sciences through conferences, publications, certifications, and educational opportunities. It enhances
laboratory capacity around the globe through training and resources and provides a network for scientists in academia, industry, and
clinical settings. Additionally, ASM promotes a deeper understanding of the microbial sciences to diverse audiences and is
committed to offering open-access materials through their new journals, American Academy of Microbiology reports, and
textbooks.
ASM Recommended Curriculum Guidelines for Undergraduate Microbiology Education
PART 1: Concepts and Statements Evolution

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1. Cells, organelles (e.g., mitochondria and chloroplasts) and all major metabolic pathways evolved from early prokaryotic cells.
2. Mutations and horizontal gene transfer, with the immense variety of microenvironments, have selected for a huge diversity of
microorganisms.
3. Human impact on the environment influences the evolution of microorganisms (e.g., emerging diseases and the selection of
antibiotic resistance).
4. The traditional concept of species is not readily applicable to microbes due to asexual reproduction and the frequent occurrence
of horizontal gene transfer.
5. The evolutionary relatedness of organisms is best reflected in phylogenetic trees.
Cell Structure and Function
6. The structure and function of microorganisms have been revealed by the use of microscopy (including bright field, phase
contrast, fluorescent, and electron).
7. Bacteria have unique cell structures that can be targets for antibiotics, immunity and phage infection.
8. Bacteria and Archaea have specialized structures (e.g., flagella, endospores, and pili) that often confer critical capabilities.
9. While microscopic eukaryotes (for example, fungi, protozoa and algae) carry out some of the same processes as bacteria, many
of the cellular properties are fundamentally different.
10 . The replication cycles of viruses (lytic and lysogenic) differ among viruses and are determined by their unique structures and
genomes.
Metabolic Pathways
11 . Bacteria and Archaea exhibit extensive, and often unique, metabolic diversity (e.g., nitrogen fixation, methane
production, anoxygenic photosynthesis) .
12. The interactions of microorganisms among themselves and with their environment are determined by their metabolic abilities
(e.g., quorum sensing, oxygen consumption, nitrogen transformations).
13. . The survival and growth of any microorganism in a given environment depends on its metabolic characteristics.
14. . The growth of microorganisms can be controlled by physical, chemical, mechanical, or biological means.
Information Flow and Genetics
15. Genetic variations can impact microbial functions (e.g., in biofilm formation, pathogenicity and drug resistance).
16 . Although the central dogma is universal in all cells, the processes of replication, transcription, and translation differ in
Bacteria, Archaea, and Eukaryotes.
17 . The regulation of gene expression is influenced by external and internal molecular cues and/or signals. 18 . The synthesis of
viral genetic material and proteins is dependent on host cells.
19. . Cell genomes can be manipulated to alter cell function.
Microbial Systems
20. Microorganisms are ubiquitous and live in diverse and dynamic ecosystems.
21. Most bacteria in nature live in biofilm communities.
22. Microorganisms and their environment interact with and modify each other.
23. Microorganisms, cellular and viral, can interact with both human and nonhuman hosts in beneficial, neutral or detrimental
ways.
Impact of Microorganisms
24. Microbes are essential for life as we know it and the processes that support life (e.g., in biogeochemical cycles and plant and/or
animal microbiota).
25. Microorganisms provide essential models that give us fundamental knowledge about life processes.

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26. Humans utilize and harness microorganisms and their products.
27. Because the true diversity of microbial life is largely unknown , its effects and potential benefits have not been fully explored.
PART 2: Competencies and Skills Scientific Thinking
28. Ability to apply the process of science
a. Demonstrate an ability to formulate hypotheses and design experiments based on the scientific method.
b. Analyze and interpret results from a variety of microbiological methods and apply these methods to analogous situations.
29. Ability to use quantitative reasoning
a. Use mathematical reasoning and graphing skills to solve problems in microbiology.
30. . Ability to communicate and collaborate with other disciplines
a. Effectively communicate fundamental concepts of microbiology in written and oral format.
b. Identify credible scientific sources and interpret and evaluate the information therein.
31. Ability to understand the relationship between science and society
a. Identify and discuss ethical issues in microbiology.
Microbiology Laboratory Skills
32. Properly prepare and view specimens for examination using microscopy (bright field and, if possible, phase contrast).
33. Use pure culture and selective techniques to enrich for and isolate microorganisms.
34. Use appropriate methods to identify microorganisms (media-based, molecular and serological).
35. Estimate the number of microorganisms in a sample (using, for example, direct count, viable plate count, and
spectrophotometric methods).
36. Use appropriate microbiological and molecular lab equipment and methods.
37. Practice safe microbiology, using appropriate protective and emergency procedures.
38. Document and report on experimental protocols, results and conclusions.

About the Authors

Senior Contributing Authors
Nina Parker (Content Lead), Shenandoah University
Dr. Nina Parker received her BS and MS from the University of Michigan, and her PhD in Immunology from Ohio University. She
joined Shenandoah University's Department of Biology in 1995 and serves as Associate Professor, teaching general microbiology,
medical microbiology, immunology, and epidemiology to biology majors and allied health students . Prior to her academic career,
Dr. Parker was trained as a Medical Technologist and received ASCP certification, experiences that drive her ongoing passion for
training health professionals and those preparing for clinical laboratory work. Her areas of specialization include infectious disease,
immunology, microbial pathogenesis, and medical microbiology. Dr. Parker is also deeply interested in the history of medicine and
science, and pursues information about diseases often associated with regional epidemics in Virginia.
Mark Schneegurt (Lead Writer), Wichita State University
Dr. Mark A. Schneegurt is a Professor of Biological Sciences at Wichita State University and maintains joint appointments in
Curriculum and Instruction and Biomedical Engineering. Dr. Schneegurt holds degrees from Rensselaer Polytechnic Institute and a
Ph.D. from Brown University. He was a postdoctoral fellow at Eli Lilly and has taught and researched at Purdue University and the
University of Notre Dame. His research focuses on applied and environmental microbiology, resulting in 70+ scientific
publications and 150+ presentations.
Anh-Hue Thi Tu (Senior Reviewer), Georgia Southwestern State University
Dr. Anh-Hue Tu (born in Saigon, Vietnam) earned a BS in Chemistry from Baylor University and a PhD in Medical Sciences from
Texas A & M Health Science Center. At the University of Alabama-Birmingham, she completed postdoctoral appointments in the
areas of transcriptional regulation in Escherichia coli and characterization of virulence factors in Streptococcus pneumoniae and

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then became a research assistant professor working in the field of mycoplasmology. In 2004, Dr. Tu joined Georgia Southwestern
State University where she currently serves as Professor, teaching various biology courses and overseeing undergraduate student
research. Her areas of research interest include gene regulation, bacterial genetics, and molecular biology. Dr. Tu's teaching
philosophy is to instill in her students the love of science by using critical thinking. As a teacher, she believes it is important to take
technical information and express it in a way that is understandable to any student.
Brian M. Forster, Saint Joseph's University
Dr. Brian M. Forster received his BS in Biology from Binghamton University and his PhD in Microbiology from Cornell
University. In 2011, he joined the faculty of Saint Joseph's University. Dr. Forster is the laboratory coordinator for the natural
science laboratory-based classes designed for students who are not science majors. He teaches courses in general biology, heredity
and evolution, environmental science, and microbiology for students wishing to enter nursing or allied health programs. He has
publications in the Journal of Bacteriology, the Journal of Microbiology & Biology Education and Tested Studies for Laboratory
Education (ABLE Proceedings).
Philip Lister, Central New Mexico Community College
Dr. Philip Lister earned his BS in Microbiology (1986) from Kansas State University and PhD in Medical Microbiology (1992)
from Creighton University. He was a Professor of Medical Microbiology and Immunology at Creighton University (1994-2011),
with appointments in the Schools of Medicine and Pharmacy. He also served as Associate Director of the Center for Research in
Anti-Infectives and Biotechnology. He has published research articles, reviews, and book chapters related to antimicrobial
resistance and pharmacodynamics, and has served as an Editor for the Journal of Antimicrobial Chemotherapy. He is currently
serving as Chair of Biology and Biotechnology at Central New Mexico Community College.
Contributing Authors
Summer Allen, Brown University
Ann Auman, Pacific Lutheran University
Graciela Brelles-Marifio, Universidad Nacional de la Plata
Myriam Alhadeff Feldman, Lake Washington Institute of Technology Paul Flowers, University of North Carolina-Pembroke
Clifton Franklund, Ferris State University Ann Paterson, Williams Baptist University
George Pinchuk, Mississippi University for Women Ben Rowley, University of Central Arkansas
Mark Sutherland, Hendrix College
Reviewers
Michael Angell, Eastern Michigan University Roberto Anitori, Clark College
James Bader, Case Western Reserve University Amy Beumer, College of William and Mary Gilles Bolduc, Massasoit Community
College Susan Bornstein-Forst, Marian University Nancy Boury, Iowa State University
Jennifer Brigati, Maryville College Harold Bull, University of Saskatchewan Evan Burkala, Oklahoma State University Bernadette
Connors, Dominican College
Richard J. Cristiano, Houston Community College-Northwest AnnMarie DelliPizzi, Dominican College
Elisa M. LaBeau DiMenna, Central New Mexico Community College Diane Dixon, Southeastern Oklahoma State University
Randy Durren, Longwood University Elizabeth A. B. Emmert, Salisbury University Karen Frederick, Marygrove College
Sharon Gusky, Northwestern Connecticut Community College Deborah V. Harbour, College of Southern Nevada
Randall Harris, William Carey University Diane Hartman, Baylor University Angela Hartsock, University of Akron
Nazanin Zarabadi Hebel, Houston Community College Heather Klenovich, Community College of Alleghany County Kathleen
Lavoie, Plattsburgh State University
Toby Mapes, Blue Ridge Community College Barry Margulies, Towson University
Kevin M. McCabe, Columbia Gorge Community College Karin A. Melkonian, Long Island University
Jennifer Metzler, Ball State University

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Ellyn R. Mulcahy, Johnson County Community College Jonas Okeagu, Fayetteville State University
Randall Kevin Pegg, Florida State College-Jacksonville Judy Penn, Shoreline Community College
Lalitha Ramamoorthy, Marian University Drew Rholl, North Park University
Hilda Rodriguez, Miami Dade College Sean Rollins, Fitchburg State University Sameera Sayeed, University of Pittsburgh Pramila
Sen, Houston Community College
Brian Robert Shmaefsky, Kingwood College Janie Sigmon, York Technical College
Denise Signorelli, College of Southern Nevada Molly Smith, South Georgia State College-Waycross Paula Steiert, Southwest
Baptist University
Robert Sullivan, Fairfield University Suzanne Wakim, Butte Community College Anne Weston, Francis Crick Institute Valencia L.
Williams, West Coast University James Wise, Chowan State University Virginia Young, Mercer University

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 CHAPTER OVERVIEW

1: Depth and Breadth of Microbiology

Microorganisms (or microbes, as they are also called) are small organisms. Most are so small that they cannot be seen without a
microscope. Most microorganisms are harmless to humans and, in fact, many are helpful. They play fundamental roles in
ecosystems everywhere on earth, forming the backbone of many food webs. People use them to make biofuels, medicines, and
even foods. Without microbes, there would be no bread, cheese, or beer. Our bodies are filled with microbes, and our skin alone is
home to trillions of them. Some of them we can’t live without; others cause diseases that can make us sick or even kill us. Although
much more is known today about microbial life than ever before, the vast majority of this invisible world remains unexplored.
Microbiologists continue to identify new ways that microbes benefit and threaten humans.
1.1: What Our Ancestors Knew
1.2: Spontaneous Generation
1.3: Foundations of Modern Cell Theory
1.4: A Systematic Approach
1.5: Types of Microorganisms
1.6: Tools and Media Used for Bacterial Growth
Chapter 1 Exercises

Thumbnail: A cluster of Escherichia coli bacteria magnified 10,000 times. (Public Domain; Eric Erbe, digital colorization by
Christopher Pooley, both of USDA, ARS, EMU).

This page titled 1: Depth and Breadth of Microbiology is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

1
1.1: What Our Ancestors Knew
Learning Objectives
Describe how our ancestors improved food with the use of invisible microbes
Describe how the causes of sickness and disease were explained in ancient times, prior to the invention of the microscope

Clinical Focus - part 1

Cora, a 41-year-old lawyer and mother of two, has recently been experiencing severe headaches, a high fever, and a stiff neck.
Her husband, who has accompanied Cora to see a doctor, reports that Cora also seems confused at times and unusually drowsy.
Based on these symptoms, the doctor suspects that Cora may have meningitis, a potentially life-threatening infection of the
tissue that surrounds the brain and spinal cord.
Meningitis has several potential causes. It can be brought on by bacteria, fungi, viruses, or even a reaction to medication or
exposure to heavy metals. Although people with viral meningitis usually heal on their own, bacterial and fungal meningitis are
quite serious and require treatment.

Figure 1.1.1 : (a) A lumbar puncture is used to take a sample of a patient’s cerebrospinal fluid (CSF) for testing. A needle is
inserted between two vertebrae of the lower back, called the lumbar region. (b) CSF should be clear, as in this sample.
Abnormally cloudy CSF may indicate an infection but must be tested further to confirm the presence of microorganisms.
(credit b: modification of work by James Heilman)
Cora’s doctor orders a lumbar puncture (spinal tap) to take three samples of cerebrospinal fluid (CSF) from around the spinal
cord (Figure 1.1.1). The samples will be sent to laboratories in three different departments for testing: clinical chemistry,
microbiology, and hematology. The samples will first be visually examined to determine whether the CSF is abnormally
colored or cloudy; then the CSF will be examined under a microscope to see if it contains a normal number of red and white
blood cells and to check for any abnormal cell types. In the microbiology lab, the specimen will be centrifuged to concentrate
any cells in a sediment; this sediment will be smeared on a slide and stained with a Gram stain. Gram staining is a procedure
used to differentiate between two different types of bacteria (gram-positive and gram-negative).
About 80% of patients with bacterial meningitis will show bacteria in their CSF with a Gram stain.1 Cora’s Gram stain did not
show any bacteria, but her doctor decides to prescribe her antibiotics just in case. Part of the CSF sample will be cultured—put
in special dishes to see if bacteria or fungi will grow. It takes some time for most microorganisms to reproduce in sufficient
quantities to be detected and analyzed.

Exercise 1.1.1

What types of microorganisms would be killed by antibiotic treatment?

Most people today, even those who know very little about microbiology, are familiar with the concept of microbes, or “germs,” and
their role in human health. Schoolchildren learn about bacteria, viruses, and other microorganisms, and many even view specimens
under a microscope. But a few hundred years ago, before the invention of the microscope, the existence of many types of microbes

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was impossible to prove. By definition, microorganisms, or microbes, are very small organisms; many types of microbes are too
small to see without a microscope, although some parasites and fungi are visible to the naked eye.
Humans have been living with—and using—microorganisms for much longer than they have been able to see them. Historical
evidence suggests that humans have had some notion of microbial life since prehistoric times and have used that knowledge to
develop foods as well as prevent and treat disease. In this section, we will explore some of the historical applications of
microbiology as well as the early beginnings of microbiology as a science.

Fermented Foods and Beverages

People across the world have enjoyed fermented foods and beverages like beer, wine, bread, yogurt, cheese, and pickled vegetables
for all of recorded history. Discoveries from several archeological sites suggest that even prehistoric people took advantage of
fermentation to preserve and enhance the taste of food. Archaeologists studying pottery jars from a Neolithic village in China found
that people were making a fermented beverage from rice, honey, and fruit as early as 7000 BC.2
Production of these foods and beverages requires microbial fermentation, a process that uses bacteria, mold, or yeast to convert
sugars (carbohydrates) to alcohol, gases, and organic acids (Figure 1.1.12). While it is likely that people first learned about
fermentation by accident—perhaps by drinking old milk that had curdled or old grape juice that had fermented—they later learned
to harness the power of fermentation to make products like bread, cheese, and wine.

Figure 1.1.2 : A microscopic view of Saccharomyces cerevisiae, the yeast responsible for making bread rise (left). Yeast is a
microorganism. Its cells metabolize the carbohydrates in flour (middle) and produce carbon dioxide, which causes the bread to rise
(right). (credit middle: modification of work by Janus Sandsgaard; credit right: modification of work by “MDreibelbis”/Flickr)

The Iceman Treateth

Prehistoric humans had a very limited understanding of the causes of disease, and various cultures developed different beliefs and
explanations. While many believed that illness was punishment for angering the gods or was simply the result of fate,
archaeological evidence suggests that prehistoric people attempted to treat illnesses and infections. One example of this is Ötzi the
Iceman, a 5300-year-old mummy found frozen in the ice of the Ötzal Alps on the Austrian-Italian border in 1991. Because Ötzi
was so well preserved by the ice, researchers discovered that he was infected with the eggs of the parasite Trichuris trichiura,
which may have caused him to have abdominal pain and anemia. Researchers also found evidence of Borrelia burgdorferi, a
bacterium that causes Lyme disease.3 Some researchers think Ötzi may have been trying to treat his infections with the woody fruit
of the Piptoporus betulinus fungus, which was discovered tied to his belongings.4 This fungus has both laxative and antibiotic
properties. Ötzi was also covered in tattoos that were made by cutting incisions into his skin, filling them with herbs, and then
burning the herbs.5 There is speculation that this may have been another attempt to treat his health ailments.

Early Notions of Disease, Contagion, and Containment

Several ancient civilizations appear to have had some understanding that disease could be transmitted by things they could not see.
This is especially evident in historical attempts to contain the spread of disease. For example, the Bible refers to the practice of
quarantining people with leprosy and other diseases, suggesting that people understood that diseases could be communicable.
Ironically, while leprosy is communicable, it is also a disease that progresses slowly. This means that people were likely
quarantined after they had already spread the disease to others.
The ancient Greeks attributed disease to bad air, mal’aria, which they called “miasmatic odors.” They developed hygiene practices
that built on this idea. The Romans also believed in the miasma hypothesis and created a complex sanitation infrastructure to deal
with sewage. In Rome, they built aqueducts, which brought fresh water into the city, and a giant sewer, the Cloaca Maxima, which
carried waste away and into the river Tiber (Figure 1.1.3). Some researchers believe that this infrastructure helped protect the
Romans from epidemics of waterborne illnesses.

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 Figure 1.1.3 : (a) The Cloaca Maxima, or “Greatest Sewer” (shown in red), ran through ancient Rome. It was an engineering marvel
that carried waste away from the city and into the river Tiber. (b) These ancient latrines emptied into the Cloaca Maxima.
Even before the invention of the microscope, some doctors, philosophers, and scientists made great strides in understanding the
invisible forces—what we now know as microbes—that can cause infection, disease, and death.
The Greek physician Hippocrates (460–370 BC) is considered the “father of Western medicine” (Figure 1.1.4a). Unlike many of
his ancestors and contemporaries, he dismissed the idea that disease was caused by supernatural forces. Instead, he posited that
diseases had natural causes from within patients or their environments. Hippocrates and his heirs are believed to have written the
Hippocratic Corpus, a collection of texts that make up some of the oldest surviving medical books.6 Hippocrates is also often
credited as the author of the Hippocratic Oath, taken by new physicians to pledge their dedication to diagnosing and treating
patients without causing harm.
While Hippocrates is considered the father of Western medicine, the Greek philosopher and historian Thucydides (460–395 BC) is
considered the father of scientific history because he advocated for evidence-based analysis of cause-and-effect reasoning (Figure
1.1.4b). Among his most important contributions are his observations regarding the Athenian plague that killed one-third of the

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population of Athens between 430 and 410 BC. Having survived the epidemic himself, Thucydides made the important observation
that survivors did not get re-infected with the disease, even when taking care of actively sick people.7 This observation shows an
early understanding of the concept of immunity.
Marcus Terentius Varro (116–27 BC) was a prolific Roman writer who was one of the first people to propose the concept that
things we cannot see (what we now call microorganisms) can cause disease (Figure 1.1.4c). In Res Rusticae (On Farming),
published in 36 BC, he said that “precautions must also be taken in neighborhood swamps . . . because certain minute creatures
[animalia minuta] grow there which cannot be seen by the eye, which float in the air and enter the body through the mouth and
nose and there cause serious diseases.”8

Figure 1.1.4: (a) the physician Hippocrates, (b) philosopher and historian Thucydides and (c) writer Marcus Terentius Varro.

Exercise 1.1.2

Give two examples of foods that have historically been produced by humans with the aid of microbes.
Explain how historical understandings of disease contributed to attempts to treat and contain disease.

Key Concepts and Summary

Microorganisms (or microbes) are living organisms that are generally too small to be seen without a microscope.
Throughout history, humans have used microbes to make fermented foods such as beer, bread, cheese, and wine.
Long before the invention of the microscope, some people theorized that infection and disease were spread by living things that
were too small to be seen. They also correctly intuited certain principles regarding the spread of disease and immunity.

Footnotes
1. Rebecca Buxton. “Examination of Gram Stains of Spinal Fluid—Bacterial Meningitis.” American Society for Microbiology.
2007. www.microbelibrary.org/librar...ial-meningitis
2. P.E. McGovern et al. “Fermented Beverages of Pre- and Proto-Historic China.” Proceedings of the National Academy of
Sciences of the United States of America 1 no. 51 (2004):17593–17598. doi:10.1073/pnas.0407921102.
3. A. Keller et al. “New Insights into the Tyrolean Iceman's Origin and Phenotype as Inferred by Whole-Genome
Sequencing.”Nature Communications, 3 (2012): 698. doi:10.1038/ncomms1701.
4. L. Capasso. “5300 Years Ago, the Ice Man Used Natural Laxatives and Antibiotics.” The Lancet, 352 (1998) 9143: 1864. doi:
10.1016/s0140-6736(05)79939-6.
5. L. Capasso, L. “5300 Years Ago, the Ice Man Used Natural Laxatives and Antibiotics.” The Lancet, 352 no. 9143 (1998): 1864.
doi: 10.1016/s0140-6736(05)79939-6.

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 6. G. Pappas et al. “Insights Into Infectious Disease in the Era of Hippocrates.” International Journal of Infectious Diseases 12
(2008) 4:347–350. doi: http://dx.doi.org/10.1016/j.ijid.2007.11.003.
7. Thucydides. The History of the Peloponnesian War. The Second Book. 431 BC. Translated by Richard Crawley.
http://classics.mit.edu/Thucydides/p....2.second.html.
8. Plinio Prioreschi. A History of Medicine: Roman Medicine. Lewiston, NY: Edwin Mellen Press, 1998: p. 215.

Glossary
microbe
generally, an organism that is too small to be seen without a microscope; also known as a microorganism

microorganism
generally, an organism that is too small to be seen without a microscope; also known as a microbe

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 1.1: What Our Ancestors Knew is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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1.2: Spontaneous Generation
Learning Objectives
Explain the theory of spontaneous generation and why people once accepted it as an explanation for the existence of certain
types of organisms
Explain how certain individuals (van Helmont, Redi, Needham, Spallanzani, and Pasteur) tried to prove or disprove
spontaneous generation

Humans have been asking for millennia: Where does new life come from? Religion, philosophy, and science have all wrestled with
this question. One of the oldest explanations was the theory of spontaneous generation, which can be traced back to the ancient
Greeks and was widely accepted through the Middle Ages.

The Theory of Spontaneous Generation

The Greek philosopher Aristotle (384–322 BC) was one of the earliest recorded scholars to articulate the theory of spontaneous
generation, the notion that life can arise from nonliving matter. Aristotle proposed that life arose from nonliving material if the
material contained pneuma (“vital heat”). As evidence, he noted several instances of the appearance of animals from environments
previously devoid of such animals, such as the seemingly sudden appearance of fish in a new puddle of water.1
This theory persisted into the 17th century, when scientists undertook additional experimentation to support or disprove it. By this
time, the proponents of the theory cited how frogs simply seem to appear along the muddy banks of the Nile River in Egypt during
the annual flooding. Others observed that mice simply appeared among grain stored in barns with thatched roofs. When the roof
leaked and the grain molded, mice appeared. Jan Baptista van Helmont, a 17th century Flemish scientist, proposed that mice could
arise from rags and wheat kernels left in an open container for 3 weeks. In reality, such habitats provided ideal food sources and
shelter for mouse populations to flourish.
However, one of van Helmont’s contemporaries, Italian physician Francesco Redi (1626–1697), performed an experiment in 1668
that was one of the first to refute the idea that maggots (the larvae of flies) spontaneously generate on meat left out in the open air.
He predicted that preventing flies from having direct contact with the meat would also prevent the appearance of maggots. Redi left
meat in each of six containers (Figure 1.2.1). Two were open to the air, two were covered with gauze, and two were tightly sealed.
His hypothesis was supported when maggots developed in the uncovered jars, but no maggots appeared in either the gauze-covered
or the tightly sealed jars. He concluded that maggots could only form when flies were allowed to lay eggs in the meat, and that the
maggots were the offspring of flies, not the product of spontaneous generation.

Figure 1.2.1 : Francesco Redi’s experimental setup consisted of an open container, a container sealed with a cork top, and a
container covered in mesh that let in air but not flies. Maggots only appeared on the meat in the open container. However, maggots
were also found on the gauze of the gauze-covered container.
In 1745, John Needham (1713–1781) published a report of his own experiments, in which he briefly boiled broth infused with plant
or animal matter, hoping to kill all preexisting microbes.2 He then sealed the flasks. After a few days, Needham observed that the

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broth had become cloudy and a single drop contained numerous microscopic creatures. He argued that the new microbes must have
arisen spontaneously. In reality, however, he likely did not boil the broth enough to kill all preexisting microbes.
Lazzaro Spallanzani (1729–1799) did not agree with Needham’s conclusions, however, and performed hundreds of carefully
executed experiments using heated broth.3 As in Needham’s experiment, broth in sealed jars and unsealed jars was infused with
plant and animal matter. Spallanzani’s results contradicted the findings of Needham: Heated but sealed flasks remained clear,
without any signs of spontaneous growth, unless the flasks were subsequently opened to the air. This suggested that microbes were
introduced into these flasks from the air. In response to Spallanzani’s findings, Needham argued that life originates from a “life
force” that was destroyed during Spallanzani’s extended boiling. Any subsequent sealing of the flasks then prevented new life force
from entering and causing spontaneous generation (Figure 1.2.2).

Figure 1.2.2 : (a) Francesco Redi, who demonstrated that maggots were the offspring of flies, not products of spontaneous
generation. (b) John Needham, who argued that microbes arose spontaneously in broth from a “life force.” (c) Lazzaro Spallanzani,
whose experiments with broth aimed to disprove those of Needham.

Exercise 1.2.1

1. Describe the theory of spontaneous generation and some of the arguments used to support it.
2. Explain how the experiments of Redi and Spallanzani challenged the theory of spontaneous generation.

Disproving Spontaneous Generation

The debate over spontaneous generation continued well into the 19th century, with scientists serving as proponents of both sides. To
settle the debate, the Paris Academy of Sciences offered a prize for resolution of the problem. Louis Pasteur, a prominent French
chemist who had been studying microbial fermentation and the causes of wine spoilage, accepted the challenge. In 1858, Pasteur
filtered air through a gun-cotton filter and, upon microscopic examination of the cotton, found it full of microorganisms, suggesting
that the exposure of a broth to air was not introducing a “life force” to the broth but rather airborne microorganisms.
Later, Pasteur made a series of flasks with long, twisted necks (“swan-neck” flasks), in which he boiled broth to sterilize it (Figure
1.2.3). His design allowed air inside the flasks to be exchanged with air from the outside, but prevented the introduction of any

airborne microorganisms, which would get caught in the twists and bends of the flasks’ necks. If a life force besides the airborne
microorganisms were responsible for microbial growth within the sterilized flasks, it would have access to the broth, whereas the
microorganisms would not. He correctly predicted that sterilized broth in his swan-neck flasks would remain sterile as long as the
swan necks remained intact. However, should the necks be broken, microorganisms would be introduced, contaminating the flasks
and allowing microbial growth within the broth.
Pasteur’s set of experiments irrefutably disproved the theory of spontaneous generation and earned him the prestigious Alhumbert
Prize from the Paris Academy of Sciences in 1862. In a subsequent lecture in 1864, Pasteur articulated “Omne vivum ex vivo”
(“Life only comes from life”). In this lecture, Pasteur recounted his famous swan-neck flask experiment, stating that “…life is a
germ and a germ is life. Never will the doctrine of spontaneous generation recover from the mortal blow of this simple
experiment.”4 To Pasteur’s credit, it never has.

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 Figure 1.2.3 : (a) French scientist Louis Pasteur, who definitively refuted the long-disputed theory of spontaneous generation. (b)
The unique swan-neck feature of the flasks used in Pasteur’s experiment allowed air to enter the flask but prevented the entry of
bacterial and fungal spores. (c) Pasteur’s experiment consisted of two parts. In the first part, the broth in the flask was boiled to
sterilize it. When this broth was cooled, it remained free of contamination. In the second part of the experiment, the flask was
boiled and then the neck was broken off. The broth in this flask became contaminated. (credit b: modification of work by
“Wellcome Images”/Wikimedia Commons)

Exercise 1.2.2

1. How did Pasteur’s experimental design allow air, but not microbes, to enter, and why was this important?
2. What was the control group in Pasteur’s experiment and what did it show?

Summary
The theory of spontaneous generation states that life arose from nonliving matter. It was a long-held belief dating back to
Aristotle and the ancient Greeks.
Experimentation by Francesco Redi in the 17th century presented the first significant evidence refuting spontaneous generation
by showing that flies must have access to meat for maggots to develop on the meat. Prominent scientists designed experiments
and argued both in support of (John Needham) and against (Lazzaro Spallanzani) spontaneous generation.
Louis Pasteur is credited with conclusively disproving the theory of spontaneous generation with his famous swan-neck flask
experiment. He subsequently proposed that “life only comes from life.”
Footnotes
1. K. Zwier. “Aristotle on Spontaneous Generation.” www.sju.edu/int/academics/cas...R.%20Zwier.pdf
2. E. Capanna. “Lazzaro Spallanzani: At the Roots of Modern Biology.” Journal of Experimental Zoology 285 no. 3 (1999):178–
196.
3. R. Mancini, M. Nigro, G. Ippolito. “Lazzaro Spallanzani and His Refutation of the Theory of Spontaneous Generation.” Le
Infezioni in Medicina 15 no. 3 (2007):199–206.
4. R. Vallery-Radot. The Life of Pasteur, trans. R.L. Devonshire. New York: McClure, Phillips and Co, 1902, 1:142.

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Contributors and Attributions
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 1.2: Spontaneous Generation is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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1.3: Foundations of Modern Cell Theory
Learning Objectives
Explain the key points of cell theory and the individual contributions of Hooke, Schleiden, Schwann, Remak, and Virchow
Explain the contributions of Semmelweis, Snow, Pasteur, Lister, and Koch to the development of germ theory

While some scientists were arguing over the theory of spontaneous generation, other scientists were making discoveries leading to
a better understanding of what we now call the cell theory. Modern cell theory has three basic tenets:
All organisms are made of cells
All cells only come from other cells (the principle of biogenesis).
Cells are the fundamental units of structure and function in organisms.
Today, these tenets are fundamental to our understanding of life on earth. However, modern cell theory grew out of the collective
work of many scientists.

The Origins of Cell Theory

The English scientist Robert Hooke first used the term “cells” in 1665 to describe the small chambers within cork that he observed
under a microscope of his own design. To Hooke, thin sections of cork resembled “Honey-comb,” or “small Boxes or Bladders of
Air.” He noted that each “Cavern, Bubble, or Cell” was distinct from the others (Figure 1.3.1). At the time, Hooke was not aware
that the cork cells were long dead and, therefore, lacked the internal structures found within living cells.

Figure 1.3.1 : Robert Hooke (1635–1703) was the first to describe cells based upon his microscopic observations of cork. This
illustration was published in his work Micrographia.
Despite Hooke’s early description of cells, their significance as the fundamental unit of life was not yet recognized. Around the
same time, a Dutch cloth merchant named Antonie van Leeuwenhoek (1632–1723) was the first to develop a lens powerful enough
to view microbes. In 1675, using a simple but powerful microscope of his own making, Leeuwenhoek was able to observe single-
celled organisms, which he described as “animalcules” or “wee little beasties,” swimming in a drop of rain water. From his
drawings of these little organisms, we now know he was looking at bacteria and protists. For this reason he is known as the "Father
of Microbiology."
Nearly 200 years later, in 1838, Matthias Schleiden (1804–1881), a German botanist who made extensive microscopic observations
of plant tissues, described them as being composed of cells. Visualizing plant cells was relatively easy because plant cells are
clearly separated by their thick cell walls. Schleiden believed that cells formed through crystallization, rather than cell division.
Theodor Schwann (1810–1882), a noted German physiologist, made similar microscopic observations of animal tissue. In 1839,
after a conversation with Schleiden, Schwann realized that similarities existed between plant and animal tissues. This laid the
foundation for the idea that cells are the fundamental components of plants and animals.

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In the 1850's, two Polish scientists living in Germany pushed this idea further, culminating in what we recognize today as the
modern cell theory. In 1852, Robert Remak (1815–1865), a prominent neurologist and embryologist, published convincing
evidence that cells are derived from other cells as a result of cell division. However, this idea was questioned by many in the
scientific community. Three years later, Rudolf Virchow (1821–1902), a well-respected pathologist, published an editorial essay
entitled “Cellular Pathology,” which popularized the concept of cell theory using the Latin phrase omnis cellula a cellula (“all cells
arise from cells”), which is essentially the second tenet of modern cell theory.1 Given the similarity of Virchow’s work to Remak’s,
there is some controversy as to which scientist should receive credit for articulating cell theory. See the following Eye on Ethics
feature for more about this controversy.

Science and Plagiarism

Rudolf Virchow, a prominent, Polish-born, German scientist, is often remembered as the “Father of Pathology.” Well known
for innovative approaches, he was one of the first to determine the causes of various diseases by examining their effects on
tissues and organs. He was also among the first to use animals in his research and, as a result of his work, he was the first to
name numerous diseases and created many other medical terms. Over the course of his career, he published more than 2,000
papers and headed various important medical facilities, including the Charité – Universitätsmedizin Berlin, a prominent Berlin
hospital and medical school. But he is, perhaps, best remembered for his 1855 editorial essay titled “Cellular Pathology,”
published in Archiv für Pathologische Anatomie und Physiologie, a journal that Virchow himself cofounded and still exists
today.
Despite his significant scientific legacy, there is some controversy regarding this essay, in which Virchow proposed the central
tenet of modern cell theory—that all cells arise from other cells. Robert Remak, a former colleague who worked in the same
laboratory as Virchow at the University of Berlin, had published the same idea three years before. Though it appears Virchow
was familiar with Remak’s work, he neglected to credit Remak’s ideas in his essay. When Remak wrote a letter to Virchow
pointing out similarities between Virchow’s ideas and his own, Virchow was dismissive. In 1858, in the preface to one of his
books, Virchow wrote that his 1855 publication was just an editorial piece, not a scientific paper, and thus there was no need to
cite Remak’s work.

Figure 1.3.2 : (a) Rudolf Virchow (1821–1902) popularized the cell theory in an 1855 essay entitled “Cellular Pathology.” (b)
The idea that all cells originate from other cells was first published in 1852 by his contemporary and former colleague Robert
Remak (1815–1865).
By today’s standards, Virchow’s editorial piece would certainly be considered an act of plagiarism, since he presented Remak’s
ideas as his own. However, in the 19th century, standards for academic integrity were much less clear. Virchow’s strong
reputation, coupled with the fact that Remak was a Jew in a somewhat anti-Semitic political climate, shielded him from any
significant repercussions. Today, the process of peer review and the ease of access to the scientific literature help discourage
plagiarism. Although scientists are still motivated to publish original ideas that advance scientific knowledge, those who would
consider plagiarizing are well aware of the serious consequences.
In academia, plagiarism represents the theft of both individual thought and research—an offense that can destroy reputations
and end careers.2 3 4 5

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 Exercise 1.3.1

1. What are the key points of the cell theory?

2. What contributions did Rudolf Virchow and Robert Remak make to the development of the cell theory?

The Germ Theory of Disease

Prior to the discovery of microbes during the 17th century, other theories circulated about the origins of disease. For example, the
ancient Greeks proposed the miasma theory, which held that disease originated from particles emanating from decomposing matter,
such as that in sewage or cesspits. Such particles infected humans in close proximity to the rotting material. Diseases including the
Black Death, which ravaged Europe’s population during the Middle Ages, were thought to have originated in this way.
In 1546, Italian physician Girolamo Fracastoro proposed, in his essay De Contagione et Contagiosis Morbis, that seed-like spores
may be transferred between individuals through direct contact, exposure to contaminated clothing, or through the air. We now
recognize Fracastoro as an early proponent of the germ theory of disease, which states that diseases may result from microbial
infection. However, in the 16th century, Fracastoro’s ideas were not widely accepted and would be largely forgotten until the 19th
century.
In 1847, Hungarian obstetrician Ignaz Semmelweis (Figure 1.3.3) observed that mothers who gave birth in hospital wards staffed
by physicians and medical students were more likely to suffer and die from puerperal fever after childbirth (10%–20% mortality
rate) than were mothers in wards staffed by midwives (1% mortality rate). Semmelweis observed medical students performing
autopsies and then subsequently carrying out vaginal examinations on living patients without washing their hands in between. He
suspected that the students carried disease from the autopsies to the patients they examined. His suspicions were supported by the
untimely death of a friend, a physician who contracted a fatal wound infection after a postmortem examination of a woman who
had died of a puerperal infection. The dead physician’s wound had been caused by a scalpel used during the examination, and his
subsequent illness and death closely paralleled that of the dead patient.
Although Semmelweis did not know the true cause of puerperal fever, he proposed that physicians were somehow transferring the
causative agent to their patients. He suggested that the number of puerperal fever cases could be reduced if physicians and medical
students simply washed their hands with chlorinated lime water before and after examining every patient. When this practice was
implemented, the maternal mortality rate in mothers cared for by physicians dropped to the same 1% mortality rate observed
among mothers cared for by midwives. This demonstrated that handwashing was a very effective method for preventing disease
transmission. Despite this great success, many discounted Semmelweis’s work at the time, and physicians were slow to adopt the
simple procedure of handwashing to prevent infections in their patients because it contradicted established norms for that time
period.

Figure 1.3.3 : Ignaz Semmelweis (1818–1865) was a proponent of the importance of handwashing to prevent transfer of disease
between patients by physicians.
Around the same time Semmelweis was promoting handwashing, in 1848, British physician John Snow conducted studies to track
the source of cholera outbreaks in London. By tracing the outbreaks to two specific water sources, both of which were
contaminated by sewage, Snow ultimately demonstrated that cholera bacteria were transmitted via drinking water. Snow’s work is
influential in that it represents the first known epidemiological study, and it resulted in the first known public health response to an
epidemic. The work of both Semmelweis and Snow clearly refuted the prevailing miasma theory of the day, showing that disease is
not only transmitted through the air but also through contaminated items. Although the work of Semmelweis and Snow

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successfully showed the role of sanitation in preventing infectious disease, the cause of disease was not fully understood. The
subsequent work of Louis Pasteur, Robert Koch, and Joseph Lister would further substantiate the germ theory of disease.
While studying the causes of beer and wine spoilage in 1856, Pasteur discovered properties of fermentation by microorganisms. He
had demonstrated with his swan-neck flask experiments that airborne microbes, not spontaneous generation, were the cause of food
spoilage. He also suggested that if microbes were responsible for food spoilage and fermentation, they could also be responsible for
causing infection. This laid the foundation for the germ theory of disease.
Meanwhile, British surgeon Joseph Lister (Figure 1.3.4) was trying to determine the causes of post-surgical infections. Many
physicians did not give credence to the idea that microbes on their hands, on their clothes, or in the air could infect patients’
surgical wounds, despite the fact that 50% of surgical patients, on average, were dying of post-surgical infections.10 Lister,
however, was familiar with the work of Semmelweis and Pasteur; therefore, he insisted on handwashing and extreme cleanliness
during surgery. In 1867, to further decrease the incidence of post-surgical wound infections, Lister began using carbolic acid
(phenol) spray disinfectant/antiseptic during surgery. His extremely successful efforts to reduce post-surgical infection caused his
techniques to become a standard medical practice.
A few years later, Robert Koch (Figure 1.3.4) proposed a series of postulates (Koch’s postulates) based on the idea that the cause
of a specific disease could be attributed to a specific microbe. Using these postulates, Koch and his colleagues were able to
definitively identify the causative pathogens of specific diseases, including anthrax, tuberculosis, and cholera.11 Koch’s “one
microbe, one disease” concept was the culmination of the 19th century’s paradigm shift away from miasma theory and toward the
germ theory of disease.

Figure 1.3.4 : (a) Joseph Lister developed procedures for the proper care of surgical wounds and the sterilization of surgical
equipment. (b) Robert Koch established a protocol to determine the cause of infectious disease. Both scientists contributed
significantly to the acceptance of the germ theory of disease.

Exercise 1.3.3
1. Compare and contrast the miasma theory of disease with the germ theory of disease.
2. How did Joseph Lister’s work contribute to the debate between the miasma theory and germ theory and how did this
increase the success of medical procedures?
3. How did the discovery of microbes change human understanding of disease?

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 Figure 1.3.5 : (credit “swan-neck flask”: modification of work by Wellcome Images)

Key Concepts and Summary

Although cells were first observed in the 1660's by Robert Hooke, cell theory was not well accepted for another 200 years. The
work of scientists such as Schleiden, Schwann, Remak, and Virchow contributed to its acceptance.
The miasma theory of disease was widely accepted until the 19th century, when it was replaced by the germ theory of disease
thanks to the work of Semmelweis, Snow, Pasteur, Lister, and Koch, and others.

Footnotes
1. M. Schultz. “Rudolph Virchow.” Emerging Infectious Diseases 14 no. 9 (2008):1480–1481.
2. B. Kisch. “Forgotten Leaders in Modern Medicine, Valentin, Gouby, Remak, Auerbach.” Transactions of the American
Philosophical Society 44 (1954):139–317.
3. H. Harris. The Birth of the Cell. New Haven, CT: Yale University Press, 2000:133.
4. C. Webster (ed.). Biology, Medicine and Society 1840-1940. Cambridge, UK; Cambridge University Press, 1981:118–119.
5. C. Zuchora-Walske. Key Discoveries in Life Science. Minneapolis, MN: Lerner Publishing, 2015:12–13.
6. T. Embley, W. Martin. “Eukaryotic Evolution, Changes, and Challenges.” Nature Vol. 440 (2006):623–630.
7. O.G. Berg, C.G. Kurland. “Why Mitochondrial Genes Are Most Often Found in Nuclei.” Molecular Biology and Evolution 17
no. 6 (2000):951–961.
8. L. Sagan. “On the Origin of Mitosing Cells.” Journal of Theoretical Biology 14 no. 3 (1967):225–274.
9. A.E. Douglas. “The Microbial Dimension in Insect Nutritional Ecology.” Functional Ecology 23 (2009):38–47.
10. Alexander, J. Wesley. “The Contributions of Infection Control to a Century of Progress” Annals of Surgery 201:423-428, 1985.
11. S.M. Blevins and M.S. Bronze. “Robert Koch and the ‘Golden Age’ of Bacteriology.” International Journal of Infectious
Diseases. 14 no. 9 (2010): e744-e751. doi:10.1016/j.ijid.2009.12.003.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 1.3: Foundations of Modern Cell Theory is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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1.4: A Systematic Approach
Learning Objectives
Describe how microorganisms are classified and distinguished as unique species
Compare historical and current systems of taxonomy used to classify microorganisms

Once microbes became visible to humans with the help of microscopes, scientists began to realize their enormous diversity.
Microorganisms vary in all sorts of ways, including their size, their appearance, and their rates of reproduction. To study this
incredibly diverse new array of organisms, researchers needed a way to systematically organize them.

The Science of Taxonomy

Taxonomy is the classification, description, identification, and naming of living organisms. Classification is the practice of
organizing organisms into different groups based on their shared characteristics. The most famous early taxonomist was a Swedish
botanist, zoologist, and physician named Carolus Linnaeus (1701–1778). In 1735, Linnaeus published Systema Naturae, an 11-
page booklet in which he proposed the Linnaean taxonomy, a system of categorizing and naming organisms using a standard
format so scientists could discuss organisms using consistent terminology. He continued to revise and add to the book, which grew
into multiple volumes (Figure 1.4.1).

Figure 1.4.1 : Swedish botanist, zoologist, and physician Carolus Linnaeus developed a new system for categorizing plants and
animals. In this 1853 portrait by Hendrik Hollander, Linnaeus is holding a twinflower, named Linnaea borealis in his honor.
In his taxonomy, Linnaeus divided the natural world into three kingdoms: animal, plant, and mineral (the mineral kingdom was
later abandoned). Within the animal and plant kingdoms, he grouped organisms using a hierarchy of increasingly specific levels
and sublevels based on their similarities. The names of the levels in Linnaeus’s original taxonomy were kingdom, class, order,
family, genus (plural: genera), and species. Species was, and continues to be, the most specific and basic taxonomic unit.

Evolving Trees of Life (Phylogenies)

With advances in technology, other scientists gradually made refinements to the Linnaean system and eventually created new
systems for classifying organisms. In the 1800's, there was a growing interest in developing taxonomies that took into account the
evolutionary relationships, or phylogenies, of all different species of organisms on earth. One way to depict these relationships is
via a diagram called a phylogenetic tree (or tree of life). In these diagrams, groups of organisms are arranged by how closely
related they are thought to be. In early phylogenetic trees, the relatedness of organisms was inferred by their visible similarities,
such as the presence or absence of hair or the number of limbs. Now, the analysis is more complicated. Today, phylogenic analyses
include genetic, biochemical, and embryological comparisons, as will be discussed later in this chapter.
Linnaeus’s tree of life contained just two main branches for all living things: the animal and plant kingdoms. In 1866, Ernst
Haeckel, a German biologist, philosopher, and physician, proposed another kingdom, Protista, for unicellular organisms (Figure
1.4.2). He later proposed a fourth kingdom, Monera, for unicellular organisms whose cells lack nuclei, like bacteria.

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 Figure 1.4.2 : Ernst Haeckel’s rendering of the tree of life, from his 1866 book General Morphology of Organisms, contained three
kingdoms: Plantae, Protista, and Animalia. He later added a fourth kingdom, Monera, for unicellular organisms lacking a nucleus.
Nearly 100 years later, in 1969, American ecologist Robert Whittaker (1920–1980) proposed adding another kingdom—Fungi—in
his tree of life. Whittaker’s tree also contained a level of categorization above the kingdom level—the empire or superkingdom
level—to distinguish between organisms that have membrane-bound nuclei in their cells (eukaryotes) and those that do not
(prokaryotes). Empire Prokaryota contained just the Kingdom Monera. The Empire Eukaryota contained the other four kingdoms:
Fungi, Protista, Plantae, and Animalia. Whittaker’s five-kingdom tree was considered the standard phylogeny for many years.
Figure 1.4.3 shows how the tree of life has changed over time. Note that viruses are not found in any of these trees. That is because
they are not made up of cells and thus it is difficult to determine where they would fit into a tree of life.

Figure 1.4.3 : This timeline shows how the shape of the tree of life has changed over the centuries. Even today, the taxonomy of
living organisms is continually being reevaluated and refined with advances in technology.

Exercise 1.4.1

Briefly summarize how our evolving understanding of microorganisms has contributed to changes in the way that organisms
are classified.

Clinical Focus: part 2

Antibiotic drugs are specifically designed to kill or inhibit the growth of bacteria. But after a couple of days on antibiotics,
Cora shows no signs of improvement. Also, her CSF cultures came back from the lab negative. Since bacteria or fungi were
not isolated from Cora’s CSF sample, her doctor rules out bacterial and fungal meningitis. Viral meningitis is still a possibility.
However, Cora now reports some troubling new symptoms. She is starting to have difficulty walking. Her muscle stiffness has
spread from her neck to the rest of her body, and her limbs sometimes jerk involuntarily. In addition, Cora’s cognitive

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 symptoms are worsening. At this point, Cora’s doctor becomes very concerned and orders more tests on the CSF samples.

Exercise 1.4.2

What types of microorganisms could be causing Cora’s symptoms?

The Role of Genetics in Modern Taxonomy

Haeckel’s and Whittaker’s trees presented hypotheses about the phylogeny of different organisms based on readily observable
characteristics. But the advent of molecular genetics in the late 20th century revealed other ways to organize phylogenetic trees.
Genetic methods allow for a standardized way to compare all living organisms without relying on observable characteristics that
can often be subjective. Modern taxonomy relies heavily on comparing the nucleic acids (deoxyribonucleic acid [DNA] or
ribonucleic acid [RNA]) or proteins from different organisms. The more similar the nucleic acids and proteins are between two
organisms, the more closely related they are considered to be.
In the 1970's, American microbiologist Carl Woese discovered what appeared to be a “living record” of the evolution of organisms.
He and his collaborator George Fox created a genetics-based tree of life based on similarities and differences they observed in the
small subunit ribosomal RNA (rRNA) of different organisms. In the process, they discovered that a certain type of bacteria, called
archaebacteria (now known simply as archaea), were significantly different from other bacteria and eukaryotes in terms of the
sequence of small subunit rRNA. To accommodate this difference, they created a tree with three Domains above the level of
Kingdom: Archaea, Bacteria, and Eukarya (Figure 1.4.4). Genetic analysis of the small subunit rRNA suggests archaea, bacteria,
and eukaryotes all evolved from a common ancestral cell type. The tree is skewed to show a closer evolutionary relationship
between Archaea and Eukarya than they have to Bacteria.

Figure 1.4.4 : Scientists continue to use analysis of RNA, DNA, and proteins to determine how organisms are related. One
interesting, and complicating, discovery is that of horizontal gene transfer—when a gene of one species is absorbed into another
organism’s genome. Horizontal gene transfer is especially common in microorganisms and can make it difficult to determine how
organisms are evolutionarily related. Consequently, some scientists now think in terms of “webs of life” rather than “trees of life.”

Exercise 1.4.3

1. In modern taxonomy, how do scientists determine how closely two organisms are related?
2. Explain why the branches on the “tree of life” all originate from a single “trunk.”

Naming Microbes
In developing his taxonomy, Linnaeus used a system of binomial nomenclature, a two-word naming system for identifying
organisms by genus and species. For example, modern humans are in the genus Homo and have the species name sapiens, so their

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scientific name in binomial nomenclature is Homo sapiens. In binomial nomenclature, the genus part of the name is always
capitalized; it is followed by the species name, which is not capitalized. Both names are italicized (or you can use the less popular
underlining).
Taxonomic names in the 18th through 20th centuries were typically derived from Latin, since that was the common language used
by scientists when taxonomic systems were first created. Today, newly discovered organisms can be given names derived from
Latin, Greek, or English. Sometimes these names reflect some distinctive trait of the organism; in other cases, microorganisms are
named after the scientists who discovered them. The archaeon Haloquadratum walsbyi is an example of both of these naming
schemes. The genus, Haloquadratum, describes the microorganism’s saltwater habitat (halo is derived from the Greek word for
“salt”) as well as the arrangement of its square cells, which are arranged in square clusters of four cells (quadratum is Latin for
“foursquare”). The species, walsbyi, is named after Anthony Edward Walsby, the microbiologist who discovered Haloquadratum
walsbyi in in 1980. While it might seem easier to give an organism a common descriptive name—like a red-headed woodpecker—
we can imagine how that could become problematic. What happens when another species of woodpecker with red head coloring is
discovered? The systematic nomenclature scientists use eliminates this potential problem by assigning each organism a single,
unique two-word name that is recognized by scientists all over the world.
In this text, we will typically abbreviate an organism’s genus and species after its first mention. The abbreviated form is simply the
first initial of the genus, followed by a period and the full name of the species. For example, the bacterium Escherichia coli is
shortened to E. coli in its abbreviated form. You will encounter this same convention in other scientific texts as well.

Bergey’s Manuals
Whether in a tree or a web, microbes can be difficult to identify and classify. Without easily observable macroscopic features like
feathers, feet, or fur, scientists must capture, grow, and devise ways to study their biochemical properties to differentiate and
classify microbes. Despite these hurdles, a group of microbiologists created and updated a set of manuals for identifying and
classifying microorganisms. First published in 1923 and since updated many times, Bergey’s Manual of Determinative Bacteriology
and Bergey’s Manual of Systematic Bacteriology are the standard references for identifying and classifying different prokaryotes.
Because so many bacteria look identical, methods based on nonvisual characteristics must be used to identify them. For example,
biochemical tests can be used to identify chemicals unique to certain species. Likewise, serological tests can be used to identify
specific antibodies that will react against the proteins found in certain species. Ultimately, DNA and rRNA sequencing can be used
both for identifying a particular bacterial species and for classifying newly discovered species.

Exercise 1.4.4

1. What is binomial nomenclature and why is it a useful tool for naming organisms?
2. Explain why a resource like one of Bergey’s manuals would be helpful in identifying a microorganism in a sample.

Same Name, Different Strain

Within one species of microorganism, there can be several subtypes called strains. While different strains may be nearly
identical genetically, they can have very different attributes. The bacterium Escherichia coli is infamous for causing food
poisoning and traveler’s diarrhea. However, there are actually many different strains of E. coli, and they vary in their ability to
cause disease.
One pathogenic (disease-causing) E. coli strain that you may have heard of is E. coli O157:H7. In humans, infection from E.
coli O157:H7 can cause abdominal cramps and diarrhea. Infection usually originates from contaminated water or food,
particularly raw vegetables and undercooked meat. In the 1990's, there were several large outbreaks of E. coli O157:H7
thought to have originated in undercooked hamburgers.
While E. coli O157:H7 and some other strains have given E. coli a bad name, most E. coli strains do not cause disease. In fact,
some can be helpful. Different strains of E. coli found naturally in our gut help us digest our food, provide us with some
needed chemicals, and fight against pathogenic microbes.

Key Concepts and Summary

Carolus Linnaeus developed a taxonomic system for categorizing organisms into related groups.
Binomial nomenclature assigns organisms Latinized scientific names with a genus and species designation.

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 A phylogenetic tree is a way of showing how different organisms are thought to be related to one another from an evolutionary
standpoint.
The first phylogenetic tree contained kingdoms for plants and animals; Ernst Haeckel proposed adding kingdom for protists.
Robert Whittaker’s tree contained five kingdoms: Animalia, Plantae, Protista, Fungi, and Monera.
Carl Woese used small subunit ribosomal RNA to create a phylogenetic tree that groups organisms into three domains based on
their genetic similarity.
Bergey’s manuals of determinative and systemic bacteriology are the standard references for identifying and classifying
bacteria, respectively.
Bacteria can be identified through biochemical tests, DNA/RNA analysis, and serological testing methods.

Glossary
binomial nomenclature
a universal convention for the scientific naming of organisms using Latinized names for genus and species

eukaryote
an organism made up of one or more cells that contain a membrane-bound nucleus and organelles

phylogeny
the evolutionary history of a group of organisms

prokaryote
an organism whose cell structure does not include a membrane-bound nucleus

taxonomy
the classification, description, identification, and naming of living organisms
strain
organisms which vary even though they are within the same species that, particularly common with bacteria due to horizontal
gene transfer

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 1.4: A Systematic Approach is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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1.5: Types of Microorganisms
Learning Objectives
List the various types of microorganisms and describe their defining characteristics
Give examples of different types of cellular and viral microorganisms and infectious agents
Provide an overview of the field of microbiology

Most microbes are unicellular and small enough that they require artificial magnification to be seen. However, there are some
unicellular microbes that are visible to the naked eye, and some multicellular organisms that are microscopic. An object must
measure about 100 micrometers (µm) to be visible without a microscope, but most microorganisms are many times smaller than
that. For some perspective, consider that a typical animal cell measures roughly 10 µm across but is still microscopic. Bacterial
cells are typically about 1 µm, and viruses can be 10 times smaller than bacteria (Figure 1.5.1). See Table 1.5.1 for units of length
used in microbiology.

Figure 1.5.1 : The relative sizes of various microscopic and nonmicroscopic objects. Note that a typical virus measures about 100
nm, 10 times smaller than a typical bacterium (~1 µm), which is at least 10 times smaller than a typical plant or animal cell (~10–
100 µm). An object must measure about 100 µm to be visible without a microscope.
Figure 1.5.1 : Units of Length Commonly Used in Microbiology
Metric Unit Meaning of Prefix Metric Equivalent

meter (m) — 1 m = 100 m

decimeter (dm) 1/10 1 dm = 0.1 m = 10−1 m

centimeter (cm) 1/100 1 cm = 0.01 m = 10−2 m

millimeter (mm) 1/1000 1 mm = 0.001 m = 10−3 m

micrometer (μm) 1/1,000,000 1 μm = 0.000001 m = 10−6 m

nanometer (nm) 1/1,000,000,000 1 nm = 0.000000001 m = 10−9 m

Microorganisms differ from each other not only in size, but also in structure, habitat, metabolism, and many other characteristics.
While we typically think of microorganisms as being unicellular, there are also many multicellular organisms that are too small to
be seen without a microscope. Some microbes, such as viruses, are even acellular (not composed of cells).
Microorganisms are found in each of the three domains of life: Archaea, Bacteria, and Eukarya. Microbes within the domains
Bacteria and Archaea are all prokaryotes (their cells lack a nucleus), whereas microbes in the domain Eukarya are eukaryotes (their
cells have a nucleus). Other microbes, such as viruses, do not fall within any of the three domains of life. In this section, we will

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briefly introduce each of the broad groups of microbes. Later chapters will go into greater depth about the diverse species within
each group.

Prokaryotic Microorganisms: Bacteria

Bacteria are found in nearly every habitat on earth, including within and on humans. Most bacteria are harmless or helpful, but
some are pathogens, causing disease in humans and other animals. Bacteria are prokaryotic because their genetic material (DNA) is
not housed within a true nucleus. Most bacteria have cell walls that contain peptidoglycan. Bacteria are often described in terms of
their general shape. Common shapes include spherical (coccus), rod-shaped (bacillus), or curved (spirillum, spirochete, or vibrio).
Figure 1.5.2 shows examples of these shapes.

Figure 1.5.2 : Common bacterial shapes. Note how coccobacillus is a combination of spherical (coccus) and rod-shaped (bacillus).
(credit “Coccus”: modification of work by Janice Haney Carr, Dr. Richard Facklam, Centers for Disease Control and Prevention;
credit “Bacillus”: modification of work by “Elapied”/Wikimedia Commons)
They have a wide range of metabolic capabilities and can grow in a variety of environments, using different combinations of
nutrients. Some bacteria are photosynthetic, such as oxygenic cyanobacteria and anoxygenic green sulfur and green nonsulfur
bacteria; these bacteria use energy derived from sunlight, and fix carbon dioxide for growth. Other types of bacteria are
nonphotosynthetic, obtaining their energy from organic or inorganic compounds in their environment.

Prokaryotic Microorganisms: Archaea

Archaea are also unicellular prokaryotic organisms. Archaea and bacteria have different evolutionary histories, as well as
significant differences in genetics, metabolic pathways, and the composition of their cell walls and membranes. Unlike most
bacteria, archaeal cell walls do not contain peptidoglycan, but their cell walls are often composed of a similar substance called
pseudopeptidoglycan. Like bacteria, archaea are found in nearly every habitat on earth, even extreme environments that are very
cold, very hot, very basic, or very acidic (Figure 1.5.3). Some archaea live in the human body, but none have been shown to be
human pathogens.

Figure 1.5.3 : Some archaea live in extreme environments, such as the Morning Glory pool, a hot spring in Yellowstone National
Park. The color differences in the pool result from the different communities of microbes that are able to thrive at various water
temperatures.

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 Exercise 1.5.1

1. What are the two main types of prokaryotic organisms?

2. Name some of the defining characteristics of each type.

Eukaryotic Microorganisms
The domain Eukarya contains all eukaryotes, including uni- or multicellular eukaryotes such as protists, fungi, plants, and animals.
The major defining characteristic of eukaryotes is that their cells contain a nucleus.

Protists
Protists are unicellular eukaryotes that are not plants, animals, or fungi. This is a highly diverse group. Algae and protozoa are
examples of protists groups.
Algae (singular: alga) are plant-like protists that can be either unicellular or multicellular (Figure 1.5.4). Their cells are surrounded
by cell walls made of cellulose, a type of carbohydrate. Algae are photosynthetic organisms that extract energy from the sun and
release oxygen and carbohydrates into their environment. Because other organisms can use their waste products for energy, algae
are important parts of many ecosystems. Many consumer products contain ingredients derived from algae, such as carrageenan or
alginic acid, which are found in some brands of ice cream, salad dressing, beverages, lipstick, and toothpaste. A derivative of algae
also plays a prominent role in the microbiology laboratory. Agar, a gel derived from algae, can be mixed with various nutrients and
used to grow microorganisms in a Petri dish. Algae are also being developed as a possible source for biofuels.

Figure 1.5.4 : Assorted diatoms, a kind of algae, live in annual sea ice in McMurdo Sound, Antarctica. Diatoms range in size from 2
μm to 200 μm and are visualized here using light microscopy. (credit: National Oceanic and Atmospheric Administration)
Protozoa (singular: protozoan) are protists that make up the backbone of many food webs by providing nutrients for other
organisms. Protozoa are very diverse, though often more animal-like. Some protozoa move with help from hair-like structures
called cilia or whip-like structures called flagella. Others extend part of their cell membrane and cytoplasm to propel themselves
forward. These cytoplasmic extensions are called pseudopods (“false feet”). A few protozoa are photosynthetic; many others feed
on organic material. Some are free-living, whereas others are parasitic, only able to survive by extracting nutrients from a host
organism. Most protozoa are harmless, but some are pathogens that can cause disease in animals or humans (Figure 1.5.5).

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 Figure 1.5.5 : Giardia lamblia, an intestinal protozoan parasite that infects humans and other mammals, causing severe diarrhea.
(credit: modification of work by Centers for Disease Control and Prevention)

Fungi
Fungi (singular: fungus) are also eukaryotes. Some multicellular fungi, such as mushrooms, resemble plants, but they are actually
quite different. Fungi are not photosynthetic, and their cell walls are usually made out of chitin rather than cellulose. The
microscopic fungi broadly get split into two groups: yeasts and molds.
Yeasts are unicellular fungi included within the study of microbiology. There are more than 1000 known species. Yeasts are found
in many different environments, from the deep sea to the human navel. Some yeasts have beneficial uses, such as causing bread to
rise and beverages to ferment; but yeasts can also cause food to spoil. Some even cause diseases, such as vaginal yeast infections
and oral thrush (Figure 1.5.6).

Figure 1.5.6 : Candida albicans is a unicellular fungus, or yeast. It is the causative agent of vaginal yeast infections as well as oral
thrush, a yeast infection of the mouth that commonly afflicts infants. C. albicans has a morphology similar to that of coccus
bacteria; however, yeast is a eukaryotic organism (note the nuclei) and is much larger. (credit: modification of work by Centers for
Disease Control and Prevention)
Molds are colonial (many attached single cells) that make long filaments which form visible colonies (Figure 1.5.7). Molds are
found in many different environments, from soil to rotting food to dank bathroom corners. Molds play a critical role in the
decomposition of dead plants and animals. Some molds can cause allergies, and others produce disease-causing metabolites called
mycotoxins. Molds have been used to make pharmaceuticals, including penicillin, which is one of the most commonly prescribed
antibiotics, and cyclosporine, used to prevent organ rejection following a transplant.

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 Figure 1.5.7 : Large colonies of microscopic fungi can often be observed with the naked eye, as seen on the surface of these moldy
oranges.

Exercise 1.5.2

1. Name two types of protists and two types of fungi.

2. Name some of the defining characteristics of each type.

Helminths
Multicellular parasitic worms called helminths are not technically microorganisms, as most are large enough to see without a
microscope. However, these worms fall within the field of microbiology because diseases caused by helminths involve microscopic
eggs and larvae. These features place them in the animal kingdom like us. One example of a helminth is the guinea worm, or
Dracunculus medinensis, which causes dizziness, vomiting, diarrhea, and painful ulcers on the legs and feet when the worm works
its way out of the skin (Figure 1.5.8). Infection typically occurs after a person drinks water containing water fleas infected by
guinea-worm larvae. In the mid-1980's, there were an estimated 3.5 million cases of guinea-worm disease, but the disease has been
largely eradicated. In 2014, there were only 126 cases reported, thanks to the coordinated efforts of the World Health Organization
(WHO) and other groups committed to improvements in drinking water sanitation.1,2

Figure 1.5.8 : The beef tapeworm, Taenia saginata, infects both cattle and humans. T. saginata eggs are microscopic (around 50
µm), but adult worms like the one shown here can reach 4–10 m, taking up residence in the digestive system. (b) An adult guinea
worm, Dracunculus medinensis, is removed through a lesion in the patient’s skin by winding it around a matchstick. (credit b:
modification of work by Centers for Disease Control and Prevention)

Acellular, Infectious Particles

Viruses are acellular microorganisms, which means they are not composed of cells. Essentially, a virus consists of proteins and
genetic material—either DNA or RNA, but never both—that are inert outside of a host organism. However, by incorporating
themselves into a host cell, viruses are able to co-opt the host’s cellular mechanisms to multiply and infect other hosts. Viruses can
infect all types of cells, from human cells to the cells of other microorganisms. In humans, viruses are responsible for numerous
diseases, from the common cold to deadly Ebola (Figure 1.5.9). However, many viruses do not cause disease. There are also a few
other examples of non-virus infectious particles, like the prions seen with the clinial focus for this chapter.

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 Figure 1.5.9 : (a) Members of the Coronavirus family can cause respiratory infections like the common cold, severe acute
respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). Here they are viewed under a transmission electron
microscope (TEM). (b) Ebolavirus, a member of the Filovirus family, as visualized using a TEM. (credit b: modification of work
by Thomas W. Geisbert).

Exercise 1.5.3

1. Are helminths microorganisms? Explain why or why not.

2. How are viruses different from other microorganisms?

Microbiology as a Field of Study

Microbiology is a broad term that encompasses the study of all different types of microorganisms. But in practice, microbiologists
tend to specialize in one of several subfields. For example: bacteriology is the study of bacteria; mycology is the study of fungi;
protozoology is the study of protozoa; parasitology is the study of helminths and other parasites; and virology is the study of
viruses (Figure 1.5.10). Immunology, the study of the immune system, is often included in the study of microbiology because host–
pathogen interactions are central to our understanding of infectious disease processes. Microbiologists can also specialize in certain
areas of microbiology, such as clinical microbiology, environmental microbiology, applied microbiology, or food microbiology. In
this textbook, we are primarily concerned with clinical applications of microbiology, but since the various subfields of
microbiology are highly interrelated, we will often discuss applications that are not strictly clinical.

Figure 1.5.10 : A virologist samples eggs from this nest to be tested for the influenza A virus, which causes avian flu in birds.
(credit: Don Becker)

Bio-ethics in Microbiology
In the 1940's, the U.S. government was looking for a solution to a medical problem: the prevalence of sexually transmitted
diseases (STDs) among soldiers. Several now-infamous government-funded studies used human subjects to research common
STDs and treatments. In one such study, American researchers intentionally exposed more than 1300 human subjects in
Guatemala to syphilis, gonorrhea, and chancroid to determine the ability of penicillin and other antibiotics to combat these
diseases. Subjects of the study included Guatemalan soldiers, prisoners, prostitutes, and psychiatric patients—none of whom

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 were informed that they were taking part in the study. Researchers exposed subjects to STDs by various methods, from
facilitating intercourse with infected prostitutes to inoculating subjects with the bacteria known to cause the diseases. This
latter method involved making a small wound on the subject’s genitals or elsewhere on the body, and then putting bacteria
directly into the wound.3 In 2011, a U.S. government commission tasked with investigating the experiment revealed that only
some of the subjects were treated with penicillin, and 83 subjects died by 1953, likely as a result of the study.4
Unfortunately, this is one of many horrific examples of microbiology experiments that have violated basic ethical standards.
Even if this study had led to a life-saving medical breakthrough (it did not), few would argue that its methods were ethically
sound or morally justifiable. But not every case is so clear cut. Professionals working in clinical settings are frequently
confronted with ethical dilemmas, such as working with patients who decline a vaccine or life-saving blood transfusion. These
are just two examples of life-and-death decisions that may intersect with the religious and philosophical beliefs of both the
patient and the health-care professional.
No matter how noble the goal, microbiology studies and clinical practice must be guided by a certain set of ethical principles.
Studies must be done with integrity. Patients and research subjects provide informed consent (not only agreeing to be treated or
studied but demonstrating an understanding of the purpose of the study and any risks involved). Patients’ rights must be
respected. Procedures must be approved by an institutional review board. When working with patients, accurate record-
keeping, honest communication, and confidentiality are paramount. Animals used for research must be treated humanely, and
all protocols must be approved by an institutional animal care and use committee. These are just a few of the ethical principles
explored in the Eye on Ethics boxes throughout this book.

Clinical Focus - Resolution

Cora’s CSF samples show no signs of inflammation or infection, as would be expected with a viral infection. However, there is
a high concentration of a particular protein, 14-3-3 protein, in her CSF. An electroencephalogram (EEG) of her brain function
is also abnormal. The EEG resembles that of a patient with a neurodegenerative disease like Alzheimer’s or Huntington’s, but
Cora’s rapid cognitive decline is not consistent with either of these. Instead, her doctor concludes that Cora has Creutzfeldt-
Jakob disease (CJD), a type of transmissible spongiform encephalopathy (TSE).
CJD is an extremely rare disease, with only about 300 cases in the United States each year. It is not caused by a bacterium,
fungus, or virus, but rather by prions—which do not fit neatly into any particular category of microbe. Like viruses, prions are
not found on the tree of life because they are acellular. Prions are extremely small, about one-tenth the size of a typical virus.
They contain no genetic material and are composed solely of a type of abnormally folded protein.
CJD can have several different causes. It can be acquired through exposure to the brain or nervous-system tissue of an infected
person or animal. Consuming meat from an infected animal is one way such exposure can occur. There have also been rare
cases of exposure to CJD through contact with contaminated surgical equipment5 and from cornea and growth-hormone donors
who unknowingly had CJD.6,7 In rare cases, the disease results from a specific genetic mutation that can sometimes be
hereditary. However, in approximately 85% of patients with CJD, the cause of the disease is spontaneous (or sporadic) and has
no identifiable cause.8 Based on her symptoms and their rapid progression, Cora is diagnosed with sporadic CJD.
Unfortunately for Cora, CJD is a fatal disease for which there is no approved treatment. Approximately 90% of patients die
within 1 year of diagnosis.9 Her doctors focus on limiting her pain and cognitive symptoms as her disease progresses. Eight
months later, Cora dies. Her CJD diagnosis is confirmed with a brain autopsy.

Key Concepts and Summary

Microorganisms are very diverse and are found in all three domains of life: Archaea, Bacteria, and Eukarya.
Archaea and bacteria are classified as prokaryotes because they lack a cellular nucleus. Archaea differ from bacteria in
evolutionary history, genetics, metabolic pathways, and cell wall and membrane composition.
Archaea inhabit nearly every environment on earth, but no archaea have been identified as human pathogens.
Eukaryotes studied in microbiology include algae, protozoa, fungi, and helminths.
Algae are plant-like organisms that can be either unicellular or multicellular, and derive energy via photosynthesis.
Protozoa are unicellular organisms with complex cell structures; most are motile.
Microscopic fungi include molds and yeasts.

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 Helminths are multicellular parasitic worms. They are included in the field of microbiology because their eggs and larvae are
often microscopic.
Viruses are acellular microorganisms that require a host to reproduce.
The field of microbiology is extremely broad. Microbiologists typically specialize in one of many subfields, but all health
professionals need a solid foundation in clinical microbiology.

Footnotes
1. C. Greenaway “Dracunculiasis (Guinea Worm Disease).” Canadian Medical Association Journal 170 no. 4 (2004):495–500.
2. World Health Organization. “Dracunculiasis (Guinea-Worm Disease).” WHO. 2015.
http://www.who.int/mediacentre/factsheets/fs359/en/. Accessed October 2, 2015.
3. Kara Rogers. “Guatemala Syphilis Experiment: American Medical Research Project”. Encylopaedia Britannica.
www.britannica.com/event/Guat...lis-experiment. Accessed June 24, 2015.
4. Susan Donaldson James. “Syphilis Experiments Shock, But So Do Third-World Drug Trials.” ABC World News. August 30,
2011. http://abcnews.go.com/Health/guatema...ry?id=14414902. Accessed June 24, 2015.
5. Greg Botelho. “Case of Creutzfeldt-Jakob Disease Confirmed in New Hampshire.” CNN. 2013.
http://www.cnn.com/2013/09/20/health...brain-disease/.
6. P. Rudge et al. “Iatrogenic CJD Due to Pituitary-Derived Growth Hormone With Genetically Determined Incubation Times of
Up to 40 Years.” Brain 138 no. 11 (2015): 3386–3399.
7. J.G. Heckmann et al. “Transmission of Creutzfeldt-Jakob Disease via a Corneal Transplant.” Journal of Neurology,
Neurosurgery & Psychiatry 63 no. 3 (1997): 388–390.
8. National Institute of Neurological Disorders and Stroke. “Creutzfeldt-Jakob Disease Fact Sheet.” NIH. 2015.
http://www.ninds.nih.gov/disorders/c....htm#288133058.
9. National Institute of Neurological Disorders and Stroke. “Creutzfeldt-Jakob Disease Fact Sheet.” NIH. 2015.
http://www.ninds.nih.gov/disorders/c....htm#288133058. Accessed June 22, 2015.

Glossary
acellular
not consisting of a cell or cells

algae
(singular: alga) any of various unicellular and multicellular photosynthetic eukaryotic organisms; distinguished from plants by
their lack of vascular tissues and organs

archaea
any of various unicellular prokaryotic microorganisms, typically having cell walls containing pseudopeptidoglycan

bacteria
(singular: bacterium) any of various unicellular prokaryotic microorganisms typically (but not always) having cell wells that
contain peptidoglycan

eukarya
the domain of life that includes all unicellular and multicellular organisms with cells that contain membrane-bound nuclei and
organelles

fungi
(singular: fungus) any of various unicellular or multicellular eukaryotic organisms, typically having cell walls made out of
chitin and lacking photosynthetic pigments, vascular tissues, and organs

helminth
a multicellular parasitic worm

immunology
the study of the immune system

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infectious particle
any type of acellular microorganism

mold
a multicellular fungus, typically made up of long filaments

mycology
the study of fungi

parasitology
the study of parasites

pathogen
a disease-causing microorganism

protist
a unicellular eukaryotic microorganism, usually a type of algae or protozoa

protozoan
(plural: protozoa) a unicellular eukaryotic organism, usually motile

protozoology
the study of protozoa

virology
the study of viruses

virus
an acellular microorganism, consisting of proteins and genetic material (DNA or RNA), that can replicate itself by infecting a
host cell

yeast
any unicellular fungus

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 1.5: Types of Microorganisms is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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1.6: Tools and Media Used for Bacterial Growth
Learning Objectives
Identify and describe tools used in the laboratory setting.
Describe general protocols for the inoculation, incubation and description of microbes in or on media
Identify the safety precautions we need to take in a general lab setting.
Identify and describe culture media for the growth of bacteria, including examples of all-purpose media, enriched, selective,
differential, defined, and enrichment media

The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive
under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and
environmental requirements and the diversity of these requirements among different species.

Microbiology Toolbox
Because individual microbes are generally too small to be seen with the naked eye, the science of microbiology is dependent on
technology that can artificially enhance the capacity of our natural senses of perception. Early microbiologists like Pasteur and
Koch had fewer tools at their disposal than are found in modern laboratories, making their discoveries and innovations that much
more impressive. Later chapters of this text will explore many applications of technology in depth, but for now, here is a brief
overview of some of the fundamental tools of the microbiology lab.
Growth media are used to grow microorganisms in a lab setting. Some media are liquids; others are more solid or gel-like. A
growth medium provides nutrients, including water, various salts, a source of carbon (like glucose), and a source of nitrogen
and amino acids (like yeast extract) so microorganisms can grow and reproduce. Ingredients in a growth medium can be
modified to grow unique types of microorganisms, or to help separate or identify several microbes grown next to each other.
Test tubes are cylindrical plastic or glass tubes with rounded bottoms and open tops. They can be used to grow microbes in
broth, or semisolid or solid growth media.(Figure 1.6.1

Figure 1.6.1 : The caps are missing, but this image shows the bottom of several possible options. (credit Nazzy Pakpour and
Sharon Horgan, California State University)

A Petri dish is a flat-lidded dish that is typically 10–11 centimeters (cm) in diameter and 1–1.5 cm high. Petri dishes made out
of either plastic or glass are used to hold thin layers of solid media, usually solidified with agar. (Figure 1.6.2)
A Bunsen burner is a metal apparatus that creates a flame that can be used to sterilize pieces of equipment. A rubber tube
carries gas (fuel) to the burner. In many labs, Bunsen burners are being phased out in favor of infrared microincinerators, which
serve a similar purpose without the safety risks of an open flame.
An inoculation loop is a handheld tool that ends in a small wire loop (Figure 1.6.2). The loop can be used to streak
microorganisms on agar in a Petri dish or to transfer them from one test tube to another. Before each use, the inoculation loop
must be sterilized so cultures do not become contaminated, and re-sterilized after the transfer.
An inoculation needle is another handheld tool that is used for specific solid or semi-solid inoculations. It looks similar to the
loop except that it is a straight point. It can help to determine whether an organism is motile (moves). Like the inoculation
needle, it needs to be sterilized before every use, and afterwards too.
Sterile cotton swabs are another common tool of microbiology, useful for collecting samples from surfaces. However they are
not able to be reused.
Microscopes produce magnified images of microorganisms, human cells and tissues, and many other types of specimens too
small to be observed with the naked eye. Microscopes will be discussed more in chapter 3.

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 Stains and dyes are used to add color to microbes so they can be better observed under a microscope. Some dyes can be used on
living microbes, whereas others require that the specimens be fixed with chemicals or heat before staining. Some stains only
work on certain types of microbes because of differences in their cellular chemical composition. Staining will be discussed
more in chapter 3.

Figure 1.6.2 : (a) This Petri dish filled with agar has been streaked with Legionella, the bacterium responsible for causing
Legionnaire’s disease. (b) An inoculation loop like this one can be used to streak bacteria on agar in a Petri dish. (credit a:
modification of work by Centers for Disease Control and Prevention; credit b: modification of work by Jeffrey M. Vinocur)

Inoculating and Incubating Media

Aseptic technique is always observed when transferring and inoculating media after applying a label. The inoculation is the
introduction of a microbe into "clean" media (media free from other microbes). The instrument used is always sterile or sterilized
in a microincincerator or Bunsen Burner first, then used to pick up the sample and applied to the new media. The instrument should
either be disposed of or re-sterilized after each inoculation. This is the best procedure to try to ensure a pure culture- a culture with
only one microbe growing. Mixed cultures (purposefully adding multiple organisms) are occasionally use to show how isolation
streaking can be used to separate organisms. The goal is to avoid a contaminated culture, where unwanted microbes are growing
next to or on top of the desired organism.
After the inoculation, the microbe will need to be incubated at its preferred temperature and with its preferred oxygen gas exposure
for a number of hours or days. Generally the incubation times are shorter for prokaryotes than for eukaryotes. Test tubes are always
incubated in a rack or other holder upright, while plates are incubated upside down.

Figure 1.6.3 : Examples of how to use a broth culture to inoculate other types of media. You could also use a agar plate with
isolated colonies as a sample. (credit Nazzy Pakpour and Sharon Horgan, California State University)

Describing the Growth of Microbes in Media

Once a microbe has been grown on a plate or in a test tube, they need to be observed for various characteristics. Broth cultures are
the easiest to describe as they have the fewest options. Clear broth generally indicates no growth. Cloudy (turbid) broth has growth
in it, obscuring the light coming through. Sometimes this growth is only at the top of the tube (pellicle) or only at the bottom
(sediment). The last possibility is that the broth has flakes of growth throughout.

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 Figure 1.6.4 : The caps are missing, but this image shows the bottom of several possible growth patterns in broth. (credit:
Microbiology Lab I, Libretext Ancillary material)

For growth on plates, the technique used to inoculate the plate does make a difference. If the plate was inoculated using a single
line inoculation, generally the optical property (opaque, translucent or transparent), overall texture (dry, moist or oily) and the
colors of both the microbe and the media are observed. For isolated colonies, especially when identification is desired, more
observations are required. When observing isolated colonies we generally describe the following:
general size (small, medium or large can be used or you can measure its diameter in mm)
general shape/form (circular, irregular, filamentous, rhizoid)
optical properties (opaque, translucent or transparent)
the color of the colony (or colors)
any discoloration of the agar under or around the colony
the edge or margin of the colony is described in terms of how smooth it is.
the surface should be described in terms of both texture (dry, moist or oily) and smoothness of the surface (smooth, contoured,
radiating, concentric, rugose)
elevation as seen from a side-on or profile (raised, convex, flat, umbonate, crateriform)

Figure 1.6.5 : Common terms used to describe isolated colonies. (credit form, elevation and margin: McLaughlin and Peterson,
Queensborough Community College; credit surface of colony: Gray Kaiser, Community College of Baltimore County)

Laboratory Biological Safety Levels

For researchers or laboratory personnel working with pathogens, the risks associated with specific pathogens determine the levels
of cleanliness and control required. The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health
(NIH) have established four classification levels, called “biological safety levels” (BSLs). Each BSL requires a different level of
biocontainment to prevent contamination and spread of infectious agents to laboratory personnel and, ultimately, the community.
BSL-1 agents are those that generally do not cause infection in healthy human adults. These include noninfectious bacteria, such as
nonpathogenic strains of Escherichia coli and Bacillus subtilis, and viruses known to infect bacteria (phages) or animals other than
humans, such as baculoviruses (insect viruses). Because working with BSL-1 agents poses very little risk, few precautions are
necessary. Laboratory workers use standard aseptic technique and may work with these agents at an open laboratory bench or table,
wearing personal protective equipment (PPE) such as a laboratory coat, goggles, and gloves, as needed. Cleaning the area before

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and after working with a disinfectant like bleach is generally required. Other than a sink for handwashing and a door separating the
laboratory from the rest of the building, no additional modifications are needed.
BSL-2 through BSL-4 organism require higher amounts of containment. These will be discussed further in chapter 11.

Handwashing the Right Way

Handwashing is critical for public health and should be emphasized in a clinical setting. For the general public, the CDC
recommends handwashing before, during, and after food handling; before eating; before and after interacting with someone
who is ill; before and after treating a wound; after using the toilet or changing diapers; after coughing, sneezing, or blowing the
nose; after handling garbage; and after interacting with an animal, its feed, or its waste. Figure 1.6.6 illustrates the five steps of
proper handwashing recommended by the CDC.
Handwashing is even more important for health-care workers, who should wash their hands thoroughly between every patient
contact, after the removal of gloves, after contact with bodily fluids and potentially infectious fomites, and before and after
assisting a surgeon with invasive procedures. Even with the use of proper surgical attire, including gloves, scrubbing for
surgery is more involved than routine handwashing. The goal of surgical scrubbing is to reduce the normal microbiota on the
skin’s surface to prevent the introduction of these microbes into a patient’s surgical wounds.
There is no single widely accepted protocol for surgical scrubbing. Protocols for length of time spent scrubbing may depend on
the antimicrobial used; health-care workers should always check the manufacturer’s recommendations. According to the
Association of Surgical Technologists (AST), surgical scrubs may be performed with or without the use of brushes (Figure
1.6.6).

Figure 1.6.6 : (a) The CDC recommends five steps as part of typical handwashing for the general public. (b) Surgical scrubbing
is more extensive, requiring scrubbing starting from the fingertips, extending to the hands and forearms, and then up beyond
the elbows, as shown here. (credit a: modification of work by World Health Organization)

To learn more about proper handwashing, visit the CDC’s website.

Nutritional Requirements
The number of available media to grow bacteria is considerable. Some media are considered general all-purpose media and support
growth of a large variety of organisms. A prime example of an all-purpose medium is trypticase soy broth (TSB). Specialized
media are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. One type, enriched
media, contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms, organisms
that cannot make certain nutrients and require them to be added to the medium.

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When the complete chemical composition of a medium is known, it is called a chemically defined medium. For example, in EZ
medium, all individual chemical components are identified and the exact amounts of each is known. In complex media, which
contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. Amounts of
individual components are undetermined and variable. Nutrient broth, such as trypticase soy broth, and brain heart infusion, are all
examples of complex media.
Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying
nutrients and reducing competition are called selective media. An example of a selective medium is MacConkey agar. It contains
bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative
bacteria, particularly the Enterobacteriaceae family. The species within this family are commonly named enterics, reside in the
intestine, and are adapted to the presence of bile salts. The enrichment cultures foster the preferential growth of a desired
microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want to isolate bacteria that
break down crude oil, hydrocarbonoclastic bacteria, sequential subculturing (taking a isolated colony and continue to grow it) in a
medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria.
Media that is differential makes it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the
color of the medium. Color changes are the result of end products created by interaction of bacterial enzymes with differential
substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium. In Figure 1.6.7, the
differential fermentation of lactose can be observed on MacConkey agar. The lactose fermenters produce acid, which turns the
medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns
to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria
by biochemical methods.

Figure 1.6.7 : On this MacConkey agar plate, the lactose-fermenter E. coli colonies are bright pink. Serratia marcescens, which
does not ferment lactose, forms a cream-colored streak on the tan medium. (credit: American Society for Microbiology)

Exercise 1.6.1
1. Distinguish general, specialized and enriched media.
2. Distinguish complex and chemically defined media.
3. Distinguish selective and differential media.
4. Compare the compositions of EZ medium and sheep blood agar.

The End-of-Year Picnic

The microbiology department is celebrating the end of the school year in May by holding its traditional picnic on the green.
The speeches drag on for a couple of hours, but finally all the faculty and students can dig into the food: chicken salad,
tomatoes, onions, salad, and custard pie. By evening, the whole department, except for two vegetarian students who did not eat
the chicken salad, is stricken with nausea, vomiting, retching, and abdominal cramping. Several individuals complain of
diarrhea. One patient shows signs of shock (low blood pressure). Blood and stool samples are collected from patients, and an
analysis of all foods served at the meal is conducted.
Bacteria can cause gastroenteritis (inflammation of the stomach and intestinal tract) either by colonizing and replicating in the
host, which is considered an infection, or by secreting toxins, which is considered intoxication. Signs and symptoms of

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 infections are typically delayed, whereas intoxication manifests within hours, as happened after the picnic.
Blood samples from the patients showed no signs of bacterial infection, which further suggests that this was a case of
intoxication. Since intoxication is due to secreted toxins, bacteria are not usually detected in blood or stool samples.
MacConkey agar and sorbitol-MacConkey agar plates and xylose-lysine-deoxycholate (XLD) plates were inoculated with stool
samples and did not reveal any unusually colored colonies, and no black colonies or white colonies were observed on XLD. All
lactose fermenters on MacConkey agar also ferment sorbitol. These results ruled out common agents of food-borne illnesses:
E. coli, Salmonella spp., and Shigella spp.
Analysis of the chicken salad revealed an abnormal number of gram-positive cocci arranged in clusters (Figure 1.6.8). A
culture of the gram-positive cocci releases bubbles when mixed with hydrogen peroxide. The culture turned mannitol salt agar
yellow after a 24-hour incubation.
All the tests point to Staphylococcus aureus as the organism that secreted the toxin. Samples from the salad showed the
presence of gram-positive cocci bacteria in clusters. The colonies were positive for catalase. The bacteria grew on mannitol salt
agar fermenting mannitol, as shown by the change to yellow of the medium. The pH indicator in mannitol salt agar is phenol
red, which turns to yellow when the medium is acidified by the products of fermentation.
The toxin secreted by S. aureus is known to cause severe gastroenteritis. The organism was probably introduced into the salad
during preparation by the food handler and multiplied while the salad was kept in the warm ambient temperature during the
speeches.

Figure 1.6.8 : Gram-positive cocci in clusters. (credit: Centers for Disease Control and Prevention)

Exercise 1.6.2
1. What are some other factors that might have contributed to rapid growth of S. aureus in the chicken salad?
2. Why would S. aureus not be inhibited by the presence of salt in the chicken salad?

Identifying Bacteria by using API Testing Panels

Identification of a microbial isolate is essential for the proper diagnosis and appropriate treatment of patients. Scientists have
developed techniques that identify bacteria according to their biochemical characteristics. Typically, they either examine the
use of specific carbon sources as substrates for fermentation or other metabolic reactions, or they identify fermentation
products or specific enzymes present in reactions. In the past, microbiologists have used individual test tubes and plates to
conduct biochemical testing. However, scientists, especially those in clinical laboratories, now more frequently use plastic,
disposable, multi-test panels that contain a number of miniature reaction tubes, each typically including a specific substrate and
pH indicator. After inoculation of the test panel with a small sample of the microbe in question and incubation, scientists can
compare the results to a database that includes the expected results for specific biochemical reactions for known microbes, thus
enabling rapid identification of a sample microbe. These test panels have allowed scientists to reduce costs while improving
efficiency and reproducibility by performing a larger number of tests simultaneously.

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 Many commercial, miniaturized biochemical test panels cover a number of clinically important groups of bacteria and yeasts.
One of the earliest and most popular test panels is the Analytical Profile Index (API) panel invented in the 1970's. Once some
basic laboratory characterization of a given strain has been performed, such as determining the strain’s Gram morphology, an
appropriate test strip that contains 10 to 20 different biochemical tests for differentiating strains within that microbial group can
be used. Currently, the various API strips can be used to quickly and easily identify more than 600 species of bacteria, both
aerobic and anaerobic, and approximately 100 different types of yeasts. Based on the colors of the reactions when metabolic
end products are present, due to the presence of pH indicators, a metabolic profile is created from the results (Figure 1.6.9).
Microbiologists can then compare the sample’s profile to the database to identify the specific microbe.

Figure 1.6.9 : This example API 20NE test strip is used to identify specific strains of gram-negative bacteria outside the
Enterobacteriaceae. Here is an API 20NE test strip result for Photobacterium damselae ssp. piscicida.

Key Concepts and Summary

Chemically defined media contain only chemically known components.
Selective media favor the growth of some microorganisms while inhibiting others.
Enriched media contain added essential nutrients a specific organism needs to grow
Differential media help distinguish bacteria by the color of the colonies or the change in the medium.

Footnotes
1. Centers for Disease Control and Prevention, World Health Organization. “CDC Laboratory Methods for the Diagnosis of
Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenza. WHO Manual, 2nd
edition.” 2011. http://www.cdc.gov/meningitis/lab-ma...ull-manual.pdf

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 1.6: Tools and Media Used for Bacterial Growth is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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Chapter 1 Exercises
Review Questions for Chapter 1:

Multiple Choice

1) Which of the following foods is NOT made by fermentation?

1. beer
2. bread
3. cheese
4. orange juice

2) Who is considered the “father of Western medicine”?

1. Marcus Terentius Varro
2. Thucydides
3. Antonie van Leeuwenhoek
4. Hippocrates

3) Who proposed that swamps might harbor tiny, disease-causing animals too small to see?
1. Thucydides
2. Marcus Terentius Varro
3. Hippocrates
4. Louis Pasteur

4) Which of the following individuals argued in favor of the theory of spontaneous generation?
1. Francesco Redi
2. Louis Pasteur
3. John Needham
4. Lazzaro Spallanzani

5) Which of the following scientists experimented with raw meat, maggots, and flies in an attempt to disprove the theory of
spontaneous generation?
1. Aristotle
2. Lazzaro Spallanzani
3. Antonie van Leeuwenhoek
4. Francesco Redi

6) Which of the following individuals is credited for definitively refuting the theory of spontaneous generation using broth in swan-
neck flask?
1. Aristotle
2. Jan Baptista van Helmont
3. John Needham
4. Louis Pasteur

1 https://bio.libretexts.org/@go/page/79163
7) Who was the first to describe “cells” in dead cork tissue?
1. Hans Janssen
2. Zaccharias Janssen
3. Antonie van Leeuwenhoek
4. Robert Hooke

8) Which of the following individuals did not contribute to the establishment of cell theory?
1. Girolamo Fracastoro
2. Matthias Schleiden
3. Robert Remak
4. Robert Hooke

9) Who is the probable inventor of the compound microscope?

1. Girolamo Fracastoro
2. Zaccharias Janssen
3. Antonie van Leeuwenhoek
4. Robert Hooke

10) Who was the first to observe “animalcules” under the microscope?
1. Antonie van Leeuwenhoek
2. Ötzi the Iceman
3. Marcus Terentius Varro
4. Robert Koch

11) Which of the following developed a set of postulates for determining whether a particular disease is caused by a particular
pathogen?
1. John Snow
2. Robert Koch
3. Joseph Lister
4. Louis Pasteur

12) Which of the following was NOT a kingdom in Linnaeus’s taxonomy?

1. animal
2. mineral
3. protist
4. plant

13) Which of the following is a correct usage of binomial nomenclature?

1. Homo Sapiens
2. homo sapiens
3. Homo sapiens
4. Homo Sapiens

14) Which scientist proposed adding a kingdom for protists?

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 1. Carolus Linnaeus
2. Carl Woese
3. Robert Whittaker
4. Ernst Haeckel

15) Which of the following types of microorganisms is photosynthetic?

1. yeast
2. virus
3. helminth
4. alga

16) Which of the following is a prokaryotic microorganism?

1. helminth
2. protozoan
3. cyanobacterium
4. mold

17) Which of the following is acellular?

1. virus
2. bacterium
3. fungus
4. protozoan

18) Which of the following is a type of fungal microorganism?

1. bacterium
2. protozoan
3. alga
4. yeast

19) Which of the following is not a subfield of microbiology?

1. bacteriology
2. botany
3. clinical microbiology
4. virology

20) EMB agar is a medium used in the identification and isolation of pathogenic bacteria. It contains digested meat proteins as a
source of organic nutrients. Two indicator dyes, eosin and methylene blue, inhibit the growth of gram-positive bacteria and
distinguish between lactose fermenting and nonlactose fermenting organisms. Lactose fermenters form metallic green or deep
purple colonies, whereas the nonlactose fermenters form completely colorless colonies. EMB agar is an example of which of the
following?
A. a selective medium only
B. a differential medium only
C. a selective medium and a chemically defined medium
D. a selective medium, a differential medium, and a complex medium

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21) Haemophilus influenzae must be grown on chocolate agar, which is blood agar treated with heat to release growth factors in the
medium. H. influenzae is described as ________.
A. an acidophile
B. a thermophile
C. an obligate anaerobe
D. fastidious

Fill-In-The-Blank

22) Thucydides is known as the father of _______________.

23) Researchers think that Ötzi the Iceman may have been infected with _____ disease.

24) The process by which microbes turn grape juice into wine is called _______________.

25) A microscope that uses multiple lenses is called a _________ microscope.

26) The assertion that “life only comes from life” was stated by Louis Pasteur in regard to his experiments that definitively refuted
the theory of ___________.

27) John Snow is known as the Father of _____________.

28) The ____________ theory states that disease may originate from proximity to decomposing matter and is not due to person-to-
person contact.

29) The scientist who first described cells was _____________.

30) In binomial nomenclature, an organism’s scientific name includes its ________ and __________.

31) Whittaker proposed adding the kingdoms ________ and ________ to his phylogenetic tree.

32) __________ are organisms without membrane-bound nuclei.

33) ______ are microorganisms that are not included in phylogenetic trees because they are acellular.

34) A ________ is a disease-causing microorganism.

35) Multicellular parasitic worms studied by microbiologists are called ___________.

36) The study of viruses is ___________.

37) The cells of prokaryotic organisms lack a _______.

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38) Staphylococcus aureus can be grown on multipurpose growth medium or on mannitol salt agar that contains 7.5% NaCl. The
bacterium is ________.

39) Blood agar contains many unspecified nutrients, supports the growth of a large number of bacteria, and allows differentiation of
bacteria according to hemolysis (breakdown of blood). The medium is ________ and ________.

40) Rogosa agar contains yeast extract. The pH is adjusted to 5.2 and discourages the growth of many microorganisms; however,
all the colonies look similar. The medium is ________ and ________.

Short Answer

41) What did Thucydides learn by observing the Athenian plague?

42) Why was the invention of the microscope important for microbiology?

43) What are some ways people use microbes?

44) Why is Antonie van Leeuwenhoek’s work much better known than that of Zaccharias Janssen?

45) Why did the cork cells observed by Robert Hooke appear to be empty, as opposed to being full of other structures?

46) Explain in your own words Pasteur’s swan-neck flask experiment.

47) Explain why the experiments of Needham and Spallanzani yielded in different results even though they used similar
methodologies.

48) How did the explanation of Virchow and Remak for the origin of cells differ from that of Schleiden and Schwann?

49) What were the differences in mortality rates due to puerperal fever that Ignaz Semmelweis observed? How did he propose to
reduce the occurrence of puerperal fever? Did it work?

50) What is a phylogenetic tree?

51) Which of the five kingdoms in Whittaker’s phylogenetic tree are prokaryotic, and which are eukaryotic?

52) What molecule did Woese and Fox use to construct their phylogenetic tree?

53) Name some techniques that can be used to identify and differentiate species of bacteria.

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54) Describe the differences between bacteria and archaea.

55) Name three structures that various protozoa use for locomotion.

56) Describe the actual and relative sizes of a virus, a bacterium, and a plant or animal cell.

57) What is the major difference between an enrichment culture and a selective culture?

Critical Thinking

58) Explain how the discovery of fermented foods likely benefited our ancestors.

59) What evidence would you use to support this statement: Ancient people thought that disease was transmitted by things they
could not see.

60) What would the results of Pasteur’s swan-neck flask experiment have looked like if they supported the theory of spontaneous
generation?

61) Why was the work of Snow so important in supporting the germ theory?

62) Why is using binomial nomenclature more useful than using common names?

63) Label the three Domains found on modern phylogenetic trees.

64) Contrast the behavior of a virus outside versus inside a cell.

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65) Where would a virus, bacterium, animal cell, and a prion belong on this chart?

66) A microbiology instructor prepares cultures for a gram-staining practical laboratory by inoculating growth medium with a
gram-positive coccus (nonmotile) and a gram-negative rod (motile). The goal is to demonstrate staining of a mixed culture. The
flask is incubated at 35 °C for 24 hours without aeration. A sample is stained and reveals only gram-negative rods. Both cultures
are known facultative anaerobes. Give a likely reason for success of the gram-negative rod. Assume that the cultures have
comparable intrinsic growth rates.

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 CHAPTER OVERVIEW

2: Chemistry and Biochemistry

2.1: Atoms, Isotopes, Ions, and Molecules - The Building Blocks
2.2: Water
2.3: Carbon and Organic Molecules
2.4: Carbohydrates
2.5: Lipids
2.6: Proteins
2.7: Nucleic Acids
Chapter 2 Exercises

2: Chemistry and Biochemistry is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

1
2.1: Atoms, Isotopes, Ions, and Molecules - The Building Blocks
Learning Objectives
Define matter and elements
Describe the interrelationship between protons, neutrons, and electrons
Compare the ways in which electrons can be donated or shared between atoms
Explain the ways in which naturally occurring elements combine to create molecules, cells, tissues, organ systems, and
organisms

All biological processes follow the laws of physics and chemistry, so in order to understand how biological systems work, it is
important to understand the underlying physics and chemistry. For example, the flow of blood within the circulatory system follows
the laws of physics that regulate the modes of fluid flow. The breakdown of the large, complex molecules of food into smaller
molecules—and the conversion of these to release energy to be stored in adenosine triphosphate (ATP)—is a series of chemical
reactions that follow chemical laws. The properties of water and the formation of hydrogen bonds are key to understanding living
processes. Recognizing the properties of acids and bases is important, for example, to our understanding of the digestive process.
Therefore, the fundamentals of physics and chemistry are important for gaining insight into biological processes.
At its most fundamental level, life is made up of matter. Matter is any substance that occupies space and has mass. Elements are
unique forms of matter with specific chemical and physical properties that cannot be broken down into smaller substances by
ordinary chemical reactions. There are 118 elements (more unnamed), but only 92 occur naturally. The remaining elements are
synthesized in laboratories and are unstable.
Each element is designated by its chemical symbol, which is a single capital letter or, when the first letter is already “taken” by
another element, a combination of two letters. Some elements follow the English term for the element, such as C for carbon and Ca
for calcium. Other elements’ chemical symbols derive from their Latin names; for example, the symbol for sodium is Na, referring
to natrium, the Latin word for sodium.
The four elements common to all living organisms are oxygen (O), carbon (C), hydrogen (H), and nitrogen (N). In the non-living
world, elements are found in different proportions, and some elements common to living organisms are relatively rare on the earth
as a whole, as shown in Table 2.1.1. For example, the atmosphere is rich in nitrogen and oxygen but contains little carbon and
hydrogen, while the earth’s crust, although it contains oxygen and a small amount of hydrogen, has little nitrogen and carbon. In
spite of their differences in abundance, all elements and the chemical reactions between them obey the same chemical and physical
laws regardless of whether they are a part of the living or non-living world.

Table 2.1.1 : Approximate Percentage of Elements in Living Organisms (Humans) Compared to the Non-living World
Element Life (Humans) Atmosphere Earth’s Crust

Oxygen (O) 65% 21% 46%

Carbon (C) 18% trace trace
Hydrogen (H) 10% trace 0.1%
Nitrogen (N) 3% 78% trace

Clinical Focus: part 1

Penny is a 16-year-old student who visited her doctor, complaining about an itchy skin rash. She had a history of allergic
episodes. The doctor looked at her sun-tanned skin and asked her if she switched to a different sunscreen. She said she had, so
the doctor diagnosed an allergic eczema. The symptoms were mild so the doctor told Penny to avoid using the sunscreen that
caused the reaction and prescribed an over-the-counter moisturizing cream to keep her skin hydrated and to help with itching.

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 Exercise 2.1.1

1. What kinds of substances would you expect to find in a moisturizing cream?

2. What physical or chemical properties of these substances would help alleviate itching and inflammation of the skin?

The Structure of the Atom

To understand how elements come together, we must first discuss the smallest component or building block of an element, the
atom. An atom is the smallest unit of matter that retains all of the chemical properties of an element. For example, one gold atom
has all of the properties of gold in that it is a solid metal at room temperature. A gold coin is simply a very large number of gold
atoms molded into the shape of a coin and containing small amounts of other elements known as impurities. Gold atoms cannot be
broken down into anything smaller while still retaining the properties of gold.
An atom is composed of two regions: the nucleus, which is in the center of the atom and contains protons and neutrons, and the
outermost region of the atom which holds its electrons in orbit around the nucleus, as illustrated in Figure 2.1.1. Atoms contain
protons, electrons, and neutrons, among other subatomic particles. The only exception is hydrogen (H), which is made of one
proton and one electron with no neutrons.

Figure 2.1.1 : Elements, such as helium, depicted here, are made up of atoms. Atoms are made up of protons and neutrons located
within the nucleus, with electrons in orbitals surrounding the nucleus.
Protons and neutrons have approximately the same mass, about 1.67 × 10-24 grams. Scientists arbitrarily define this amount of mass
as one atomic mass unit (amu) or one Dalton, as shown in Table 2.1.2. Although similar in mass, protons and neutrons differ in
their electric charge. A proton is positively charged whereas a neutron is uncharged. Therefore, the number of neutrons in an atom
contributes significantly to its mass, but not to its charge. Electrons are much smaller in mass than protons, weighing only 9.11 ×
10-28 grams, or about 1/1800 of an atomic mass unit. Hence, they do not contribute much to an element’s overall atomic mass.
Therefore, when considering atomic mass, it is customary to ignore the mass of any electrons and calculate the atom’s mass based
on the number of protons and neutrons alone. Although not significant contributors to mass, electrons do contribute greatly to the
atom’s charge, as each electron has a negative charge equal to the positive charge of a proton. In uncharged, neutral atoms, the
number of electrons orbiting the nucleus is equal to the number of protons inside the nucleus. In these atoms, the positive and
negative charges cancel each other out, leading to an atom with no net charge.
Table 2.1.2 : Protons, Neutrons, and Electrons
Charge Mass (amu) Location

Proton +1 1 nucleus
Neutron 0 1 nucleus
Electron –1 0 orbitals

Accounting for the sizes of protons, neutrons, and electrons, most of the volume of an atom—greater than 99 percent—is, in fact,
empty space. With all this empty space, one might ask why so-called solid objects do not just pass through one another. The reason
they do not is that the electrons that surround all atoms are negatively charged and negative charges repel each other.

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Atomic Number and Mass
Atoms of each element contain a characteristic number of protons and electrons. The number of protons determines an element’s
atomic number and is used to distinguish one element from another. The number of neutrons is variable, resulting in isotopes,
which are different forms of the same atom that vary only in the number of neutrons they possess. Together, the number of protons
and the number of neutrons determine an element’s mass number, as illustrated in Figure 2.1.2. Note that the small contribution of
mass from electrons is disregarded in calculating the mass number. This approximation of mass can be used to easily calculate how
many neutrons an element has by simply subtracting the number of protons from the mass number. Since an element’s isotopes will
have slightly different mass numbers, scientists also determine the atomic mass, which is the calculated mean of the mass number
for its naturally occurring isotopes. Often, the resulting number contains a fraction. For example, the atomic mass of chlorine (Cl)
is 35.45 because chlorine is composed of several isotopes, some (the majority) with atomic mass 35 (17 protons and 18 neutrons)
and some with atomic mass 37 (17 protons and 20 neutrons).

Figure 2.1.2 : Carbon has an atomic number of six, and two stable isotopes with mass numbers of twelve and thirteen, respectively.
Its atomic mass is 12.11.

Exercise 2.1.2

1. How many neutrons do carbon-12 and carbon-13 have, respectively?

Isotopes
Isotopes are different forms of an element that have the same number of protons but a different number of neutrons. Some elements
—such as carbon, potassium, and uranium—have naturally occurring isotopes. Carbon-12 contains six protons, six neutrons, and
six electrons; therefore, it has a mass number of 12 (six protons and six neutrons). Carbon-14 contains six protons, eight neutrons,
and six electrons; its atomic mass is 14 (six protons and eight neutrons). These two alternate forms of carbon are isotopes. Some
isotopes may emit neutrons, protons, and electrons, and attain a more stable atomic configuration (lower level of potential energy);
these are the radioactive isotopes, or radioisotopes. Radioactive decay (carbon-14 losing neutrons to eventually become carbon-12)
describes the energy loss that occurs when an unstable atom’s nucleus releases radiation.

The Periodic Table

The different elements are organized and displayed in the periodic table. Devised by Russian chemist Dmitri Mendeleev (1834–
1907) in 1869, the table groups elements that, although unique, share certain chemical properties with other elements. The
properties of elements are responsible for their physical state at room temperature: they may be gases, solids, or liquids. Elements
also have specific chemical reactivity, the ability to combine and to chemically bond with each other.
In the periodic table, shown in Figure 2.1.3, the elements are organized and displayed according to their atomic number and are
arranged in a series of rows and columns based on shared chemical and physical properties. In addition to providing the atomic
number for each element, the periodic table also displays the element’s atomic mass. Looking at carbon, for example, its symbol
(C) and name appear, as well as its atomic number of six (in the upper left-hand corner) and its atomic mass of 12.11.

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 Figure 2.1.3 : The periodic table shows the atomic mass and atomic number of each element. The atomic number appears above the
symbol for the element and the approximate atomic mass appears below it.
The periodic table groups elements according to chemical properties. The differences in chemical reactivity between the elements
are based on the number and spatial distribution of an atom’s electrons. Atoms that chemically react and bond to each other form
molecules. Molecules are simply two or more atoms chemically bonded together. Logically, when two atoms chemically bond to
form a molecule, their electrons, which form the outermost region of each atom, come together first as the atoms form a chemical
bond.

Electron Shells and the Bohr Model

It should be stressed that there is a connection between the number of protons in an element, the atomic number that distinguishes
one element from another, and the number of electrons it has. In all electrically neutral atoms, the number of electrons is the same
as the number of protons. Thus, each element, at least when electrically neutral, has a characteristic number of electrons equal to its
atomic number.
An early model of the atom was developed in 1913 by Danish scientist Niels Bohr (1885–1962). The Bohr model shows the atom
as a central nucleus containing protons and neutrons, with the electrons in circular orbitals at specific distances from the nucleus, as
illustrated in Figure 2.1.4. These orbits form electron shells or energy levels, which are a way of visualizing the number of
electrons in the outermost shells. These energy levels are designated by a number and the symbol “n.” For example, 1n represents
the first energy level located closest to the nucleus.

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 Figure 2.1.4 : The Bohr model was developed by Niels Bohrs in 1913. In this model, electrons exist within principal shells. An
electron normally exists in the lowest energy shell available, which is the one closest to the nucleus. Energy from a photon of light
can bump it up to a higher energy shell, but this situation is unstable, and the electron quickly decays back to the ground state. In
the process, a photon of light is released
Electrons fill orbitals in a consistent order: they first fill the orbitals closest to the nucleus, then they continue to fill orbitals of
increasing energy further from the nucleus. If there are multiple orbitals of equal energy, they will be filled with one electron in
each energy level before a second electron is added. The electrons of the outermost energy level determine the energetic stability of
the atom and its tendency to form chemical bonds with other atoms to form molecules.
Under standard conditions, atoms fill the inner shells first, often resulting in a variable number of electrons in the outermost shell.
The innermost shell has a maximum of two electrons but the next two electron shells can each have a maximum of eight electrons.
This is known as the octet rule, which states, with the exception of the innermost shell, that atoms are more stable energetically
when they have eight electrons in their valence shell, the outermost electron shell. Examples of some neutral atoms and their
electron configurations are shown in Figure 2.1.5. Notice that in this figure, helium has a complete outer electron shell, with two
electrons filling its first and only shell. Similarly, neon has a complete outer 2n shell containing eight electrons. In contrast,
chlorine and sodium have seven and one in their outer shells, respectively, but theoretically they would be more energetically stable
if they followed the octet rule and had eight.

Figure 2.1.5 : Bohr diagrams indicate how many electrons fill each principal shell. Group 18 elements (helium, neon, and argon are
shown) have a full outer, or valence, shell. A full valence shell is the most stable electron configuration. Elements in other groups
have partially filled valence shells and gain or lose electrons to achieve a stable electron configuration.
An atom may give, take, or share electrons with another atom to achieve a full valence shell, the most stable electron configuration.
Looking at this figure, how many electrons do elements in group 1 need to lose in order to achieve a stable electron configuration?
How many electrons do elements in groups 14 and 17 need to gain to achieve a stable configuration?
Understanding that the organization of the periodic table is based on the total number of protons (and electrons) helps us know how
electrons are distributed among the outer shell. The periodic table is arranged in columns and rows based on the number of
electrons and where these electrons are located. Take a closer look at the some of the elements in the table’s far right column in
Figure 2.1.6. The group 18 atoms helium (He), neon (Ne), and argon (Ar) all have filled outer electron shells, making it
unnecessary for them to share electrons with other atoms to attain stability; they are highly stable as single atoms. Their non-
reactivity has resulted in their being named the inert gases (or noble gases). Compare this to the group 1 elements in the left-hand

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column. These elements, including hydrogen (H), lithium (Li), and sodium (Na), all have one electron in their outermost shells.
That means that they can achieve a stable configuration and a filled outer shell by donating or sharing one electron with another
atom or a molecule such as water. Hydrogen will donate or share its electron to achieve this configuration, while lithium and
sodium will donate their electron to become stable. As a result of losing a negatively charged electron, they become positively
charged ions. Group 17 elements, including fluorine and chlorine, have seven electrons in their outmost shells, so they tend to fill
this shell with an electron from other atoms or molecules, making them negatively charged ions. Group 14 elements, of which
carbon is the most important to living systems, have four electrons in their outer shell allowing them to make several covalent
bonds (discussed below) with other atoms. Thus, the columns of the periodic table represent the potential shared state of these
elements’ outer electron shells that is responsible for their similar chemical characteristics.

Chemical Reactions and Molecules

All elements are most stable when their outermost shell is filled with electrons according to the octet rule. This is because it is
energetically favorable for atoms to be in that configuration and it makes them stable. However, since not all elements have enough
electrons to fill their outermost shells, atoms form chemical bonds with other atoms thereby obtaining the electrons they need to
attain a stable electron configuration. When two or more atoms chemically bond with each other, the resultant chemical structure is
a molecule. The familiar water molecule, H2O, consists of two hydrogen atoms and one oxygen atom; these bond together to form
water, as illustrated in Figure 2.1.6. Atoms can form molecules by donating, accepting, or sharing electrons to fill their outer shells.

Figure 2.1.6 : Two or more atoms may bond with each other to form a molecule. When two hydrogens and an oxygen share
electrons via covalent bonds, a water molecule is formed.
Chemical reactions occur when two or more atoms bond together to form molecules or when bonded atoms are broken apart. The
substances used in the beginning of a chemical reaction are called the reactants (usually found on the left side of a chemical
equation), and the substances found at the end of the reaction are known as the products (usually found on the right side of a
chemical equation). An arrow is typically drawn between the reactants and products to indicate the direction of the chemical
reaction; this direction is not always a “one-way street.” For the creation of the water molecule shown above, the chemical equation
would be:
2H + O → H2 O (2.1.1)

An example of a simple chemical reaction is the breaking down of hydrogen peroxide molecules, each of which consists of two
hydrogen atoms bonded to two oxygen atoms (H2O2). The reactant hydrogen peroxide is broken down into water, containing one
oxygen atom bound to two hydrogen atoms (H2O), and oxygen, which consists of two bonded oxygen atoms (O2). In the equation
below, the reaction includes two hydrogen peroxide molecules and two water molecules. This is an example of a balanced chemical
equation, wherein the number of atoms of each element is the same on each side of the equation. According to the law of
conservation of matter, the number of atoms before and after a chemical reaction should be equal, such that no atoms are, under
normal circumstances, created or destroyed.
2 H2 O2 (hydrogen peroxide) → 2 H2 O(water) + O2 (oxygen) (2.1.2)

Even though all of the reactants and products of this reaction are molecules (each atom remains bonded to at least one other atom),
in this reaction only hydrogen peroxide and water are representatives of compounds: they contain atoms of more than one type of

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element. Molecular oxygen, on the other hand, as shown in Figure 2.1.7, consists of two doubly bonded oxygen atoms and is not
classified as a compound but as a mononuclear molecule.

Figure 2.1.7 : The oxygen atoms in an O2 molecule are joined by a double bond.
Some chemical reactions, such as the one shown above, can proceed in one direction until the reactants are all used up. The
equations that describe these reactions contain a unidirectional arrow and are irreversible. Reversible reactions are those that can go
in either direction. In reversible reactions, reactants are turned into products, but when the concentration of product goes beyond a
certain threshold (characteristic of the particular reaction), some of these products will be converted back into reactants; at this
point, the designations of products and reactants are reversed. This back and forth continues until a certain relative balance between
reactants and products occurs—a state called equilibrium. These situations of reversible reactions are often denoted by a chemical
equation with a double headed arrow pointing towards both the reactants and products.
For example, in human blood, excess hydrogen ions (H+) bind to bicarbonate ions (HCO3-) forming an equilibrium state with
carbonic acid (H2CO3). If carbonic acid were added to this system, some of it would be converted to bicarbonate and hydrogen
ions.
− +
HC O +H ↔ H2 C O3 (2.1.3)
3

In biological reactions, however, equilibrium is rarely obtained because the concentrations of the reactants or products or both are
constantly changing, often with a product of one reaction being a reactant for another. To return to the example of excess hydrogen
ions in the blood, the formation of carbonic acid will be the major direction of the reaction. However, the carbonic acid can also
leave the body as carbon dioxide gas (via exhalation) instead of being converted back to bicarbonate ion, thus driving the reaction
to the right by the chemical law known as law of mass action. These reactions are important for maintaining the homeostasis of our
blood.
− +
HC O +H ↔ H2 C O3 ↔ C O2 + H2 O (2.1.4)
3

Another common type of chemical reaction in biological systems are combinations of oxidation and reduction reactions, which
occur at the same time. An oxidation reaction strips an electron from an atom in a compound, and the addition of this electron to
another compound is a reduction reaction. Because oxidation and reduction usually occur together, these pairs of reactions are
called oxidation reduction reactions, or redox reactions. This type of reaction serves to move energy instead of changing the
general properties of its reactants.
The removal of an electron from a molecule (oxidizing it), results in a decrease in potential energy in the oxidized compound. The
electron (sometimes as part of a hydrogen atom) does not remain unbonded, however, in the cytoplasm of a cell. Rather, the
electron is shifted to a second compound, reducing the second compound. The shift of an electron from one compound to another
removes some potential energy from the first compound (the oxidized compound) and increases the potential energy of the second
compound (the reduced compound). The transfer of electrons between molecules is important because most of the energy stored in
atoms and used to fuel cell functions is in the form of high-energy electrons. The transfer of energy in the form of high-energy
electrons allows the cell to transfer and use energy in an incremental fashion—in small packages rather than in a single, destructive
burst. This will be discussed further in chapter 7.

Ions and Ionic Bonds

Some atoms are more stable when they gain or lose an electron (or possibly two) and form ions. This fills their outermost electron
shell and makes them energetically more stable. Because the number of electrons does not equal the number of protons, each ion
has a net charge. Cations are positive ions that are formed by losing electrons. Negative ions are formed by gaining electrons and
are called anions. Anions are designated by their elemental name being altered to end in “-ide”: the anion of chlorine is called
chloride, and the anion of sulfur is called sulfide, for example.

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This movement of electrons from one element to another is referred to as electron transfer. As Figure 2.1.8 illustrates, sodium (Na)
only has one electron in its outer electron shell. It takes less energy for sodium to donate that one electron than it does to accept
seven more electrons to fill the outer shell. If sodium loses an electron, it now has 11 protons, 11 neutrons, and only 10 electrons,
leaving it with an overall charge of +1. It is now referred to as a sodium ion. Chlorine (Cl) in its lowest energy state (called the
ground state) has seven electrons in its outer shell. Again, it is more energy-efficient for chlorine to gain one electron than to lose
seven. Therefore, it tends to gain an electron to create an ion with 17 protons, 17 neutrons, and 18 electrons, giving it a net negative
(–1) charge. It is now referred to as a chloride ion. In this example, sodium will donate its one electron to empty its shell, and
chlorine will accept that electron to fill its shell. Both ions now satisfy the octet rule and have complete outermost shells. Because
the number of electrons is no longer equal to the number of protons, each is now an ion and has a +1 (sodium cation) or –1
(chloride anion) charge. Note that these transactions can normally only take place simultaneously: in order for a sodium atom to
lose an electron, it must be in the presence of a suitable recipient like a chlorine atom.

Figure 2.1.8 : In the formation of an ionic compound, metals lose electrons and nonmetals gain electrons to achieve an octet.
Ionic bonds are formed between ions with opposite charges. For instance, positively charged sodium ions and negatively charged
chloride ions bond together to make crystals of sodium chloride, or table salt, creating a crystalline molecule with zero net charge.
Certain salts are referred to in physiology as electrolytes contain ions (including sodium, potassium, and calcium) necessary for
nerve impulse conduction, muscle contractions and water balance. Many sports drinks and dietary supplements provide these ions
to replace those lost from the body via sweating during exercise.

Covalent Bonds and Other Bonds and Interactions

Another way the octet rule can be satisfied is by the sharing of electrons between atoms to form covalent bonds. These bonds are
stronger and much more common than ionic bonds in the molecules of living organisms. Covalent bonds are commonly found in
carbon-based organic molecules, such as our DNA and proteins. Covalent bonds are also found in inorganic molecules like H2O,
CO2, and O2. One, two, or three pairs of electrons may be shared, making single, double, and triple bonds, respectively. The more
covalent bonds between two atoms, the stronger their connection. Thus, triple bonds are the strongest.
The strength of different levels of covalent bonding is one of the main reasons living organisms have a difficult time in acquiring
nitrogen for use in constructing their molecules, even though molecular nitrogen, N2, is the most abundant gas in the atmosphere.
Molecular nitrogen consists of two nitrogen atoms triple bonded to each other and, as with all molecules, the sharing of these three
pairs of electrons between the two nitrogen atoms allows for the filling of their outer electron shells, making the molecule more
stable than the individual nitrogen atoms. This strong triple bond makes it difficult for living systems to break apart this nitrogen in
order to use it as constituents of proteins and DNA.
The formation of water molecules provides an example of covalent bonding. The hydrogen and oxygen atoms that combine to form
water molecules are bound together by covalent bonds. The electron from the hydrogen splits its time between the incomplete outer
shell of the hydrogen atoms and the incomplete outer shell of the oxygen atoms. To completely fill the outer shell of oxygen, which
has six electrons in its outer shell but which would be more stable with eight, two electrons (one from each hydrogen atom) are
needed: hence the well-known formula H2O. The electrons are shared between the two elements to fill the outer shell of each,
making both elements more stable.

Video: View this short video to see an animation of ionic and covalent bonding.

Polar Covalent Bonds

There are two types of covalent bonds: polar and nonpolar. In a polar covalent bond, shown in Figure 2.1.9, the electrons are
unequally shared by the atoms and are attracted more to one nucleus than the other. Because of the unequal distribution of electrons

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between the atoms of different elements, a slightly positive (δ+) or slightly negative (δ–) charge develops. This partial charge is an
important property of water and accounts for many of its characteristics.
Water is a polar molecule, with the hydrogen atoms acquiring a partial positive charge and the oxygen a partial negative charge.
This occurs because the nucleus of the oxygen atom is more attractive to the electrons of the hydrogen atoms than the hydrogen
nucleus is to the oxygen’s electrons. Thus oxygen has a higher electronegativity than hydrogen and the shared electrons spend more
time in the vicinity of the oxygen nucleus than they do near the nucleus of the hydrogen atoms, giving the atoms of oxygen and
hydrogen slightly negative and positive charges, respectively. Another way of stating this is that the probability of finding a shared
electron near an oxygen nucleus is more likely than finding it near a hydrogen nucleus. Either way, the atom’s relative
electronegativity contributes to the development of partial charges whenever one element is significantly more electronegative than
the other, and the charges generated by these polar bonds may then be used for the formation of hydrogen bonds based on the
attraction of opposite partial charges. (Hydrogen bonds, which are discussed in detail below, are weak bonds between slightly
positively charged hydrogen atoms to slightly negatively charged atoms in other molecules.) Since macromolecules often have
atoms within them that differ in electronegativity, polar bonds are often present in organic molecules.

Nonpolar Covalent Bonds

Nonpolar covalent bonds form between two atoms of the same element or between different elements that share electrons equally.
For example, molecular oxygen (O2) is nonpolar because the electrons will be equally distributed between the two oxygen atoms.
Another example of a nonpolar covalent bond is methane (CH4), also shown in Figure 2.1.9. Carbon has four electrons in its
outermost shell and needs four more to fill it. It gets these four from four hydrogen atoms, each atom providing one, making a
stable outer shell of eight electrons. Carbon and hydrogen do not have the same electronegativity but are similar; thus, nonpolar
bonds form. The hydrogen atoms each need one electron for their outermost shell, which is filled when it contains two electrons.
These elements share the electrons equally among the carbons and the hydrogen atoms, creating a nonpolar covalent molecule.

Figure 2.1.9 : Whether a molecule is polar or nonpolar depends both on bond type and molecular shape. Both water and carbon
dioxide have polar covalent bonds, but carbon dioxide is linear, so the partial charges on the molecule cancel each other out.

Hydrogen Bonds and van der Waals Interactions

Ionic and covalent bonds between elements require energy to break. Ionic bonds are not as strong as covalent, which determines
their behavior in biological systems. However, not all bonds are ionic or covalent bonds. Weaker bonds can also form between
molecules. Two weak bonds that occur frequently are hydrogen bonds and van der Waals interactions. Without these two types of
bonds, life as we know it would not exist. Hydrogen bonds provide many of the critical, life-sustaining properties of water and also
stabilize the structures of proteins and DNA, the building block of cells.
When polar covalent bonds containing hydrogen form, the hydrogen in that bond has a slightly positive charge because hydrogen’s
electron is pulled more strongly toward the other element and away from the hydrogen. Because the hydrogen is slightly positive, it
will be attracted to neighboring negative charges. When this happens, a weak interaction occurs between the δ+of the hydrogen
from one molecule and the δ– charge on the more electronegative atoms of another molecule, usually oxygen or nitrogen, or within
the same molecule. This interaction is called a hydrogen bond. This type of bond is common and occurs regularly between water
molecules. Individual hydrogen bonds are weak and easily broken; however, they occur in very large numbers in water and in

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organic polymers, creating a major force in combination. Hydrogen bonds are also responsible for zipping together the DNA
double helix.
Like hydrogen bonds, van der Waals interactions are weak attractions or interactions between molecules. Van der Waals attractions
can occur between any two or more molecules and are dependent on slight fluctuations of the electron densities, which are not
always symmetrical around an atom. For these attractions to happen, the molecules need to be very close to one another. These
bonds—along with ionic, covalent, and hydrogen bonds—contribute to the three-dimensional structure of the proteins in our cells
that is necessary for their proper function.

Key Concepts and Summary

Matter is anything that occupies space and has mass.
It is made up of elements. All of the 92 elements that occur naturally have unique qualities that allow them to combine in
various ways to create molecules, which in turn combine to form cells, tissues, organ systems, and organisms.
Atoms, which consist of protons, neutrons, and electrons, are the smallest units of an element that retain all of the properties of
that element.
Electrons can be transferred, shared, or cause charge disparities between atoms to create bonds, including ionic, covalent, and
hydrogen bonds, as well as van der Waals interactions.

Glossary
anion
negative ion that is formed by an atom gaining one or more electrons

atom
the smallest unit of matter that retains all of the chemical properties of an element

atomic mass
calculated mean of the mass number for an element’s isotopes

atomic number
total number of protons in an atom

balanced chemical equation

statement of a chemical reaction with the number of each type of atom equalized for both the products and reactants

cation
positive ion that is formed by an atom losing one or more electrons

chemical bond
interaction between two or more of the same or different atoms that results in the formation of molecules

chemical reaction
process leading to the rearrangement of atoms in molecules

chemical reactivity
the ability to combine and to chemically bond with each other

compound
substance composed of molecules consisting of atoms of at least two different elements

covalent bond
type of strong bond formed between two of the same or different elements; forms when electrons are shared between atoms

electrolyte

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 ion necessary for nerve impulse conduction, muscle contractions and water balance

electron
negatively charged subatomic particle that resides outside of the nucleus in the electron orbital; lacks functional mass and has a
negative charge of –1 unit

electron configuration
arrangement of electrons in an atom’s electron shell (for example, 1s22s22p6)

electron orbital
how electrons are spatially distributed surrounding the nucleus; the area where an electron is most likely to be found

electron transfer
movement of electrons from one element to another; important in creation of ionic bonds

electronegativity
ability of some elements to attract electrons (often of hydrogen atoms), acquiring partial negative charges in molecules and
creating partial positive charges on the hydrogen atoms

element
one of 118 unique substances that cannot be broken down into smaller substances; each element has unique properties and a
specified number of protons

equilibrium
steady state of relative reactant and product concentration in reversible chemical reactions in a closed system

hydrogen bond
weak bond between slightly positively charged hydrogen atoms to slightly negatively charged atoms in other molecules

inert gas
(also, noble gas) element with filled outer electron shell that is unreactive with other atoms

ion
atom or chemical group that does not contain equal numbers of protons and electrons

ionic bond
chemical bond that forms between ions with opposite charges (cations and anions)

irreversible chemical reaction

chemical reaction where reactants proceed uni-directionally to form products

isotope
one or more forms of an element that have different numbers of neutrons

law of mass action

chemical law stating that the rate of a reaction is proportional to the concentration of the reacting substances

mass number
total number of protons and neutrons in an atom

matter
anything that has mass and occupies space

molecule
two or more atoms chemically bonded together

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neutron
uncharged particle that resides in the nucleus of an atom; has a mass of one amu

noble gas
see inert gas

nonpolar covalent bond

type of covalent bond that forms between atoms when electrons are shared equally between them

nucleus
core of an atom; contains protons and neutrons

octet rule
rule that atoms are most stable when they hold eight electrons in their outermost shells

orbital
region surrounding the nucleus; contains electrons

periodic table
organizational chart of elements indicating the atomic number and atomic mass of each element; provides key information
about the properties of the elements

polar covalent bond

type of covalent bond that forms as a result of unequal sharing of electrons, resulting in the creation of slightly positive and
slightly negative charged regions of the molecule

product
molecule found on the right side of a chemical equation

proton
positively charged particle that resides in the nucleus of an atom; has a mass of one amu and a charge of +1

radioisotope
isotope that emits radiation composed of subatomic particles to form more stable elements

reactant
molecule found on the left side of a chemical equation

reversible chemical reaction

chemical reaction that functions bi-directionally, where products may turn into reactants if their concentration is great enough

valence shell
outermost shell of an atom
van der Waals interaction
very weak interaction between molecules due to temporary charges attracting atoms that are very close together

Contributors and Attributions

Connie Rye (East Mississippi Community College), Robert Wise (University of Wisconsin, Oshkosh), Vladimir Jurukovski
(Suffolk County Community College), Jean DeSaix (University of North Carolina at Chapel Hill), Jung Choi (Georgia Institute
of Technology), Yael Avissar (Rhode Island College) among other contributing authors. Original content by OpenStax (CC BY
4.0; Download for free at http://cnx.org/contents/185cbf87-c72...f21b5eabd@9.87).

This page titled 2.1: Atoms, Isotopes, Ions, and Molecules - The Building Blocks is shared under a not declared license and was authored,
remixed, and/or curated by OpenStax.

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2.2: Water
Learning Objectives
Describe the properties of water that are critical to maintaining life
Explain why water is an excellent solvent
Provide examples of water’s cohesive and adhesive properties
Discuss the role of acids, bases, and buffers in homeostasis

Why do scientists spend time looking for water on other planets? Why is water so important? It is because water is essential to life
as we know it. Water is one of the more abundant molecules and the one most critical to life on Earth. Approximately 60–70
percent of the human body is made up of water. Without it, life as we know it simply would not exist.
The polarity of the water molecule and its resulting hydrogen bonding make water a unique substance with special properties that
are intimately tied to the processes of life. Life originally evolved in a watery environment, and most of an organism’s cellular
chemistry and metabolism occur inside the watery contents of the cell’s cytoplasm. Special properties of water are its high heat
capacity and heat of vaporization, its ability to dissolve polar molecules, its cohesive and adhesive properties, and its dissociation
into ions that leads to the generation of pH. Understanding these characteristics of water helps to elucidate its importance in
maintaining life.

Water’s Polarity
One of water’s important properties is that it is composed of polar molecules: the hydrogen and oxygen within water molecules
(H2O) form polar covalent bonds. While there is no net charge to a water molecule, the polarity of water creates a slightly positive
charge on hydrogen and a slightly negative charge on oxygen, contributing to water’s properties of attraction. Water’s charges are
generated because oxygen is more electronegative than hydrogen, making it more likely that a shared electron would be found near
the oxygen nucleus than the hydrogen nucleus, thus generating the partial negative charge near the oxygen.
As a result of water’s polarity, each water molecule attracts other water molecules because of the opposite charges between water
molecules, forming hydrogen bonds. Water also attracts or is attracted to other polar molecules and ions. A polar substance that
interacts readily with or dissolves in water is referred to as hydrophilic (hydro- = “water”; -philic = “loving”). In contrast, non-
polar molecules such as oils and fats do not interact well with water, as shown in Figure 2.2.1 and separate from it rather than
dissolve in it, as we see in salad dressings containing oil and vinegar (an acidic water solution). These nonpolar compounds are
called hydrophobic (hydro- = “water”; -phobic = “fearing”). A molecule presenting both a hydrophobic portion and a hydrophilic
portion is said to be amphipathic.

Figure 2.2.1 : Oil and water do not mix. As this macro image of oil and water shows, oil does not dissolve in water but forms
droplets instead. This is due to it being a nonpolar compound. (credit: Gautam Dogra).

Water’s States: Gas, Liquid, and Solid

The formation of hydrogen bonds is an important quality of the liquid water that is crucial to life as we know it. As water
molecules make hydrogen bonds with each other, water takes on some unique chemical characteristics compared to other liquids
and, since living things have a high water content, understanding these chemical features is key to understanding life. In liquid
water, hydrogen bonds are constantly formed and broken as the water molecules slide past each other. The breaking of these bonds
is caused by the motion (kinetic energy) of the water molecules due to the heat contained in the system. When the heat is raised as
water is boiled, the higher kinetic energy of the water molecules causes the hydrogen bonds to break completely and allows water

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molecules to escape into the air as gas (steam or water vapor). On the other hand, when the temperature of water is reduced and
water freezes, the water molecules form a crystalline structure maintained by hydrogen bonding (there is not enough energy to
break the hydrogen bonds) that makes ice less dense than liquid water, a phenomenon not seen in the solidification of other liquids.
Water’s lower density in its solid form is due to the way hydrogen bonds are oriented as it freezes: the water molecules are pushed
farther apart compared to liquid water. With most other liquids, solidification when the temperature drops includes the lowering of
kinetic energy between molecules, allowing them to pack even more tightly than in liquid form and giving the solid a greater
density than the liquid.
The lower density of ice, illustrated and pictured in Figure 2.2.2, an anomaly, causes it to float at the surface of liquid water, such
as in an iceberg or in the ice cubes in a glass of ice water. In lakes and ponds, ice will form on the surface of the water creating an
insulating barrier that protects the animals and plant life in the pond from freezing. Without this layer of insulating ice, plants and
animals living in the pond would freeze in the solid block of ice and could not survive. The detrimental effect of freezing on living
organisms is caused by the expansion of ice relative to liquid water. The ice crystals that form upon freezing rupture the delicate
membranes essential for the function of living cells, irreversibly damaging them. Cells can only survive freezing if the water in
them is temporarily replaced by another liquid like glycerol.

Figure 2.2.2 : Hydrogen bonding makes ice less dense than liquid water. The (a) lattice structure of ice makes it less dense than the
freely flowing molecules of liquid water, enabling it to (b) float on water. (credit a: modification of work by Jane Whitney, image
1
created using Visual Molecular Dynamics (VMD) software ; credit b: modification of work by Carlos Ponte)

Video: Click here to see a 3-D animation of the structure of an ice lattice. (Image credit: Jane Whitney. Image
2
created using Visual Molecular Dynamics VMD software. )

Water’s High Heat Capacity

Water’s high heat capacity is a property caused by hydrogen bonding among water molecules. Water has the highest specific heat
capacity of any liquids. Specific heat is defined as the amount of heat one gram of a substance must absorb or lose to change its
temperature by one degree Celsius. For water, this amount is one calorie. It therefore takes water a long time to heat and long time
to cool. In fact, the specific heat capacity of water is about five times more than that of sand. This explains why the land cools
faster than the sea. Due to its high heat capacity, water is used by warm blooded animals to more evenly disperse heat in their
bodies: it acts in a similar manner to a car’s cooling system, transporting heat from warm places to cool places, causing the body to
maintain a more even temperature.

Water’s Heat of Vaporization

Water also has a high heat of vaporization, the amount of energy required to change one gram of a liquid substance to a gas. A
considerable amount of heat energy (586 cal) is required to accomplish this change in water. This process occurs on the surface of
water. As liquid water heats up, hydrogen bonding makes it difficult to separate the liquid water molecules from each other, which

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is required for it to enter its gaseous phase (steam). As a result, water acts as a heat sink or heat reservoir and requires much more
heat to boil than does a liquid such as ethanol (grain alcohol), whose hydrogen bonding with other ethanol molecules is weaker
than water’s hydrogen bonding. Eventually, as water reaches its boiling point of 100° Celsius (212° Fahrenheit), the heat is able to
break the hydrogen bonds between the water molecules, and the kinetic energy (motion) between the water molecules allows them
to escape from the liquid as a gas. Even when below its boiling point, water’s individual molecules acquire enough energy from
other water molecules such that some surface water molecules can escape and vaporize: this process is known as evaporation.
The fact that hydrogen bonds need to be broken for water to evaporate means that a substantial amount of energy is used in the
process. As the water evaporates, energy is taken up by the process, cooling the environment where the evaporation is taking place.
In many living organisms, including in humans, the evaporation of sweat, which is 90 percent water, allows the organism to cool so
that homeostasis of body temperature can be maintained.

Water’s Solvent Properties

Water is one of the world's most common solvents, a substance capable of dissolving other substances. Anything mixed in with a
solvent in smaller amounts (mass, volume or concentration) is a solute, and there can be more than one solute in a solvent. Some
solvents mix well with their solutes and some do not. This ability to mix is based on the respective bonding properties between
solvent and solute. The more similar they are, the more they mix into the final solution.
Since water is a polar molecule with slightly positive and slightly negative charges, ions and polar molecules can readily dissolve
in it. When water is the solvent, we describe the mix as aqueous. The charges associated with these molecules will form hydrogen
bonds with water, surrounding the particle with water molecules. This is referred to as a sphere of hydration, or a hydration shell, as
illustrated in Figure 2.2.3 and serves to keep the particles separated or dispersed in the water.
When ionic compounds are added to water, the individual ions react with the polar regions of the water molecules and their ionic
bonds are disrupted in the process of dissociation. Dissociation occurs when atoms or groups of atoms break off from molecules
and form ions. Consider table salt (NaCl, or sodium chloride): when NaCl crystals are added to water, the molecules of NaCl
dissociate into Na+ and Cl– ions, and spheres of hydration form around the ions, illustrated in Figure 2.2.3. The positively charged
sodium ion is surrounded by the partially negative charge of the water molecule’s oxygen. The negatively charged chloride ion is
surrounded by the partially positive charge of the hydrogen on the water molecule.

Figure 2.2.3 : When table salt (NaCl) is mixed in water, spheres of hydration are formed around the ions.

Water’s Cohesive and Adhesive Properties

Have you ever filled a glass of water to the very top and then slowly added a few more drops? Before it overflows, the water forms
a dome-like shape above the rim of the glass. This water can stay above the glass because of the property of cohesion. In cohesion,
water molecules are attracted to each other (because of hydrogen bonding), keeping the molecules together at the liquid-gas (water-
air) interface, although there is no more room in the glass.
Cohesion allows for the development of surface tension, the capacity of a substance to withstand being ruptured when placed under
tension or stress. This is also why water forms droplets when placed on a dry surface rather than being flattened out by gravity.
When a small scrap of paper is placed onto the droplet of water, the paper floats on top of the water droplet even though paper is
denser (heavier) than the water. Cohesion and surface tension keep the hydrogen bonds of water molecules intact and support the
item floating on the top. It’s even possible to “float” a needle on top of a glass of water if it is placed gently without breaking the
surface tension, as shown in Figure 2.2.4.

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 Figure 2.2.4 : The weight of the needle is pulling the surface downward; at the same time, the surface tension is pulling it up,
suspending it on the surface of the water and keeping it from sinking. Notice the indentation in the water around the needle. (credit:
Cory Zanker)
These cohesive forces are related to water’s property of adhesion, or the attraction between water molecules and other molecules.
This attraction is sometimes stronger than water’s cohesive forces, especially when the water is exposed to charged surfaces such
as those found on the inside of thin glass tubes known as capillary tubes. Adhesion is observed when water “climbs” up the tube
placed in a glass of water: notice that the water appears to be higher on the sides of the tube than in the middle. This is because the
water molecules are attracted to the charged glass walls of the capillary more than they are to each other and therefore adhere to it.
This type of adhesion is called capillary action, and is illustrated in Figure 2.2.5.

Figure 2.2.5 : Capillary action in a glass tube is caused by the adhesive forces exerted by the internal surface of the glass exceeding
the cohesive forces between the water molecules themselves. (credit: modification of work by Pearson-Scott Foresman, donated to
the Wikimedia Foundation)
Why are cohesive and adhesive forces important for life? Cohesive and adhesive forces are important for the transport of water
from the roots to the leaves in plants. These forces create a “pull” on the water column. This pull results from the tendency of water
molecules being evaporated on the surface of the plant to stay connected to water molecules below them, and so they are pulled
along. Plants use this natural phenomenon to help transport water from their roots to their leaves. Without these properties of water,
plants would be unable to receive the water and the dissolved minerals they require. In another example, insects such as the water
strider, shown in Figure 2.2.6, use the surface tension of water to stay afloat on the surface layer of water and even mate there.

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 Figure 2.2.6 : Water’s cohesive and adhesive properties allow this water strider (Gerris sp.) to stay afloat. (credit: Tim Vickers)

pH, Buffers, Acids, and Bases

The pH of a solution indicates its acidity or alkalinity.
+ −
H2 O(I ) ⇋ H (aq) + OH (aq) (1)

litmus or pH paper, filter paper that has been treated with a natural water-soluble dye so it can be used as a pH indicator, to test how
much acid (acidity) or base (alkalinity) exists in a solution. You might have even used some to test whether the water in a
swimming pool is properly treated. In both cases, the pH test measures the concentration of hydrogen ions in a given solution.
Hydrogen ions are spontaneously generated in pure water by the dissociation (ionization) of a small percentage of water molecules
into equal numbers of hydrogen (H+) ions and hydroxide (OH-) ions. While the hydroxide ions are kept in solution by their
hydrogen bonding with other water molecules, the hydrogen ions, consisting of naked protons, are immediately attracted to un-
ionized water molecules, forming hydronium ions (H30+). Still, by convention, scientists refer to hydrogen ions and their
concentration as if they were free in this state in liquid water.
The concentration of hydrogen ions dissociating from pure water is 1 × 10-7 moles H+ ions per liter of water. Moles (mol) are a way
to express the amount of a substance (which can be atoms, molecules, ions, etc), with one mole being equal to 6.02 x 1023 particles
of the substance. Therefore, 1 mole of water is equal to 6.02 x 1023 water molecules. The pH is calculated as the negative of the
base 10 logarithm of this concentration. The log10 of 1 × 10-7 is -7.0, and the negative of this number (indicated by the “p” of
“pH”) yields a pH of 7.0, which is also known as neutral pH. The pH inside of human cells and blood are examples of two areas of
the body where near-neutral pH is maintained.
Non-neutral pH readings result from dissolving acids or bases in water. Using the negative logarithm to generate positive integers,
high concentrations of hydrogen ions yield a low pH number, whereas low levels of hydrogen ions result in a high pH. An acid is a
substance that increases the concentration of hydrogen ions (H+) in a solution, usually by having one of its hydrogen atoms
dissociate. A base provides either hydroxide ions (OH–) or other negatively charged ions that combine with hydrogen ions,
reducing their concentration in the solution and thereby raising the pH. In cases where the base releases hydroxide ions, these ions
bind to free hydrogen ions, generating new water molecules.
The stronger the acid, the more readily it donates H+. For example, hydrochloric acid (HCl) completely dissociates into hydrogen
and chloride ions and is highly acidic, whereas the acids in tomato juice or vinegar do not completely dissociate and are considered
weak acids. Conversely, strong bases are those substances that readily donate OH– or take up hydrogen ions. Sodium hydroxide
(NaOH) and many household cleaners are highly alkaline and give up OH– rapidly when placed in water, thereby raising the pH.
An example of a weak basic solution is seawater, which has a pH near 8.0, close enough to neutral pH that marine organisms
adapted to this saline environment are able to thrive in it.
The pH scale is, as previously mentioned, an inverse logarithm and ranges from 0 to 14 (Figure 2.2.7). Anything below 7.0
(ranging from 0.0 to 6.9) is acidic, and anything above 7.0 (from 7.1 to 14.0) is alkaline. Extremes in pH in either direction from
7.0 are usually considered inhospitable to life. The pH inside cells (6.8) and the pH in the blood (7.4) are both very close to neutral.
However, the environment in the stomach is highly acidic, with a pH of 1 to 2. So how do the cells of the stomach survive in such
an acidic environment? How do they homeostatically maintain the near neutral pH inside them? The answer is that they cannot do
it and are constantly dying. New stomach cells are constantly produced to replace dead ones, which are digested by the stomach
acids. It is estimated that the lining of the human stomach is completely replaced every seven to ten days.

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 Figure 2.2.7 : The pH scale measures the concentration of hydrogen ions (H+) in a solution. (credit: modification of work by
Edward Stevens)

Watch this video for a straightforward explanation of pH and its logarithmic scale.
So how can organisms whose bodies require a near-neutral pH ingest acidic and basic substances (a human drinking orange juice,
for example) and survive? Buffers are the key. Buffers readily absorb excess H+ or OH–, keeping the pH of the body carefully
maintained in the narrow range required for survival. Maintaining a constant blood pH is critical to a person’s well-being. The
buffer maintaining the pH of human blood involves carbonic acid (H2CO3), bicarbonate ion (HCO3–), and carbon dioxide (CO2).
When bicarbonate ions combine with free hydrogen ions and become carbonic acid, hydrogen ions are removed, moderating pH
changes. Similarly, as shown in Figure 2.2.8, excess carbonic acid can be converted to carbon dioxide gas and exhaled through the
lungs. This prevents too many free hydrogen ions from building up in the blood and dangerously reducing the blood’s pH.
Likewise, if too much OH– is introduced into the system, carbonic acid will combine with it to create bicarbonate, lowering the pH.
Without this buffer system, the body’s pH would fluctuate enough to put survival in jeopardy.

Figure 2.2.8 : This diagram shows the body’s buffering of blood pH levels. The blue arrows show the process of raising pH as more
CO2 is made. The purple arrows indicate the reverse process: the lowering of pH as more bicarbonate is created.
Other examples of buffers are antacids used to combat excess stomach acid. Many of these over-the-counter medications work in
the same way as blood buffers, usually with at least one ion capable of absorbing hydrogen and moderating pH, bringing relief to
those that suffer “heartburn” after eating. The unique properties of water that contribute to this capacity to balance pH—as well as
water’s other characteristics—are essential to sustaining life on Earth.

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 To learn more about water. Visit the U.S. Geological Survey Water Science for Schools All About Water!
website.

Key Concepts and Summary

Water has many properties that are critical to maintaining life. It is a polar molecule, allowing for the formation of hydrogen
bonds.
Hydrogen bonds allow ions and other polar molecules to dissolve in water. Therefore, water is an excellent solvent.
The hydrogen bonds between water molecules cause the water to have a high heat capacity, meaning it takes a lot of added heat
to raise its temperature. As the temperature rises, the hydrogen bonds between water continually break and form anew. This
allows for the overall temperature to remain stable, although energy is added to the system.
Water also exhibits a high heat of vaporization, which is key to how organisms cool themselves by the evaporation of sweat.
Water’s cohesive forces allow for the property of surface tension, whereas its adhesive properties are seen as water rises inside
capillary tubes.
The pH value is a measure of hydrogen ion concentration in a solution and is one of many chemical characteristics that is highly
regulated in living organisms through homeostasis. Acids and bases can change pH values, but buffers tend to moderate the
changes they cause.
These properties of water are intimately connected to the biochemical and physical processes performed by living organisms,
and life would be very different if these properties were altered, if it could exist at all.

Footnotes
1. W. Humphrey W., A. Dalke, and K. Schulten, “VMD—Visual Molecular Dynamics,” Journal of Molecular Graphics 14
(1996): 33-38.
2. W. Humphrey W., A. Dalke, and K. Schulten, “VMD—Visual Molecular Dynamics,” Journal of Molecular Graphics 14
(1996): 33-38.

Glossary
acid
molecule that donates hydrogen ions and increases the concentration of hydrogen ions in a solution

adhesion
attraction between water molecules and other molecules

base
molecule that donates hydroxide ions or otherwise binds excess hydrogen ions and decreases the concentration of hydrogen ions
in a solution

buffer
substance that prevents a change in pH by absorbing or releasing hydrogen or hydroxide ions

calorie
amount of heat required to change the temperature of one gram of water by one degree Celsius

capillary action
occurs because water molecules are attracted to charges on the inner surfaces of narrow tubular structures such as glass tubes,
drawing the water molecules to the sides of the tubes

cohesion
intermolecular forces between water molecules caused by the polar nature of water; responsible for surface tension

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dissociation
release of an ion from a molecule such that the original molecule now consists of an ion and the charged remains of the original,
such as when water dissociates into H+ and OH-

evaporation
separation of individual molecules from the surface of a body of water, leaves of a plant, or the skin of an organism

heat of vaporization of water

high amount of energy required for liquid water to turn into water vapor

hydrophilic
describes ions or polar molecules that interact well with other polar molecules such as water

hydrophobic
describes uncharged non-polar molecules that do not interact well with polar molecules such as water

litmus paper
(also, pH paper) filter paper that has been treated with a natural water-soluble dye that changes its color as the pH of the
environment changes so it can be used as a pH indicator

pH paper
see litmus paper

pH scale
scale ranging from zero to 14 that is inversely proportional to the concentration of hydrogen ions in a solution

solvent
substance capable of dissolving another substance

specific heat capacity

the amount of heat one gram of a substance must absorb or lose to change its temperature by one degree Celsius

sphere of hydration
when a polar water molecule surrounds charged or polar molecules thus keeping them dissolved and in solution

surface tension
tension at the surface of a body of liquid that prevents the molecules from separating; created by the attractive cohesive forces
between the molecules of the liquid

Contributors and Attributions

Connie Rye (East Mississippi Community College), Robert Wise (University of Wisconsin, Oshkosh), Vladimir Jurukovski
(Suffolk County Community College), Jean DeSaix (University of North Carolina at Chapel Hill), Jung Choi (Georgia Institute
of Technology), Yael Avissar (Rhode Island College) among other contributing authors. Original content by OpenStax (CC BY
4.0; Download for free at http://cnx.org/contents/185cbf87-c72...f21b5eabd@9.87).

This page titled 2.2: Water is shared under a not declared license and was authored, remixed, and/or curated by OpenStax.

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2.3: Carbon and Organic Molecules
Learning Objectives
Explain why carbon is important for life
Identify common elements and structures found in organic molecules
Explain the concept of isomerism
Understand the synthesis of macromolecules
Explain the importance and use of functional groups
Explain dehydration (or condensation) and hydrolysis reactions

Cells are made of many complex molecules called macromolecules, such as proteins, nucleic acids (RNA and DNA),
carbohydrates, and lipids. The macromolecules are a subset of organic molecules (any carbon-containing liquid, solid, or gas) that
are especially important for life. The fundamental component for all of these macromolecules is carbon. The carbon atom has
unique properties that allow it to form covalent bonds to as many as four different atoms, making this versatile element ideal to
serve as the basic structural component, or “backbone,” of the macromolecules.
Individual carbon atoms have an incomplete outermost electron shell. With an atomic number of 6 (six electrons and six protons),
the first two electrons fill the inner shell, leaving four in the second shell. Therefore, carbon atoms can form up to four covalent
bonds with other atoms to satisfy the octet rule. The methane molecule provides an example: it has the chemical formula CH4.
Each of its four hydrogen atoms forms a single covalent bond with the carbon atom by sharing a pair of electrons. This results in a
filled outermost shell.

Hydrocarbons
Hydrocarbons are organic molecules consisting entirely of carbon and hydrogen, such as methane (CH4) described above. We often
use hydrocarbons in our daily lives as fuels—like the propane in a gas grill or the butane in a lighter. The many covalent bonds
between the atoms in hydrocarbons store a great amount of energy, which is released when these molecules are burned (oxidized).
Methane, an excellent fuel, is the simplest hydrocarbon molecule, with a central carbon atom bonded to four different hydrogen
atoms, as illustrated in Figure 2.3.1. The geometry of the methane molecule, where the atoms reside in three dimensions, is
determined by the shape of its electron orbitals. The carbons and the four hydrogen atoms form a shape known as a tetrahedron,
with four triangular faces; for this reason, methane is described as having tetrahedral geometry.

Figure 2.3.1 : Methane has a tetrahedral geometry, with each of the four hydrogen atoms spaced 109.5° apart.
As the backbone of the large molecules of living things, hydrocarbons may exist as linear carbon chains, carbon rings, or
combinations of both. Furthermore, individual carbon-to-carbon bonds may be single, double, or triple covalent bonds, and each
type of bond affects the geometry of the molecule in a specific way. This three-dimensional shape or conformation of the large
molecules of life (macromolecules) is critical to how they function.

Hydrocarbon Chains
Hydrocarbon chains are formed by successive bonds between carbon atoms and may be branched or unbranched. Furthermore, the
overall geometry of the molecule is altered by the different geometries of single, double, and triple covalent bonds, illustrated in
Figure 2.3.2. The hydrocarbons ethane, ethene, and ethyne serve as examples of how different carbon-to-carbon bonds affect the
geometry of the molecule. The names of all three molecules start with the prefix “eth-,” which is the prefix for two carbon
hydrocarbons. The suffixes “-ane,” “-ene,” and “-yne” refer to the presence of single, double, or triple carbon-carbon bonds,
respectively. Thus, propane, propene, and propyne follow the same pattern with three carbon molecules, butane, butane, and butyne

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for four carbon molecules, and so on. Double and triple bonds change the geometry of the molecule: single bonds allow rotation
along the axis of the bond, whereas double bonds lead to a planar configuration and triple bonds to a linear one. These geometries
have a significant impact on the shape a particular molecule can assume.

Figure 2.3.2 : When carbon forms single bonds with other atoms, the shape is tetrahedral. When two carbon atoms form a double
bond, the shape is planar, or flat. Single bonds, like those found in ethane, are able to rotate. Double bonds, like those found in
ethene cannot rotate, so the atoms on either side are locked in place.

Hydrocarbon Rings
So far, the hydrocarbons we have discussed have been aliphatic hydrocarbons, which consist of linear chains of carbon atoms.
Another type of hydrocarbon, aromatic hydrocarbons, consists of closed rings of carbon atoms. Ring structures are found in
hydrocarbons, sometimes with the presence of double bonds, which can be seen by comparing the structure of cyclohexane to
benzene in Figure 2.3.3. Examples of biological molecules that incorporate the benzene ring include some amino acids and
cholesterol and its derivatives, including the hormones estrogen and testosterone. The benzene ring is also found in the herbicide
2,4-D. Benzene is a natural component of crude oil and has been classified as a carcinogen. Some hydrocarbons have both aliphatic
and aromatic portions; beta-carotene is an example of such a hydrocarbon.

Figure 2.3.3 : Carbon can form five-and six membered rings. Single or double bonds may connect the carbons in the ring, and
nitrogen may be substituted for carbon.
Biochemistry is the discipline that studies the chemistry of life, and its objective is to explain form and function based on chemical
principles. Organic chemistry is the discipline devoted to the study of carbon-based chemistry, which is the foundation for the study
of biomolecules and the discipline of biochemistry.

Other Elements in Living Cells

The most abundant element in cells is hydrogen (H), followed by carbon (C), oxygen (O), nitrogen (N), phosphorous (P), and
sulfur (S). We call these elements macronutrients, and they account for about 99% of the dry weight of cells. Some elements, such
as sodium (Na), potassium (K), magnesium (Mg), zinc (Zn), iron (Fe), calcium (Ca), molybdenum (Mo), copper (Cu), cobalt (Co),
manganese (Mn), or vanadium (Va), are required by some cells in very small amounts and are called micronutrients or trace
elements. All of these elements are essential to the function of many biochemical reactions, and, therefore, are essential to life.
The four most abundant elements in living matter (C, N, O, and H) have low atomic numbers and are thus light elements capable of
forming strong bonds with other atoms to produce molecules (Figure 2.3.4). Unlike carbon, nitrogen forms up to three bonds,
oxygen forms up to two, and hydrogen forms one. When bonded together within molecules, oxygen, sulfur, and nitrogen often have
one or more “lone pairs” of electrons that play important roles in determining many of the molecules’ physical and chemical
properties. These traits in combination permit the formation of a vast number of diverse molecular species necessary to form the
structures and enable the functions of living organisms.

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 Figure 2.3.4 : Some common molecules include carbon dioxide, ammonia, and oxygen, which consist of combinations of oxygen
atoms (red spheres), carbon atoms (gray spheres), hydrogen atoms (white spheres), or nitrogen atoms (blue spheres).

Living organisms contain inorganic compounds (mainly water and salts) and organic molecules. Organic molecules contain carbon;
inorganic compounds do not. Carbon oxides and carbonates are exceptions; they contain carbon but are considered inorganic
because they do not contain hydrogen. The atoms of an organic molecule are typically organized around chains of carbon atoms.
Inorganic compounds make up 1%–1.5% of a living cell’s mass. They are small, simple compounds that play important roles in the
cell, although they do not form cell structures. Most of the carbon found in organic molecules originates from inorganic carbon
sources such as carbon dioxide captured via carbon fixation by microorganisms.

Exercise 2.3.1

1. Describe the most abundant elements in nature.

2. Describe the most abundant elements in nature.
3. What are the differences between organic and inorganic molecules?

Organic Molecules
Organic molecules in organisms are generally larger and more complex than inorganic molecules. Their carbon skeletons are held
together by covalent bonds. They form the cells of an organism and perform the chemical reactions that facilitate life. All of these
molecules, called biomolecules because they are part of living matter, contain carbon, which is the building block of life. Carbon is
a very unique element in that it has four valence electrons in its outer orbitals and can form four single covalent bonds with up to
four other atoms at the same time. These atoms are usually oxygen, hydrogen, nitrogen, sulfur, phosphorous, and carbon itself; the
simplest organic compound is methane, in which carbon binds only to hydrogen (Figure 2.3.5).
As a result of carbon’s unique combination of size and bonding properties, carbon atoms can bind together in large numbers, thus
producing a chain or carbon skeleton. The carbon skeleton of organic molecules can be straight, branched, or ring shaped (cyclic).
Organic molecules are built on chains of carbon atoms of varying lengths; most are typically very long, which allows for a huge
number and variety of compounds. No other element has the ability to form so many different molecules of so many different sizes
and shapes.

Figure 2.3.5 : A carbon atom can bond with up to four other atoms. The simplest organic molecule is methane (CH4), depicted here.
Molecules with the same atomic makeup but different structural arrangement of atoms are called isomers. The concept of
isomerism is very important in chemistry because the structure of a molecule is always directly related to its function. Slight
changes in the structural arrangements of atoms in a molecule may lead to very different properties. Chemists represent molecules
by their structural formula, which is a graphic representation of the molecular structure, showing how the atoms are arranged.
Compounds that have identical molecular formulas but differ in the bonding sequence of the atoms are called structural isomers.

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The monosaccharides glucose, galactose, and fructose all have the same molecular formula, C6H12O6, but we can see from Figure
2.3.6 that the atoms are bonded together differently.

The molecular structures of the linear forms of glucose, galactose, and fructose are shown. Glucose and galactose
are both aldoses with a carbonyl group (carbon double-bonded to oxygen) at one end of the molecule. A hydroxyl
(OH) group is attached to each of the other residues. In glucose, the hydroxyl group attached to the second carbon is on
the left side of the molecular structure and all other hydroxyl groups are on the right. In galactose, the hydroxyl groups
attached to the third and fourth carbons are on the left, and the hydroxyl groups attached to the second, fifth and sixth
carbon are on the right. Frucose is a ketose with C doubled bonded to O at the second carbon. All other carbons have
hydroxyl groups associated with them. The hydroxyl group associated with the third carbon is on the left, and all the
other hydroxyl groups are on the right.

Figure 2.3.6 : Glucose, galactose, and fructose have the same chemical formula (C6H12O6), but these structural isomers differ in
their physical and chemical properties.
Isomers that differ in the spatial arrangements of atoms are called stereoisomers; one unique type is enantiomers. The properties of
enantiomers were originally discovered by Louis Pasteur in 1848 while using a microscope to analyze crystallized fermentation
products of wine. Enantiomers are molecules that have the characteristic of chirality, in which their structures are
nonsuperimposable mirror images of each other. Chirality is an important characteristic in many biologically important molecules,
as illustrated by the examples of structural differences in the enantiomeric forms of the monosaccharide glucose or the amino acid
alanine (Figure 2.3.7).
Many organisms are only able to use one enantiomeric form of certain types of molecules as nutrients and as building blocks to
make structures within a cell. Some enantiomeric forms of amino acids have distinctly different tastes and smells when consumed
as food. For example, L-aspartame, commonly called aspartame, tastes sweet, whereas D-aspartame is tasteless. Drug enantiomers
can have very different pharmacologic affects. For example, the compound methorphan exists as two enantiomers, one of which
acts as an antitussive (dextromethorphan, a cough suppressant), whereas the other acts as an analgesic (levomethorphan, a drug
similar in effect to codeine).

Figure 2.3.7 : Enantiomers are stereoisomers that exhibit chirality. Their chemical structures are nonsuperimposable mirror images
of each other. (a) D-glucose and L-glucose are monosaccharides that are enantiomers. (b) The enantiomers D-alanine and L-alanine
are enantiomers found in bacterial cell walls and human cells, respectively.
Enantiomers are also called optical isomers because they can rotate the plane of polarized light. Some of the crystals Pasteur
observed from wine fermentation rotated light clockwise whereas others rotated the light counterclockwise. Today, we denote
enantiomers that rotate polarized light clockwise (+) as d forms, and the mirror image of the same molecule that rotates polarized
light counterclockwise (−) as the l form. The d and l labels are derived from the Latin words dexter (on the right) and laevus (on the
left), respectively. These two different optical isomers often have very different biological properties and activities. Certain species
of molds, yeast, and bacteria, such as Rhizopus, Yarrowia, and Lactobacillus spp., respectively, can only metabolize one type of
optical isomer; the opposite isomer is not suitable as a source of nutrients. Another important reason to be aware of optical isomers
is the therapeutic use of these types of chemicals for drug treatment, because some microorganisms can only be affected by one
specific optical isomer.

Exercise 2.3.2

We say that life is carbon based. What makes carbon so suitable to be part of all the macromolecules of living organisms?
Why is the arrangement of atoms so important?

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Synthesis of Biological Molecules:
Biological macromolecules are large molecules, necessary for life, that are built from smaller organic molecules. There are four
major classes of biological macromolecules (carbohydrates, lipids, proteins, and nucleic acids); each is an important cell
component and performs a wide array of functions. Combined, these molecules make up the majority of a cell’s dry mass (recall
that water makes up the majority of its complete mass). Biological macromolecules are organic, meaning they contain carbon. In
addition, they may contain hydrogen, oxygen, nitrogen, and additional minor elements. They also share the use of functional
groups and nearly identical building and deconstructing reactions.
Table 2.3.1 : Functions of Macromolecules

Macromolecule Functions

Energy storage, receptors, food, structural role in plants, fungal cell

Carbohydrates
walls, exoskeletons of insects

Lipids Energy storage, membrane structure, insulation, hormones, pigments

Nucleic acids Storage and transfer of genetic information

Enzymes, structure, receptors, transport, structural role in the

Proteins
cytoskeleton of a cell and the extracellular matrix

Biologically Significant Functional Groups

In addition to containing carbon atoms, biomolecules also contain functional groups—groups of atoms within molecules that are
categorized by their specific chemical composition and the chemical reactions they perform, regardless of the molecule in which
the group is found. Some of the most common functional groups are listed in Figure 2.3.8. In the formulas, the symbol R stands for
“residue” and represents the remainder of the molecule. R might symbolize just a single hydrogen atom or it may represent a group
of many atoms. Notice that some functional groups are relatively simple, consisting of just one or two atoms, while some comprise
two of these simpler functional groups. For example, a carbonyl group is a functional group composed of a carbon atom double
bonded to an oxygen atom: C=O. It is present in several classes of organic compounds as part of larger functional groups such as
ketones, aldehydes, carboxylic acids, and amides. In ketones, the carbonyl is present as an internal group, whereas in aldehydes it is
a terminal group.

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 Figure 2.3.8 : Common functional groups.
Hydrogen bonds between functional groups (within the same molecule or between different molecules) are important to the
function of many macromolecules and help them to fold properly into and maintain the appropriate shape for functioning.
Hydrogen bonds are also involved in various recognition processes, such as DNA complementary base pairing and the binding of
an enzyme to its substrate, as illustrated in Figure 2.3.9.

Figure 2.3.9 : Hydrogen bonds connect two strands of DNA together to create the double-helix structure.

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Dehydration Synthesis
Most macromolecules are made from single subunits, or building blocks, called monomers. The monomers combine with each
other using covalent bonds to form larger molecules known as polymers. In doing so, monomers release water molecules as
byproducts. This type of reaction is known as dehydration synthesis, which means “to put together while losing water.”
In a dehydration synthesis reaction (Figure 2.3.10), the hydrogen of one monomer combines with the hydroxyl group of another
monomer, releasing a molecule of water. At the same time, the monomers share electrons and form covalent bonds. As additional
monomers join, this chain of repeating monomers forms a polymer. Different types of monomers can combine in many
configurations, giving rise to a diverse group of macromolecules. Even one kind of monomer can combine in a variety of ways to
form several different polymers: for example, glucose monomers are the constituents of starch, glycogen, and cellulose.

Figure 2.3.10 : In the dehydration synthesis reaction depicted above, two molecules of glucose are linked together to form the
disaccharide maltose. In the process, a water molecule is formed.

Hydrolysis
Polymers are broken down into monomers in a process known as hydrolysis, which means “to split water,” a reaction in which a
water molecule is used during the breakdown (Figure 2.3.11). During these reactions, the polymer is broken into two components:
one part gains a hydrogen atom (H+) and the other gains a hydroxyl molecule (OH–) from a split water molecule.

Figure 2.3.11 : In the hydrolysis reactions depicted above, a molecule of the disaccharide maltose is broken apart into its
component glucose molecules also breaking a water molecule apart.
Dehydration and hydrolysis reactions are catalyzed, or “sped up,” by specific enzymes; dehydration reactions involve the formation
of new bonds, requiring energy, while hydrolysis reactions break bonds and release energy. These reactions are similar for most
macromolecules, but each monomer and polymer reaction is specific for its class. For example, in our bodies, food is hydrolyzed,
or broken down, into smaller molecules by catalytic enzymes in the digestive system. This allows for easy absorption of nutrients
by cells in the intestine. Each macromolecule is broken down by a specific enzyme. For instance, carbohydrates are broken down
by amylase, sucrase, lactase, or maltase. Proteins are broken down by the enzymes pepsin and peptidase, and by hydrochloric acid.
Lipids are broken down by lipases. Breakdown of these macromolecules provides energy for cellular activities.

Visit this site to see visual representations of dehydration synthesis and hydrolysis.

Key Concepts and Summary

The unique properties of carbon make it a central part of biological molecules.Carbon binds to oxygen, hydrogen, and nitrogen
covalently to form the many molecules important for cellular function.
Carbon has four electrons in its outermost shell and can form four bonds.
Carbon and hydrogen can form hydrocarbon chains or rings. The shaping of these chains or rings define their overall chemical
characteristics and function.

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 The most abundant elements in cells are hydrogen, carbon, oxygen, nitrogen, phosphorus, and sulfur.
Life is carbon based. Each carbon atom can bind to another one producing a carbon skeleton that can be straight, branched, or
ring shaped.
The same numbers and types of atoms may bond together in different ways to yield different molecules called isomers. Isomers
may differ in the bonding sequence of their atoms (structural isomers) or in the spatial arrangement of atoms whose bonding
sequences are the same (stereoisomers), and their physical and chemical properties may vary slightly or drastically.
Proteins, carbohydrates, nucleic acids, and lipids are the four major classes of biological macromolecules—large molecules
necessary for life that are built from smaller organic molecules.
Functional groups confer specific chemical properties to molecules bearing them.
Macromolecules are made up of single units known as monomers that are joined by covalent bonds to form larger polymers.
A monomer joins with another monomer with the release of a water molecule, leading to the formation of a covalent bond.
These types of reactions are known as dehydration or condensation reactions.
When polymers are broken down into smaller units (monomers), a molecule of water is used for each bond broken by these
reactions; such reactions are known as hydrolysis reactions.
Dehydration and hydrolysis reactions are similar for all macromolecules, but each monomer and polymer reaction is specific to
its class. Dehydration reactions typically require an investment of energy for new bond formation, while hydrolysis reactions
typically release energy by breaking bonds.

Glossary
aliphatic hydrocarbon
hydrocarbon consisting of a linear chain of carbon atoms
aromatic hydrocarbon
hydrocarbon consisting of closed rings of carbon atoms
biological macromolecule
large molecule necessary for life that is built from smaller organic molecules

dehydration synthesis
(also, condensation) reaction that links monomer molecules together, releasing a molecule of water for each bond
formed

hydrocarbon
molecule that consists only of carbon and hydrogen
hydrolysis
reaction causes breakdown of larger molecules into smaller molecules with the utilization of water

monomer
smallest unit of larger molecules called polymers

organic molecule
any molecule containing carbon (except carbon dioxide)
polymer
chain of monomer residues that is linked by covalent bonds; polymerization is the process of polymer formation
from monomers by condensation

substituted hydrocarbon
hydrocarbon chain or ring containing an atom of another element in place of one of the backbone carbons

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Contributors and Attributions
Connie Rye (East Mississippi Community College), Robert Wise (University of Wisconsin, Oshkosh), Vladimir Jurukovski
(Suffolk County Community College), Jean DeSaix (University of North Carolina at Chapel Hill), Jung Choi (Georgia Institute
of Technology), Yael Avissar (Rhode Island College) among other contributing authors. Original content by OpenStax (CC BY
4.0; Download for free at http://cnx.org/contents/185cbf87-c72...f21b5eabd@9.87).

This page titled 2.3: Carbon and Organic Molecules is shared under a not declared license and was authored, remixed, and/or curated by
OpenStax.

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2.4: Carbohydrates
Learning Objectives
Give examples of monosaccharides and polysaccharides
Describe the function of monosaccharides and polysaccharides within a cell

The most abundant biomolecules on earth are carbohydrates. From a chemical viewpoint, carbohydrates are primarily a
combination of carbon and water, and many of them have the empirical formula (CH2O)n, where n is the number of repeated units.
This view represents these molecules simply as “hydrated” carbon atom chains in which water molecules attach to each carbon
atom, leading to the term “carbohydrates.” Although all carbohydrates contain carbon, hydrogen, and oxygen, there are some that
also contain nitrogen, phosphorus, and/or sulfur. Carbohydrates have myriad different functions. They are abundant in terrestrial
ecosystems, many forms of which we use as food sources. These molecules are also vital parts of macromolecular structures that
store and transmit genetic information (i.e., DNA and RNA). They are the basis of biological polymers that impart strength to
various structural components of organisms (e.g., cellulose and chitin), and they are the primary source of energy storage in the
form of starch and glycogen.

Monosaccharides: The Sweet Ones

In biochemistry, carbohydrates are often called saccharides, from the Greek sakcharon, meaning sugar, although not all the
saccharides are sweet. The simplest carbohydrates are called monosaccharides, or simple sugars. They are the building blocks
(monomers) for the synthesis of polymers or complex carbohydrates, as will be discussed further in this section. Monosaccharides
are classified based on the number of carbons in the molecule. General categories are identified using a prefix that indicates the
number of carbons and the suffix –ose, which indicates a saccharide; for example, triose (three carbons), tetrose (four carbons),
pentose (five carbons), and hexose (six carbons) (Figure 2.4.1). The hexose D-glucose is the most abundant monosaccharide in
nature. Other very common and abundant hexose monosaccharides are galactose, used to make the disaccharide milk sugar lactose,
and the fruit sugar fructose.

Figure 2.4.1 : Monosaccharides are classified based on the position of the carbonyl group and the number of carbons in the
backbone.
Monosaccharides of four or more carbon atoms are typically more stable when they adopt cyclic, or ring, structures. These ring
structures result from a chemical reaction between functional groups on opposite ends of the sugar’s flexible carbon chain, namely

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the carbonyl group and a relatively distant hydroxyl group. Glucose, for example, forms a six-membered ring (Figure 2.4.2).

Figure 2.4.2 : (a) A linear monosaccharide (glucose in this case) forms a cyclic structure. (b) This illustration shows a more realistic
depiction of the cyclic monosaccharide structure. Note in these cyclic structural diagrams, the carbon atoms composing the ring are
not explicitly shown.

Exercise 2.4.1

Why do monosaccharides form ring structures?

Disaccharides
Two monosaccharide molecules may chemically bond to form a disaccharide. The name given to the covalent bond between the
two monosaccharides is a glycosidic bond. Glycosidic bonds form between hydroxyl groups of the two saccharide molecules, an
example of the dehydration synthesis described in the previous section of this chapter:

Common disaccharides are the grain sugar maltose, made of two glucose molecules; the milk sugar lactose, made of a galactose
and a glucose molecule; and the table sugar sucrose, made of a glucose and a fructose molecule (Figure 2.4.3).

Figure 2.4.3: Common disaccharides include maltose, lactose, and sucrose.

Polysaccharides
Polysaccharides, also called glycans, are large polymers composed of hundreds of monosaccharide monomers. Unlike mono- and
disaccharides, polysaccharides are not sweet and, in general, they are not soluble in water. Like disaccharides, the monomeric units
of polysaccharides are linked together by glycosidic bonds.
Polysaccharides are very diverse in their structure. Three of the most biologically important polysaccharides—starch, glycogen,
and cellulose—are all composed of repetitive glucose units, although they differ in their structure (Figure 2.4.4). Cellulose consists
of a linear chain of glucose molecules and is a common structural component of cell walls in plants and other organisms. Glycogen
and starch are branched polymers; glycogen is the primary energy-storage molecule in animals and bacteria, whereas plants

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primarily store energy in starch. The orientation of the glycosidic linkages in these three polymers is different as well (Figure
2.4.5) and, as a consequence, linear and branched macromolecules have different properties.

Modified glucose molecules can be fundamental components of other structural polysaccharides. Examples of these types of
structural polysaccharides are N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) found in bacterial cell wall
peptidoglycan. Polymers of NAG form chitin, which is found in fungal cell walls and in the exoskeleton of insects (Figure 2.4.5.

Figure 2.4.4 : Starch, glycogen, and cellulose are three of the most important polysaccharides. In the top row, hexagons represent
individual glucose molecules. Micrographs (bottom row) show wheat starch granules stained with iodine (left), glycogen granules
(G) inside the cell of a cyanobacterium (middle), and bacterial cellulose fibers (right). (credit “iodine granules”: modification of
work by Kiselov Yuri; credit “glycogen granules”: modification of work by Stöckel J, Elvitigala TR, Liberton M, Pakrasi HB;
credit “cellulose”: modification of work by American Society for Microbiology)
Chemical structures of starch, glycogen, cellulose, and chitin.

Figure 2.4.5 : The linkages between the glucose molecules in starches or glycogen and structural carbohydrates like cellulose or
chitin make a big difference in the stability of the molecule.

Exercise 2.4.2

What are the most biologically important polysaccharides and why are they important?

Key Concepts and Summary

Carbohydrates, the most abundant biomolecules on earth, are widely used by organisms for structural and energy-storage
purposes.
Carbohydrates include individual sugar molecules (monosaccharides) as well as two or more molecules chemically linked by
glycosidic bonds. Monosaccharides are classified based on the number of carbons the molecule as trioses (3 C), tetroses (4 C),
pentoses (5 C), and hexoses (6 C). They are the building blocks for the synthesis of polymers or complex carbohydrates.
Disaccharides such as sucrose, lactose, and maltose are molecules composed of two monosaccharides linked together by a
glycosidic bond.
Polysaccharides, or glycans, are polymers composed of hundreds of monosaccharide monomers linked together by glycosidic
bonds. The energy-storage polymers starch and glycogen are examples of polysaccharides and are all composed of branched
chains of glucose molecules.
The polysaccharide cellulose is a common structural component of the cell walls of organisms. Other structural
polysaccharides, such as N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), incorporate modified glucose
molecules and are used in the construction of peptidoglycan or chitin.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

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2.5: Lipids
Learning Objectives
Describe the chemical composition of lipids
Describe the unique characteristics and diverse structures of lipids
Compare and contrast triacylglycerides (triglycerides) and phospholipids.
Describe how phospholipids are used to construct biological membranes.

Although they are composed primarily of carbon and hydrogen, lipid molecules may also contain oxygen, nitrogen, sulfur, and
phosphorous. Lipids serve numerous and diverse purposes in the structure and functions of organisms. They can be a source of
nutrients, a storage form for carbon, energy-storage molecules, or structural components of membranes and hormones. Lipids
comprise a broad class of many chemically distinct compounds, the most common of which are discussed in this section.

Fatty Acids and Triglycerides

The fatty acids are lipids that contain long-chain hydrocarbons terminated with a carboxylic acid functional group. Because the
long hydrocarbon chain, fatty acids are hydrophobic (“water fearing”) or nonpolar. A triglyceride is formed when three fatty acids
are chemically linked to a glycerol molecule (Figure 2.5.1). Triglycerides are the primary components of adipose tissue (body fat),
and are major constituents of sebum (skin oils). They play an important metabolic role, serving as efficient energy-storage
molecules that can provide more than double the caloric content of both carbohydrates and proteins.

Figure 2.5.1 : Triglycerides are composed of a glycerol molecule attached to three fatty acids by a dehydration synthesis reaction.

Saturated vs Unsaturated
Fatty acids with hydrocarbon chains that contain only single bonds are called saturated fatty acids because they have the greatest
number of hydrogen atoms possible and are, therefore, “saturated” with hydrogen. Fatty acids with hydrocarbon chains containing
at least one double bond are called unsaturated fatty acids because they have fewer hydrogen atoms. Saturated fatty acids have a
straight, flexible carbon backbone, whereas unsaturated fatty acids have “kinks” in their carbon skeleton because each double bond
causes a rigid bend of the carbon skeleton. These differences in saturated versus unsaturated fatty acid structure result in different
properties for the corresponding lipids in which the fatty acids are incorporated.
In unsaturated fatty acids the double bonds can be in either the cis or trans configuration, illustrated in Figure 2.5.2. When some of
these bonds are in the cis configuration, the resulting bend in the carbon backbone of the chain means that triglyceride molecules
cannot pack tightly, so they remain liquid (oil) at room temperature. On the other hand, triglycerides with trans double bonds
(popularly called trans fats), have relatively linear fatty acids that are able to pack tightly together at room temperature and form
solid fats. In the human diet, trans fats are linked to an increased risk of cardiovascular disease, so many food manufacturers have
reduced or eliminated their use in recent years. In contrast to unsaturated fats, triglycerides without double bonds between carbon
atoms are called saturated fats, meaning that they contain all the hydrogen atoms available. Saturated fats are a solid at room
temperature and usually of animal origin.

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 Figure 2.5.2 : These space-filling models show a cis (oleic acid) and a trans (eliadic acid) fatty acid. Notice the bend in the
molecule cause by the cis configuration.

Exercise 2.5.1

Explain why fatty acids with hydrocarbon chains that contain only single bonds are called saturated fatty acids.

Phospholipids and Biological Membranes

Triglycerides are classified as simple lipids because they are formed from just two types of compounds: glycerol and fatty acids. In
contrast, complex lipids contain at least one additional component, for example, a phosphate group (phospholipids) or a
carbohydrate moiety (glycolipids). Figure 2.5.3 depicts a typical phospholipid composed of two fatty acids linked to glycerol (a
diglyceride). The two fatty acid carbon chains may be both saturated, both unsaturated, or one of each. Instead of another fatty acid
molecule (as for triglycerides), the third binding position on the glycerol molecule is occupied by a modified phosphate group.

Figure 2.5.3 : This illustration shows a phospholipid with two different fatty acids, one saturated and one unsaturated, bonded to the
glycerol molecule. The unsaturated fatty acid has a slight kink in its structure due to the double bond.
The molecular structure of lipids results in unique behavior in aqueous environments. Figure 2.5.1 depicts the structure of a
triglyceride. Because all three substituents on the glycerol backbone are long hydrocarbon chains, these compounds are nonpolar
and not significantly attracted to polar water molecules—they are hydrophobic. Conversely, phospholipids such as the one shown
in Figure 2.5.3 have a negatively charged phosphate group. Because the phosphate is charged, it is capable of strong attraction to
water molecules and thus is hydrophilic, or “water loving.” The hydrophilic portion of the phospholipid is often referred to as a
polar “head,” and the long hydrocarbon chains as nonpolar “tails.” A molecule presenting a hydrophobic portion and a hydrophilic
moiety is said to be amphipathic. Notice the “R” designation within the hydrophilic head depicted in Figure 2.5.3, indicating that a
polar head group can be more complex than a simple phosphate moiety. Glycolipids are examples in which carbohydrates are
bonded to the lipids’ head groups.
The amphipathic nature of phospholipids enables them to form uniquely functional structures in aqueous environments. As
mentioned, the polar heads of these molecules are strongly attracted to water molecules, and the nonpolar tails are not. Because of
their considerable lengths, these tails are, in fact, strongly attracted to one another. As a result, energetically stable, large-scale
assemblies of phospholipid molecules are formed in which the hydrophobic tails congregate within enclosed regions, shielded from
contact with water by the polar heads (Figure 2.5.4). The simplest of these structures are micelles, spherical assemblies containing
a hydrophobic interior of phospholipid tails and an outer surface of polar head groups. Larger and more complex structures are

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created from lipid-bilayer sheets, or unit membranes, which are large, two-dimensional assemblies of phospholipids congregated
tail to tail. The cell membranes of nearly all organisms are made from lipid-bilayer sheets, as are the membranes of many
intracellular components. These sheets may also form lipid-bilayer spheres that are the structural basis of vesicles and liposomes,
subcellular components that play a role in numerous physiological functions.

Figure 2.5.4 : Phospholipids tend to arrange themselves in aqueous solution forming liposomes, micelles, or lipid bilayer sheets.
(credit: modification of work by Mariana Ruiz Villarreal)

Exercise 2.5.2

How is the amphipathic nature of phospholipids significant?

Isoprenoids
The isoprenoids are branched lipids, also referred to as terpenoids, that are formed by chemical modifications of the isoprene
molecule (Figure 2.5.5). These lipids play a wide variety of physiological roles in plants and animals, with many technological
uses as pharmaceuticals (capsaicin), pigments (e.g., orange beta carotene, xanthophylls), and fragrances (e.g., menthol, camphor,
limonene [lemon fragrance], and pinene [pine fragrance]). Long-chain isoprenoids are also found in hydrophobic oils and waxes.
Waxes are typically water resistant and hard at room temperature, but they soften when heated and liquefy if warmed adequately. In
humans, the main wax production occurs within the sebaceous glands of hair follicles in the skin, resulting in a secreted material
called sebum, which consists mainly of triglycerol, wax esters, and the hydrocarbon squalene. There are many bacteria in the
microbiota on the skin that feed on these lipids. One of the most prominent bacteria that feed on lipids is Propionibacterium acnes,
which uses the skin’s lipids to generate short-chain fatty acids and is involved in the production of acne.

Figure 2.5.5 : Five-carbon isoprene molecules are chemically modified in various ways to yield isoprenoids.

Steroids
Another type of lipids are steroids, complex, ringed structures that are found in cell membranes; some function as hormones. The
most common types of steroids are sterols, which are steroids containing an -OH group. These are mainly hydrophobic molecules,
but also have hydrophilic hydroxyl groups. The most common sterol found in animal tissues is cholesterol. Its structure consists of

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four rings with a double bond in one of the rings, and a hydroxyl group at the sterol-defining position. The function of cholesterol
is to strengthen cell membranes in eukaryotes and in bacteria without cell walls, such as Mycoplasma. Prokaryotes generally do not
produce cholesterol, although bacteria produce similar compounds called hopanoids, which are also multiringed structures that
strengthen bacterial membranes (Figure 2.5.6). Fungi and some protozoa produce a similar compound called ergosterol, which
strengthens the cell membranes of these organisms.

Figure 2.5.6 : Cholesterol and hopene (a hopanoid compound) are molecules that reinforce the structure of the cell membranes in
eukaryotes and prokaryotes, respectively.

Exercise 2.5.3

How are isoprenoids used in technology?

Clinical Focus: part 2

The moisturizing cream prescribed by Penny’s doctor was a topical corticosteroid cream containing hydrocortisone.
Hydrocortisone is a synthetic form of cortisol, a corticosteroid hormone produced in the adrenal glands, from cholesterol.
When applied directly to the skin, it can reduce inflammation and temporarily relieve minor skin irritations, itching, and rashes
by reducing the secretion of histamine, a compound produced by cells of the immune system in response to the presence of
pathogens or other foreign substances. Because histamine triggers the body’s inflammatory response, the ability of
hydrocortisone to reduce the local production of histamine in the skin effectively suppresses the immune system and helps
limit inflammation and accompanying symptoms such as pruritus (itching) and rashes.

Exercise 2.5.4

Does the corticosteroid cream treat the cause of Penny’s rash, or just the symptoms?

Key Concepts and Summary

Lipids are composed mainly of carbon and hydrogen, but they can also contain oxygen, nitrogen, sulfur, and phosphorous.
They provide nutrients for organisms, store carbon and energy, play structural roles in membranes, and function as hormones,
pharmaceuticals, fragrances, and pigments.
Fatty acids are long-chain hydrocarbons with a carboxylic acid functional group. Their relatively long nonpolar hydrocarbon
chains make them hydrophobic. Fatty acids with no double bonds are saturated; those with double bonds are unsaturated.
Fatty acids chemically bond to glycerol to form structurally essential lipids such as triglycerides and phospholipids.
Triglycerides comprise three fatty acids bonded to glycerol, yielding a hydrophobic molecule. Phospholipids contain both
hydrophobic hydrocarbon chains and polar head groups, making them amphipathicand capable of forming uniquely functional
large scale structures.
Biological membranes are large-scale structures based on phospholipid bilayers that provide hydrophilic exterior and interior
surfaces suitable for aqueous environments, separated by an intervening hydrophobic layer. These bilayers are the structural
basis for cell membranes in most organisms, as well as subcellular components such as vesicles.
Isoprenoids are lipids derived from isoprene molecules that have many physiological roles and a variety of commercial
applications.
A wax is a long-chain isoprenoid that is typically water resistant; an example of a wax-containing substance is sebum, produced
by sebaceous glands in the skin. Steroids are lipids with complex, ringed structures that function as structural components of

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 cell membranes and as hormones. Sterols are a subclass of steroids containing a hydroxyl group at a specific location on one of
the molecule’s rings; one example is cholesterol.
Bacteria produce hopanoids, structurally similar to cholesterol, to strengthen bacterial membranes. Fungi and protozoa produce
a strengthening agent called ergosterol.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 2.5: Lipids is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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2.6: Proteins
Learning Objectives
Describe the fundamental structure of an amino acid
Describe the chemical structures of proteins
Summarize the unique characteristics of proteins

At the beginning of this chapter, a famous experiment was described in which scientists synthesized amino acids under conditions
simulating those present on earth long before the evolution of life as we know it. These compounds are capable of bonding together
in essentially any number, yielding molecules of essentially any size that possess a wide array of physical and chemical properties
and perform numerous functions vital to all organisms. The molecules derived from amino acids can function as structural
components of cells and subcellular entities, as sources of nutrients, as atom- and energy-storage reservoirs, and in other roles such
as hormones, enzymes, receptors, and transport molecules.

Amino Acids and Peptide Bonds

An amino acid is an organic molecule in which a hydrogen atom, a carboxyl group (–COOH), and an amino group (–NH2) are all
bonded to the same carbon atom, the so-called α carbon. The fourth group bonded to the α carbon varies among the different amino
acids and is called a residue or a side chain, represented in structural formulas by the letter R. A residue is a monomer that results
when two or more amino acids combine and remove water molecules. The primary structure of a protein, a peptide chain, is made
of amino acid residues. The unique characteristics of the functional groups and R groups allow these components of the amino
acids to form hydrogen, ionic, and disulfide bonds, along with polar/nonpolar interactions needed to form secondary, tertiary, and
quaternary protein structures. These groups are composed primarily of carbon, hydrogen, oxygen, nitrogen, and sulfur, in the form
of hydrocarbons, acids, amides, alcohols, and amines. A few examples illustrating these possibilities are provided in Figure 2.6.1.

Figure 2.6.1 : Amino acids

Amino acids may chemically bond together by reaction of the carboxylic acid group of one molecule with the amine group of
another. This reaction forms a peptide bond and a water molecule and is another example of dehydration synthesis (Figure 2.6.2).

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Molecules formed by chemically linking relatively modest numbers of amino acids (approximately 50 or fewer) are called peptides,
and prefixes are often used to specify these numbers: dipeptides (two amino acids), tripeptides (three amino acids), and so forth.
More generally, the approximate number of amino acids is designated: oligopeptides are formed by joining up to approximately 20
amino acids, whereas polypeptides are synthesized from up to approximately 50 amino acids. When the number of amino acids
linked together becomes very large, or when multiple polypeptides are used as building subunits, the macromolecules that result are
called proteins. The continuously variable length (the number of monomers) of these biopolymers, along with the variety of
possible R groups on each amino acid, allows for a nearly unlimited diversity in the types of proteins that may be formed.

Figure 2.6.2 : Peptide bond formation is a dehydration synthesis reaction. The carboxyl group of the first amino acid (alanine) is
linked to the amino group of the incoming second amino acid (alanine). In the process, a molecule of water is released.
Exercise 2.6.1
How many amino acids are in polypeptides?

Protein Structure
The size (length) and specific amino acid sequence of a protein are major determinants of its shape, and the shape of a protein is
critical to its function. For example, in the process of biological nitrogen fixation, soil microorganisms collectively known as
rhizobia symbiotically interact with roots of legume plants such as soybeans, peanuts, or beans to form a novel structure called a
nodule on the plant roots. The plant then produces a carrier protein called leghemoglobin, a protein that carries nitrogen or oxygen.
Leghemoglobin binds with a very high affinity to its substrate oxygen at a specific region of the protein where the shape and amino
acid sequence are appropriate (the active site). If the shape or chemical environment of the active site is altered, even slightly, the
substrate may not be able to bind as strongly, or it may not bind at all. Thus, for the protein to be fully active, it must have the
appropriate shape for its function.
Protein structure is categorized in terms of four levels: primary, secondary, tertiary, and quaternary. The primary structure is simply
the sequence of amino acids that make up the polypeptide chain. Figure 2.6.3 depicts the primary structure of a protein. The chain
of amino acids that defines a protein’s primary structure is not rigid, but instead is flexible because of the nature of the bonds that
hold the amino acids together.
When the chain is sufficiently long, hydrogen bonding may occur between amine and carbonyl functional groups within the peptide
backbone (excluding the R side group), resulting in localized folding of the polypeptide chain into helices and sheets. These shapes
constitute a protein’s secondary structure. The most common secondary structures are the α-helix and β-pleated sheet. In the α-helix
structure, the helix is held by hydrogen bonds between the oxygen atom in a carbonyl group of one amino acid and the hydrogen
atom of the amino group that is just four amino acid units farther along the chain. In the β-pleated sheet, the pleats are formed by
similar hydrogen bonds between continuous sequences of carbonyl and amino groups that are further separated on the backbone of
the polypeptide chain (Figure 2.6.4).
The next level of protein organization is the tertiary structure, which is the large-scale three-dimensional shape of a single
polypeptide chain. Tertiary structure is determined by interactions between amino acid residues that are far apart in the chain. A
variety of interactions give rise to protein tertiary structure, such as disulfide bridges, which are bonds between the sulfhydryl (–
SH) functional groups on amino acid side groups; hydrogen bonds; ionic bonds; and hydrophobic interactions between nonpolar
side chains. All these interactions, weak and strong, combine to determine the final three-dimensional shape of the protein and its
function (Figure 2.6.5). Most proteins are complete here.
Some specialized proteins are assemblies of several separate polypeptides, also known as protein subunits. These proteins function
adequately only when all subunits are present and appropriately configured. The interactions that hold these subunits together

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constitute the quaternary structure of the protein. The overall quaternary structure is stabilized by relatively weak interactions.
Hemoglobin, for example, has a quaternary structure of four globular protein subunits: two α and two β polypeptides, each one
containing an iron-based heme (Figure 2.6.6).
The process by which a polypeptide chain assumes a large-scale, three-dimensional shape is called protein folding. Folded proteins
that are fully functional in their normal biological role are said to possess a native structure. When a protein loses its three-
dimensional shape, it may no longer be functional. These unfolded proteins are denatured. Denaturation implies the loss of the
secondary structure and tertiary structure (and, if present, the quaternary structure) without the loss of the primary structure.
Another important class of proteins is the conjugated proteins that have a nonprotein portion. If the conjugated protein has a
carbohydrate attached, it is called a glycoprotein. If it has a lipid attached, it is called a lipoprotein. These proteins are important
components of membranes. Figure 2.6.7 summarizes the four levels of protein structure.

Figure 2.6.3 : The primary structure of a protein is the sequence of amino acids. (credit: modification of work by National Human
Genome Research Institute)

Figure 2.6.4 : The secondary structure of a protein may be an α-helix or a β-pleated sheet, or both.

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 Figure 2.6.5 : The tertiary structure of proteins is determined by a variety of attractive forces, including hydrophobic interactions,
ionic bonding, hydrogen bonding, and disulfide linkages.

Figure 2.6.6 : A hemoglobin molecule has two α and two β polypeptides together with four heme groups.

Figure 2.6.7 : Protein structure has four levels of organization. (credit: modification of work by National Human Genome Research
Institute)
Exercise 2.6.2
What can happen if a protein’s primary, secondary, tertiary, or quaternary structure is changed?

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Primary Structure, Dysfunctional Proteins, and Cystic Fibrosis
Proteins associated with biological membranes are classified as extrinsic or intrinsic. Extrinsic proteins, also called peripheral
proteins, are loosely associated with one side of the membrane. Intrinsic proteins, or integral proteins, are embedded in the
membrane and often function as part of transport systems as transmembrane proteins. Cystic fibrosis (CF) is a human genetic
disorder caused by a change in the transmembrane protein. It affects mostly the lungs but may also affect the pancreas, liver,
kidneys, and intestine. CF is caused by a loss of the amino acid phenylalanine in a cystic fibrosis transmembrane protein
(CFTR). The loss of one amino acid changes the primary structure of a protein that normally helps transport salt and water in
and out of cells (Figure 2.6.8).
The change in the primary structure prevents the protein from functioning properly, which causes the body to produce unusually
thick mucus that clogs the lungs and leads to the accumulation of sticky mucus. The mucus obstructs the pancreas and stops
natural enzymes from helping the body break down food and absorb vital nutrients.
In the lungs of individuals with cystic fibrosis, the altered mucus provides an environment where bacteria can thrive. This
colonization leads to the formation of biofilms in the small airways of the lungs. The most common pathogens found in the
lungs of patients with cystic fibrosis are Pseudomonas aeruginosa (Figure 2.6.9) and Burkholderia cepacia. Pseudomonas
differentiates within the biofilm in the lung and forms large colonies, called “mucoid” Pseudomonas. The colonies have a
unique pigmentation that shows up in laboratory tests (Figure 2.6.9) and provides physicians with the first clue that the patient
has CF (such colonies are rare in healthy individuals).

Figure 2.6.8 : The normal CFTR protein is a channel protein that helps salt (sodium chloride) move in and out of cells.

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 Figure 2.6.9 : (a) A scanning electron micrograph shows the opportunistic bacterium Pseudomonas aeruginosa. (b) Pigment-
producing P. aeruginosa on cetrimide agar shows the green pigment called pyocyanin. (credit a: modification of work by the
Centers for Disease Control and Prevention)

For more information about cystic fibrosis, visit the Cystic Fibrosis Foundation website.

Key Concepts and Summary

Amino acids are small molecules essential to all life. Each has an α carbon to which a hydrogen atom, carboxyl group, and
amine group are bonded. The fourth bonded group, represented by R, varies in chemical composition, size, polarity, and charge
among different amino acids, providing variation in properties.
Peptides are polymers formed by the linkage of amino acids via dehydration synthesis. The bonds between the linked amino
acids are called peptide bonds. The number of amino acids linked together may vary from a few to many.
Proteins are polymers formed by the linkage of a very large number of amino acids. They perform many important functions in
a cell, serving as nutrients and enzymes; storage molecules for carbon, nitrogen, and energy; and structural components.
The structure of a protein is a critical determinant of its function and is described by a graduated classification: primary,
secondary, tertiary, and quaternary. The native structure of a protein may be disrupted by denaturation, resulting in loss of
its higher-order structure and its biological function.
Some proteins are formed by several separate protein subunits, the interaction of these subunits composing the quaternary
structure of the protein complex.
Conjugated proteins have a nonpolypeptide portion that can be a carbohydrate (forming a glycoprotein) or a lipid fraction
(forming a lipoprotein). These proteins are important components of membranes.

Contributors
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 2.6: Proteins is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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2.7: Nucleic Acids
Learning Objectives
Describe the structure of nucleic acids and define the types of nucleic acids
Explain the structure and role of DNA
Explain the structure and roles of RNA
Explain the structure and role of ATP

Nucleic acids are the most important macromolecules for the continuity of life. They carry the genetic blueprint of a cell and carry
instructions for the functioning of the cell.

DNA and RNA

The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the genetic material
found in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is found in the nucleus of eukaryotes
and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope.
The entire genetic content of a cell is known as its genome, and the study of genomes is genomics. This includes both the
chromosomal and extra-chromosomal DNA. Chromosomes in prokaryotes have very few differences when compared to
eukaryotes. These differences are mostly in size, number and shape. Both have packaging proteins, both contain many genes.
Eukaryotes generally have more DNA, higher numbers and a linear shape, as compared to circular in prokaryotes. Also in
eukaryotic cells, DNA forms a complex with histone proteins to form chromatin, the substance of eukaryotic chromosomes. A
chromosome may contain hundreds to tens of thousands of genes. Many genes contain the information to make protein products;
other genes code for RNA products. DNA controls all of the cellular activities by turning the genes “on” or “off.”
Another type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus (in
eukaryotes) or nucleoid (in prokaryotes) but instead use an intermediary to communicate with the rest of the cell. This intermediary
is the messenger RNA (mRNA). Other types of RNA—like rRNA, tRNA, and microRNA—are also involved in protein synthesis
and its regulation.
DNA and RNA are polymers made up of monomers known as nucleotides. The nucleotides combine can with each other to form a
polynucleotide. Each nucleotide is made up of three components: a nitrogenous base, a pentose (five-carbon) sugar, and one or
more phosphate groups (Figure 2.7.1). Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to
the phosphate group(s).
The nitrogenous bases, important components of nucleotides, are organic molecules and are so named because they contain carbon
and nitrogen. They are bases because they contain an amino group that has the potential of binding an extra hydrogen, and thus,
decreases the hydrogen ion concentration in its environment, making it more basic. Each nucleotide in DNA contains one of four
possible nitrogenous bases: adenine (A), guanine (G) cytosine (C), and thymine (T).
Adenine and guanine are classified as purines. The primary structure of a purine is two carbon-nitrogen rings. Cytosine, thymine,
and uracil are classified as pyrimidines which have a single carbon-nitrogen ring as their primary structure (Figure 2.7.1). Each of
these basic carbon-nitrogen rings has different functional groups attached to it. In molecular biology shorthand, the nitrogenous
bases are simply known by their symbols A, T, G, C, and U. DNA contains A, T, G, and C whereas RNA contains A, U, G, and C.
The pentose sugar in DNA is deoxyribose, and in RNA, the sugar is ribose (Figure 2.7.1). The difference between the sugars is the
presence of the hydroxyl group on the second carbon of the ribose and hydrogen on the second carbon of the deoxyribose. The
carbon atoms of the sugar molecule are numbered as 1′, 2′, 3′, 4′, and 5′ (1′ is read as “one prime”). The phosphate residue is
attached to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next
nucleotide, which forms a 5′–3′ phosphodiester linkage. The phosphodiester linkage is not formed by simple dehydration reaction
like the other linkages connecting monomers in macromolecules: its formation involves the removal of two phosphate groups. A
polynucleotide may have thousands of such phosphodiester linkages.

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 Figure 2.7.1 : A nucleotide is made up of three components: a nitrogenous base, a pentose sugar, and one or more phosphate
groups. Carbon residues in the pentose are numbered 1′ through 5′ (the prime distinguishes these residues from those in the base,
which are numbered without using a prime notation). The base is attached to the 1′ position of the ribose, and the phosphate is
attached to the 5′ position. When a polynucleotide is formed, the 5′ phosphate of the incoming nucleotide attaches to the 3′
hydroxyl group at the end of the growing chain. Two types of pentose are found in nucleotides, deoxyribose (found in DNA) and
ribose (found in RNA). Deoxyribose is similar in structure to ribose, but it has an H instead of an OH at the 2′ position. Bases can
be divided into two categories: purines and pyrimidines. Purines have a double ring structure, and pyrimidines have a single ring.

DNA Double-Helix Structure

DNA has a double-helix structure (Figure 2.7.2). The sugar and phosphate lie on the outside of the helix, forming the backbone of
the DNA. The nitrogenous bases are stacked in the interior, like the steps of a staircase, in pairs; the pairs are bound to each other
by hydrogen bonds. Every base pair in the double helivx is separated from the next base pair by 0.34 nm. The two strands of the
helix run in opposite directions, meaning that the 5′ carbon end of one strand will face the 3′ carbon end of its matching strand.
(This is referred to as antiparallel orientation and is important to DNA replication and in many nucleic acid interactions.)

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 Figure 2.7.2 : Native DNA is an antiparallel double helix. The phosphate backbone (indicated by the curvy lines) is on the outside,
and the bases are on the inside. Each base from one strand interacts via hydrogen bonding with a base from the opposing strand.
(credit: Jerome Walker/Dennis Myts)
Only certain types of base pairing are allowed. For example, a certain purine can only pair with a certain pyrimidine. This means A
can pair with T, and G can pair with C, as shown in Figure 2.7.3. This is known as the base complementary rule. In other words,
the DNA strands are complementary to each other. If the sequence of one strand is AATTGGCC, the complementary strand would
have the sequence TTAACCGG. During DNA replication, each strand is copied, resulting in a daughter DNA double helix
containing one parental DNA strand and a newly synthesized strand.

RNA
Ribonucleic acid, or RNA, is mainly involved in the process of protein synthesis under the direction of DNA. RNA is usually
single-stranded and is made of ribonucleotides that are linked by phosphodiester bonds. A ribonucleotide in the RNA chain
contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and the phosphate group. Even though the
RNA is single stranded, most RNA types show extensive intramolecular base pairing between complementary sequences, creating
a predictable three-dimensional structure essential for their function.
There are four major types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and microRNA
(miRNA). The first, mRNA, carries the message from DNA, which controls all of the cellular activities in a cell. If a cell requires a
certain protein to be synthesized, the gene for this product is turned “on” and the messenger RNA is synthesized in the nucleus. The
RNA base sequence is complementary to the coding sequence of the DNA from which it has been copied. However, in RNA, the
base T is absent and U is present instead. If the DNA strand has a sequence AATTGCGC, the sequence of the complementary RNA
is UUAACGCG. In the cytoplasm, the mRNA interacts with ribosomes and other cellular machinery (Figure 2.7.3). More is
described about their roles in later chapters.

Figure 2.7.3 : A ribosome has two parts: a large subunit and a small subunit. The mRNA sits in between the two subunits. A tRNA
molecule recognizes a codon on the mRNA, binds to it by complementary base pairing, and adds the correct amino acid to the
growing peptide chain.
Table 2.7.1 below summarizes features of DNA and RNA.
Table 2.7.1: Features of DNA and RNA.

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 Features of DNA and RNA

DNA RNA

Function Carries genetic information Involved in protein synthesis

Location Remains in the nucleus Leaves the nucleus

Structure Double helix Usually single-stranded

Sugar Deoxyribose Ribose

Pyrimidines Cytosine, thymine Cytosine, uracil

Purines Adenine, guanine Adenine, guanine

As you have learned, information flow in an organism takes place from DNA to RNA to protein. DNA dictates the structure of
mRNA in a process known as transcription, and RNA dictates the structure of protein in a process known as translation. This is
known as the Central Dogma of Life, which holds true for all organisms; however, exceptions to the rule occur in connection with
viral infections.

To learn more about DNA, explore the Howard Hughes Medical Institute BioInteractive animations on the topic
of DNA.

ATP
ATP is a small, relatively simple molecule (Figure 2.7.4), but within some of its bonds, it contains the potential for a quick burst of
energy that can be harnessed to perform cellular work. This molecule can be thought of as the primary energy currency of cells in
much the same way that money is the currency that people exchange for things they need. ATP is used to power the majority of
energy-requiring cellular reactions.

Figure 2.7.4 : ATP is the primary energy currency of the cell. It has an adenosine backbone with three phosphate groups attached.
As its name suggests, adenosine triphosphate is comprised of adenosine bound to three phosphate groups (Figure 2.7.5). Adenosine
is a nucleotide consisting of the nitrogenous base adenine and a five-carbon sugar, ribose. The three phosphate groups, in order of
closest to furthest from the ribose sugar, are labeled alpha, beta, and gamma. Together, these chemical groups constitute an energy
powerhouse. However, not all bonds within this molecule exist in a particularly high-energy state. Both bonds that link the
phosphates are equally high-energy bonds (phosphoanhydride bonds) that, when broken, release sufficient energy to power a
variety of cellular reactions and processes. These high-energy bonds are the bonds between the second and third (or beta and
gamma) phosphate groups and between the first and second phosphate groups. The reason that these bonds are considered “high-
energy” is because the products of such bond breaking—adenosine diphosphate (ADP) and one inorganic phosphate group (Pi)—
have considerably lower free energy than the reactants: ATP and a water molecule. Because this reaction takes place with the use of
a water molecule, it is considered a hydrolysis reaction.

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 Clinical Focus: Resolution
Penny stopped using her new sunscreen and applied the corticosteroid cream to her rash as directed. However, after several
days, her rash had not improved and actually seemed to be getting worse. She made a follow-up appointment with her doctor,
who observed a bumpy red rash and pus-filled blisters around hair follicles (Figure 2.7.3). The rash was especially
concentrated in areas that would have been covered by a swimsuit. After some questioning, Penny told the physician that she
had recently attended a pool party and spent some time in a hot tub. In light of this new information, the doctor suspected a
case of hot tub rash, an infection frequently caused by the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that
can thrive in hot tubs and swimming pools, especially when the water is not sufficiently chlorinated. P. aeruginosa is the same
bacterium that is associated with infections in the lungs of patients with cystic fibrosis.
The doctor collected a specimen from Penny’s rash to be sent to the clinical microbiology lab. Confirmatory tests were carried
out to distinguish P. aeruginosa from enteric pathogens that can also be present in pool and hot-tub water. The test included the
production of the blue-green pigment pyocyanin on cetrimide agar and growth at 42 °C. Cetrimide is a selective agent that
inhibits the growth of other species of microbial flora and also enhances the production of P. aeruginosa pigments pyocyanin
and fluorescein, which are a characteristic blue-green and yellow-green, respectively.
Tests confirmed the presence of P. aeruginosa in Penny’s skin sample, but the doctor decided not to prescribe an antibiotic.
Even though P. aeruginosa is a bacterium, Pseudomonas species are generally resistant to many antibiotics. Luckily, skin
infections like Penny’s are usually self-limiting; the rash typically lasts about 2 weeks and resolves on its own, with or without
medical treatment. The doctor advised Penny to wait it out and keep using the corticosteroid cream. The cream will not kill the
P. aeruginosa on Penny’s skin, but it should calm her rash and minimize the itching by suppressing her body’s inflammatory
response to the bacteria.

Figure 2.7.3 : Exposure to Pseudomonas aeruginosa in the water of a pool or hot tub can sometimes cause a skin infection that
manifests as “hot tub rash.” (credit: modification of work by “Lsupellmel”/Wikimedia Commons)

Key Concepts and Summary

Nucleic acids are molecules made up of nucleotides that direct cellular activities such as cell division and protein synthesis.
Each nucleotide is made up of a pentose sugar, a nitrogenous base, and a phosphate group.
DNA carries the genetic blueprint of the cell and is passed on from parents to offspring (in the form of chromosomes). It has a
double-helical structure with the two strands running in opposite directions, connected by hydrogen bonds, and complementary
to each other.
RNA is single-stranded and is made of a pentose sugar (ribose), a nitrogenous base, and a phosphate group. RNA is involved in
protein synthesis and its regulation.
ATP is a single nucleotide made of a pentose sugar (ribose), a nitrogenous base, and three phosphate groups. It helps the cell to
store and move energy.

Glossary
adenosine triphosphate (ATP)
adenosine triphosphate, the cell’s energy currency
deoxyribonucleic acid (DNA)
double-helical molecule that carries the hereditary information of the cell

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messenger RNA (mRNA)
RNA that carries information from DNA to ribosomes during protein synthesis

nucleic acid
biological macromolecule that carries the genetic blueprint of a cell and carries instructions for the functioning of the cell

nucleotide
monomer of nucleic acids; contains a pentose sugar, one or more phosphate groups, and a nitrogenous base

phosphodiester
linkage covalent chemical bond that holds together the polynucleotide chains with a phosphate group linking two pentose
sugars of neighboring nucleotides

polynucleotide
long chain of nucleotides

purine
type of nitrogenous base in DNA and RNA; adenine and guanine are purines

pyrimidine
type of nitrogenous base in DNA and RNA; cytosine, thymine, and uracil are pyrimidines

ribonucleic acid (RNA)

single-stranded, often internally base paired, molecule that is involved in protein synthesis

ribosomal RNA (rRNA)

RNA that ensures the proper alignment of the mRNA and the ribosomes during protein synthesis and catalyzes the formation of
the peptide linkage

transcription
process through which messenger RNA forms on a template of DNA

transfer RNA (tRNA)

RNA that carries activated amino acids to the site of protein synthesis on the ribosome

translation
process through which RNA directs the formation of protein

Contributors and Attributions

Connie Rye (East Mississippi Community College), Robert Wise (University of Wisconsin, Oshkosh), Vladimir Jurukovski
(Suffolk County Community College), Jean DeSaix (University of North Carolina at Chapel Hill), Jung Choi (Georgia Institute
of Technology), Yael Avissar (Rhode Island College) among other contributing authors. Original content by OpenStax (CC BY
4.0; Download for free at http://cnx.org/contents/185cbf87-c72...f21b5eabd@9.87).

This page titled 2.7: Nucleic Acids is shared under a not declared license and was authored, remixed, and/or curated by OpenStax.

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Chapter 2 Exercises
Review Questions for Chapter 2:

Multiple Choice

1) If xenon has an atomic number of 54 and a mass number of 108, how many neutrons does it have?
1. 54
2. 27
3. 100
4. 108

2) Atoms that vary in the number of neutrons found in their nuclei are called ________.
1. ions
2. neutrons
3. neutral atoms
4. isotopes

3) Potassium has an atomic number of 19. What is its electron configuration?

1. shells 1 and 2 are full, and shell 3 has nine electrons
2. shells 1, 2 and 3 are full and shell 4 has three electrons
3. shells 1, 2 and 3 are full and shell 4 has one electron
4. shells 1, 2 and 3 are full and no other electrons are present

4) Which type of bond represents a weak chemical bond?

1. hydrogen bond
2. atomic bond
3. covalent bond
4. nonpolar covalent bond

5) Which of the following statements is not true?

1. Water is polar.
2. Water stabilizes temperature.
3. Water is essential for life.
4. Water is the most abundant molecule in the Earth’s atmosphere.

6) When acids are added to a solution, the pH should ________.

1. decrease
2. increase
3. stay the same
4. cannot tell without testing

7) We call a molecule that binds up excess hydrogen ions in a solution a(n) ________.
1. acid

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 2. isotope
3. base
4. donator

8) Which of the following statements is true?

1. Acids and bases cannot mix together.
2. Acids and bases will neutralize each other.
3. Acids, but not bases, can change the pH of a solution.
4. Acids donate hydroxide ions (OH–); bases donate hydrogen ions (H+).

9) Each carbon molecule can bond with as many as________ other atom(s) or molecule(s).
1. one
2. two
3. six
4. four

10) Which of the following is not a functional group that can bond with carbon?
1. sodium
2. hydroxyl
3. phosphate
4. carbonyl

11) Which of the following is the name for molecules whose structures are nonsuperimposable mirror images?
1. structural isomers
2. monomers
3. polymers
4. enantiomers

12) By definition, carbohydrates contain which elements?

1. carbon and hydrogen
2. carbon, hydrogen, and nitrogen
3. carbon, hydrogen, and oxygen
4. carbon and oxygen

13) Monosaccharides may link together to form polysaccharides by forming which type of bond?
1. hydrogen
2. peptide
3. ionic
4. glycosidic

14) Which of the following describes lipids?

1. a source of nutrients for organisms
2. energy-storage molecules
3. molecules having structural role in membranes
4. molecules that are part of hormones and pigments

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 5. all of the above

15) Molecules bearing both polar and nonpolar groups are said to be which of the following?
1. hydrophilic
2. amphipathic
3. hydrophobic
4. polyfunctional

16) Which of the following groups varies among different amino acids?
1. hydrogen atom
2. carboxyl group
3. R group
4. amino group

17) The amino acids present in proteins differ in which of the following?
1. size
2. shape
3. side groups
4. all of the above

18) Which of the following bonds are not involved in tertiary structure?
1. peptide bonds
2. ionic bonds
3. hydrophobic interactions
4. hydrogen bonds

19) Which of the following characteristics/compounds is not considered to be a phenotypic biochemical characteristic used of
microbial identification?
1. poly-β-hydroxybutyrate
2. small-subunit (16S) rRNA gene
3. carbon utilization
4. lipid composition

20) Proteomic analysis is a methodology that deals with which of the following?
1. the analysis of proteins functioning as enzymes within the cell
2. analysis of transport proteins in the cell
3. the analysis of integral proteins of the cell membrane
4. the study of all accumulated proteins of an organism

Fill-in-the-Blanks

21) Waxes contain esters formed from long-chain __________ and saturated __________, and they may also contain substituted
hydrocarbons.

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22) Cholesterol is the most common member of the __________ group, found in animal tissues; it has a tetracyclic carbon ring
system with a __________ bond in one of the rings and one free __________group.

23) The sequence of amino acids in a protein is called its __________.

24) Denaturation implies the loss of the __________ and __________ structures without the loss of the __________ structure.

Short Answer

25) What makes ionic bonds different from covalent bonds?

26) Why are hydrogen bonds and van der Waals interactions necessary for cells?

27) Discuss how buffers help prevent drastic swings in pH.

28) Why can some insects walk on water?

29) What property of carbon makes it essential for organic life?

30) Compare and contrast saturated and unsaturated triglycerides.

31) Why are carbon, nitrogen, oxygen, and hydrogen the most abundant elements in living matter and, therefore, considered
macronutrients?

32) Identify the functional group in each of the depicted structural formulas.

33) What are monosaccharides, disaccharides, and polysaccharides?

34) Describe the structure of a typical phospholipid. Are these molecules polar or nonpolar?

Critical Thinking
35) The structural formula shown corresponds to penicillin G, a narrow-spectrum antibiotic that is given intravenously or
intramuscularly as a treatment for several bacterial diseases. The antibiotic is produced by fungi of the genus Penicillium. (a)
Identify three major functional groups in this molecule that each comprise two simpler functional groups. (b) Name the two simpler
functional groups composing each of the major functional groups identified in (a).

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36) The figure depicts the structural formulas of glucose, galactose, and fructose. (a) Circle the functional groups that classify the
sugars either an aldose or a ketose, and identify each sugar as one or the other. (b) The chemical formula of these compounds is the
same, although the structural formula is different. What are such compounds called?

37) Structural diagrams for the linear and cyclic forms of a monosaccharide are shown. (a) What is the molecular formula for this
monosaccharide? (Count the C, H and O atoms in each to confirm that these two molecules have the same formula, and report this
formula.) (b) Identify which hydroxyl group in the linear structure undergoes the ring-forming reaction with the carbonyl group.

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38) The term “dextrose” is commonly used in medical settings when referring to the biologically relevant isomer of the
monosaccharide glucose. Explain the logic of this alternative name.

39) Microorganisms can thrive under many different conditions, including high-temperature environments such as hot springs. To
function properly, cell membranes have to be in a fluid state. How do you expect the fatty acid content (saturated versus
unsaturated) of bacteria living in high-temperature environments might compare with that of bacteria living in more moderate
temperatures?

40) Heating a protein sufficiently may cause it to denature. Considering the definition of denaturation, what does this statement say
about the strengths of peptide bonds in comparison to hydrogen bonds?

41) The image shown represents a tetrapeptide. (a) How many peptide bonds are in this molecule? (b) Identify the side groups of
the four amino acids composing this peptide.

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 CHAPTER OVERVIEW

3: Microscope and the Cell

Over the past several centuries, we have learned to manipulate light to peer into previously invisible worlds—those too small or too
far away to be seen by the naked eye. Through a microscope, we can examine microbial cells and colonies, using various
techniques to manipulate color, size, and contrast in ways that help us identify species and diagnose disease. This chapter explores
how various types of microscopes manipulate light in order to provide a window into the world of microorganisms. By
understanding how various kinds of microscopes work, we can produce highly detailed images of microbes that can be useful for
both research and clinical applications.
3.1: How Microscopes Work
3.2: Staining Microscopic Specimens and Descriptions
3.3: Cells as Living Things
Chapter 3 Exercises

Thumbnail: A compound microscope in a Biology lab. (CC -BY-SA 4.0; Acagastya).

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1
3.1: How Microscopes Work
Learning Objectives
Identify and define the characteristics of electromagnetic radiation (EMR) used in microscopy
Explain how lenses are used in microscopy to manipulate visible and ultraviolet (UV) light
Describe historical developments and individual contributions that led to the invention and development of the microscope
Compare and contrast the features of simple and compound microscopes
Identify and describe the parts of a brightfield microscope
Calculate total magnification for a compound microscope
Describe the distinguishing features and typical uses for various types of light microscopes, and electron microscopes.

Clinical Focus: part 1

Cindy, a 17-year-old counselor at a summer sports camp, scraped her knee playing basketball 2 weeks ago. At the time, she
thought it was only a minor abrasion that would heal, like many others before it. Instead, the wound began to look like an
insect bite and has continued to become increasingly painful and swollen.
The camp nurse examines the lesion and observes a large amount of pus oozing from the surface. Concerned that Cindy may
have developed a potentially aggressive infection, she swabs the wound to collect a sample from the infection site. Then she
cleans out the pus and dresses the wound, instructing Cindy to keep the area clean and to come back the next day. When Cindy
leaves, the nurse sends the sample to the closest medical lab to be analyzed under a microscope.

Exercise 3.1.1

What are some things we can learn about these bacteria by looking at them under a microscope?

Visible light consists of electromagnetic waves that behave like other waves. Hence, many of the properties of light that are
relevant to microscopy can be understood in terms of light’s behavior as a wave. An important property of light waves is the
wavelength, or the distance between one peak of a wave and the next peak. The height of each peak (or depth of each trough) is
called the amplitude. In contrast, the frequency of the wave is the rate of vibration of the wave, or the number of wavelengths
within a specified time period (Figure 3.1.1).

Figure 3.1.1 : (a) The amplitude is the height of a wave, whereas the wavelength is the distance between one peak and the next. (b)
These waves have different frequencies, or rates of vibration. The wave at the top has the lowest frequency, since it has the fewest
peaks per unit time. The wave at the bottom has the highest frequency.

Interactions of Light
Light waves interact with materials by being reflected, absorbed, or transmitted. Reflection occurs when a wave bounces off of a
material. For example, a red piece of cloth may reflect red light to our eyes while absorbing other colors of light. Absorbance
occurs when a material captures the energy of a light wave. In the case of glow-in-the-dark plastics, the energy from light can be
absorbed and then later re-emitted as another form of phosphorescence. Transmission occurs when a wave travels through a
material, like light through glass (the process of transmission is called transmittance). When a material allows a large proportion of
light to be transmitted, it may do so because it is thinner, or more transparent (having more transparency and less opacity). Figure
3.1.2 illustrates the difference between transparency and opacity.

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 Figure 3.1.2 : (a) A Petri dish is made of transparent plastic or glass, which allows transmission of a high proportion of light. This
transparency allows us to see through the sides of the dish to view the contents. (b) This slice of an iron meteorite is opaque (i.e., it
has opacity). Light is not transmitted through the material, making it impossible to see the part of the hand covered by the object.
(credit a: modification of work by Umberto Salvagnin; credit b: modification of work by “Waifer X”/Flickr)

Exercise 3.1.2

1. If a light wave has a long wavelength, is it likely to have a low or high frequency?
2. If an object is transparent, does it reflect, absorb, or transmit light?

Lenses and Refraction

In the context of microscopy, refraction is perhaps the most important behavior exhibited by light waves. Refraction occurs when
light waves change direction as they enter a new medium (Figure 3.1.3). Different transparent materials transmit light at different
speeds; thus, light can change speed when passing from one material to another. This change in speed usually also causes a change
in direction (refraction), with the degree of change dependent on the angle of the incoming light.

Figure 3.1.3 : (a) Refraction occurs when light passes from one medium, such as air, to another, such as glass, changing the
direction of the light rays. (b) As shown in this diagram, light rays passing from one medium to another may be either refracted or
reflected. (credit a: modification of work by “ajizai”/Wikimedia Commons).
The extent to which a material slows transmission speed relative to empty space is called the refractive index of that material.
Large differences between the refractive indices of two materials will result in a large amount of refraction when light passes from
one material to the other. For example, light moves much more slowly through water than through air, so light entering water from
air can change direction greatly. We say that the water has a higher refractive index than air (Figure 3.1.4).

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 Figure 3.1.4 : This straight pole appears to bend at an angle as it enters the water. This optical illusion is due to the large difference
between the refractive indices of air and water.
When light crosses a boundary into a material with a higher refractive index, its direction turns to be closer to perpendicular to the
boundary (i.e., more toward a normal- the same angle- to that boundary; Figure 3.1.5). This is the principle behind lenses. We can
think of a lens as an object with a curved boundary (or a collection of prisms) that collects all of the light that strikes it and refracts
it so that it all meets at a single point called the focus point (or image point). A convex lens can be used to magnify because it can
focus at closer range than the human eye, producing a larger image. Concave lenses and mirrors can also be used in microscopes to
redirect the light path. Figure 3.1.5 shows the focal point (the image point when light entering the lens is parallel) and the focal
length (the distance to the focal point) for convex and concave lenses.

Figure 3.1.5 : (a) A lens is like a collection of prisms, such as the one shown here. (b) When light passes through a convex lens, it is
refracted toward a focal point on the other side of the lens. The focal length is the distance to the focal point. (c) Light passing
through a concave lens is refracted away from a focal point in front of the lens.
The human eye contains a lens that enables us to see images. This lens focuses the light reflecting off of objects in front of the eye
onto the surface of the retina, which is like a screen in the back of the eye. Artificial lenses placed in front of the eye (contact
lenses, glasses, or microscopic lenses) focus light before it is focused (again) by the lens of the eye, manipulating the image that
ends up on the retina (e.g., by making it appear larger).
Images are commonly manipulated by controlling the distances between the object, the lens, and the screen, as well as the
curvature of the lens. For example, for a given amount of curvature, when an object is closer to the lens, the focal points are farther
from the lens. As a result, it is often necessary to manipulate these distances to create a focused image on a screen. Similarly, more
curvature creates image points closer to the lens and a larger image when the image is in focus. This property is often described in
terms of the focal distance, or distance to the focal point.

Exercise 3.1.3

1. Explain how a lens focuses light at the image point.

2. Name some factors that affect the focal length of a lens.

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Electromagnetic Spectrum and Color
Visible light is just one form of electromagnetic radiation (EMR), a type of energy that is all around us. Other forms of EMR
include microwaves, X-rays, and radio waves, among others. The different types of EMR fall on the electromagnetic spectrum,
which is defined in terms of wavelength and frequency. The spectrum of visible light occupies a relatively small range of
frequencies between infrared and ultraviolet light (Figure 3.1.6).

Figure 3.1.6 : The electromagnetic spectrum ranges from high-frequency gamma rays to low-frequency radio waves. Visible light is
the relatively small range of electromagnetic frequencies that can be sensed by the human eye. On the electromagnetic spectrum,
visible light falls between ultraviolet and infrared light. (credit: modification of work by Johannes Ahlmann).
Whereas wavelength represents the distance between adjacent peaks of a light wave, frequency, in a simplified definition,
represents the rate of oscillation. Waves with higher frequencies have shorter wavelengths and, therefore, have more oscillations
per unit time than lower-frequency waves. Higher-frequency waves also contain more energy than lower-frequency waves. This
energy is delivered as elementary particles called photons. Higher-frequency waves deliver more energetic photons than lower-
frequency waves.
Photons with different energies interact differently with the retina. In the spectrum of visible light, each color corresponds to a
particular frequency and wavelength (Figure 3.1.6).The lowest frequency of visible light appears as the color red, whereas the
highest appears as the color violet. When the retina receives visible light of many different frequencies, we perceive this as white
light. However, white light can be separated into its component colors using refraction. If we pass white light through a prism,
different colors will be refracted in different directions, creating a rainbow-like spectrum on a screen behind the prism. This
separation of colors is called dispersion, and it occurs because, for a given material, the refractive index is different for different
frequencies of light.
Certain materials can refract nonvisible forms of EMR and, in effect, transform them into visible light. Certain fluorescent dyes, for
instance, absorb ultraviolet or blue light and then use the energy to emit photons of a different color, giving off light rather than
simply vibrating. This occurs because the energy absorption causes electrons to jump to higher energy states, after which they then
almost immediately fall back down to their ground states, emitting specific amounts of energy as photons. Not all of the energy is
emitted in a given photon, so the emitted photons will be of lower energy and, thus, of lower frequency than the absorbed ones.
Thus, a dye such as Texas red may be excited by blue light, but emit red light; or a dye such as fluorescein isothiocyanate (FITC)
may absorb (invisible) high-energy ultraviolet light and emit green light (Figure 3.1.7). In some materials, the photons may be
emitted following a delay after absorption; in this case, the process is called phosphorescence. Glow-in-the-dark plastic works by
using phosphorescent material.

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 Figure 3.1.7 : The fluorescent dyes absorbed by these bovine pulmonary artery endothelial cells emit brilliant colors when excited
by ultraviolet light under a fluorescence microscope. Various cell structures absorb different dyes. The nuclei are stained blue with
4’,6-diamidino-2-phenylindole (DAPI); microtubles are marked green by an antibody bound to FITC; and actin filaments are
labeled red with phalloidin bound to tetramethylrhodamine (TRITC).

Exercise 3.1.4

1. Which has a higher frequency: red light or green light?

2. Explain why dispersion occurs when white light passes through a prism.
3. Why do fluorescent dyes emit a different color of light than they absorb?

Magnification, Resolution, and Contrast

Microscopes magnify images and use the properties of light to create useful images of small objects. Magnification is defined as
the ability of a lens to enlarge the image of an object when compared to the real object. For example, a magnification of 10⨯
means that the image appears 10 times the size of the object as viewed with the naked eye.
Greater magnification typically improves our ability to see details of small objects, but magnification alone is not sufficient to
make the most useful images. It is often useful to enhance the resolution of objects: the ability to tell that two separate points or
objects are separate. A low-resolution image appears fuzzy, whereas a high-resolution image appears sharp. Two factors affect
resolution. The first is wavelength. Shorter wavelengths are able to resolve smaller objects; thus, an electron microscope has a
much higher resolution than a light microscope, since it uses an electron beam with a very short wavelength, as opposed to the
long-wavelength visible light used by a light microscope. The second factor that affects resolution is numerical aperture, which is a
measure of a lens’s ability to gather light. The higher the numerical aperture, the better the resolution.
Even when a microscope has high resolution, it can be difficult to distinguish small structures in many specimens because
microorganisms are relatively transparent. It is often necessary to increase contrast to detect different structures in a specimen.
Various types of microscopes use different features of light or electrons to increase contrast—visible differences between the parts
of a specimen. Additionally, dyes that bind to some structures but not others can be used to improve the contrast between images of
relatively transparent objects.

Exercise 3.1.5

1. Explain the difference between magnification and resolution.

2. Explain the difference between resolution and contrast.
3. Name two factors that affect resolution.

The Early Microscope

Some of the fundamental characteristics and functions of microscopes can be understood in the context of the history of their use.
Italian scholar Girolamo Fracastoro is regarded as the first person to formally postulate that disease was spread by tiny invisible
seminaria, or “seeds of the contagion.” In his book De Contagione (1546), he proposed that these seeds could attach themselves to
certain objects (which he called fomes [cloth]) that supported their transfer from person to person. However, since the technology
for seeing such tiny objects did not yet exist, the existence of the seminaria remained hypothetical for a little over a century—an
invisible world waiting to be revealed.

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 Who Invented the Microscope?
While Antonie van Leeuwenhoek and Robert Hooke generally receive much of the credit for early advances in microscopy,
neither can claim to be the inventor of the microscope. While van Leeuwenhoek is credited with the discovery of
microorganisms, others before him had contributed to the development of the microscope. These included the Italian
astronomer Galileo Galilei, who used a compound microscope to examine insect parts . Whereas van Leeuwenhoek used a
simple microscope, in which light is passed through just one lens, Galileo’s compound microscope was more sophisticated,
passing light through two sets of lenses.
Some argue that this designation of inventor should belong to Hans and Zaccharias Janssen, Dutch spectacle-makers who may
have invented the telescope, the simple microscope, and the compound microscope during the late 1500s or early 1600s
(Figure 3.1.8). Unfortunately, little is known for sure about the Janssens, not even the exact dates of their births and deaths.
The Janssens were secretive about their work and never published. It is also possible that the Janssens did not invent anything
at all; their neighbor, Hans Lippershey, also developed microscopes and telescopes during the same time frame, and he is often
credited with inventing the telescope. The historical records from the time are as fuzzy and imprecise as the images viewed
through those early lenses, and any archived records have been lost over the centuries.

Figure 3.1.8 : Zaccharias Janssen, along with his father Hans, may have invented the telescope, the simple microscope, and the
compound microscope during the late 1500s or early 1600s. The historical evidence is inconclusive.
By contrast, van Leeuwenhoek and Hooke can thank ample documentation of their work for their respective legacies. Like
Janssen, van Leeuwenhoek began his work in obscurity, leaving behind few records. However, his friend, the prominent
physician Reinier de Graaf, wrote a letter to the editor of the Philosophical Transactions of the Royal Society of London calling
attention to van Leeuwenhoek’s powerful microscopes. From 1673 onward, van Leeuwenhoek began regularly submitting
letters to the Royal Society detailing his observations. In 1674, his report describing single-celled organisms produced
controversy in the scientific community, but his observations were soon confirmed when the society sent a delegation to
investigate his findings. He subsequently enjoyed considerable celebrity, at one point even entertaining a visit by the czar of
Russia.
Similarly, Robert Hooke had his observations using microscopes published by the Royal Society in a book called
Micrographia in 1665. The book became a bestseller and greatly increased interest in microscopy throughout much of Europe.

Modern Microscopy
The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to
advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th
century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an
ultraviolet light source, and electron microscopy, which uses short-wavelength electron beams. These advances led to major
improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van
Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we
will focus on the most common and applications for each type of microscope.

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Light Microscopy
Many types of microscopes fall under the category of light microscopes, which use light to visualize images. Examples of light
microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast
microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. These various types of
light microscopes can be used to complement each other in diagnostics and research. We will just focus on brightfield, the most
common type to be used in labs.

Figure 3.1.9 : Components of a typical brightfield microscope.

Components of a Typical Brightfield Microscope

The item being viewed is called a specimen. The specimen is placed on a glass slide, which is then clipped into place on the stage(a
platform) of the microscope. Once the slide is secured, the specimen on the slide is positioned over the light using the x-y
mechanical stage knobs. These knobs move the slide on the surface of the stage, but do not raise or lower the stage. Once the
specimen is centered over the light, the stage position can be raised or lowered to focus the image. The coarse focusing knob is
used for large-scale movements with 4⨯ and 10⨯ objective lenses; the fine focusing knob is used for small-scale movements,
especially with 40⨯ or 100⨯ objective lenses.
When images are magnified, they become dimmer because there is less light per unit area of image. Highly magnified images
produced by microscopes, therefore, require intense lighting. In a brightfield microscope, this light is provided by an illuminator,
which is typically a high-intensity bulb below the stage. Light from the illuminator passes up through condenser lens (located
below the stage), which focuses all of the light rays on the specimen to maximize illumination. The position of the condenser can
be optimized using the attached condenser focus knob; once the optimal distance is established, the condenser should not be moved
to adjust the brightness. If less-than-maximal light levels are needed, the amount of light striking the specimen can be easily
adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be
adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator.
A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially
transmitted, absorbed, reflected, or refracted by different structures. Different colors can behave differently as they interact with
chromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are
artificially added to the specimen using stains, which serve to increase contrast and resolution. In general, structures in the
specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up
to about 1000⨯. Further magnification would create a larger image, but without increased resolution. This allows us to see objects
as small as bacteria, which are visible at about 400⨯ or so, but not smaller objects such as viruses.

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At very high magnifications, resolution may be compromised when light passes through the small amount of air between the
specimen and the lens. This is due to the large difference between the refractive indices of air and glass; the air scatters the light
rays before they can be focused by the lens. To solve this problem, a drop of oil can be used to fill the space between the specimen
and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar
to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light
collected and, thus, the resolution of the image (Figure 3.1.10). A variety of oils can be used for different types of light.

Figure 3.1.10 : (a) Oil immersion lenses like this one are used to improve resolution. (b) Because immersion oil and glass have very
similar refractive indices, there is a minimal amount of refraction before the light reaches the lens. Without immersion oil, light
scatters as it passes through the air above the slide, degrading the resolution of the image.

Brightfield Microscopes
The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more
lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece),
though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in Figure 3.1.9; in either case,
each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of
the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges
from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses
work together to create a magnified image. The total magnification is the product of the ocular magnification times the objective
magnification:

ocular magnification × objective magnification (3.1.1)

For example, if a 40⨯ objective lens is selected and the ocular lens is 10⨯, the total magnification would be
(40×)(10×)=400×

Microscope Maintenance: Best Practices

Even a very powerful microscope cannot deliver high-resolution images if it is not properly cleaned and maintained. Since lenses
are carefully designed and manufactured to refract light with a high degree of precision, even a slightly dirty or scratched lens will
refract light in unintended ways, degrading the image of the specimen. In addition, microscopes are rather delicate instruments, and
great care must be taken to avoid damaging parts and surfaces. Among other things, proper care of a microscope includes the
following:
cleaning the lenses with lens paper
not allowing lenses to contact the slide (e.g., by rapidly changing the focus)
protecting the bulb (if there is one) from breakage
not pushing an objective into a slide
not using the coarse focusing knob when using the 40⨯ or greater objective lenses
only using immersion oil with a specialized oil objective, usually the 100⨯ objective
cleaning oil from immersion lenses after using the microscope
cleaning any oil accidentally transferred from other lenses
covering the microscope and/or placing it in a cabinet when not in use

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Electron Microscopy
The maximum theoretical resolution of images created by light microscopes is ultimately limited by the wavelengths of visible
light. Most light microscopes can only magnify 1000⨯, and a few can magnify up to 1500⨯, but this does not begin to approach
the magnifying power of an electron microscope (EM), which uses short-wavelength electron beams rather than light to increase
magnification and resolution.
Electrons, like electromagnetic radiation, can behave as waves, but with wavelengths of 0.005 nm, they can produce much better
resolution than visible light. An EM can produce a sharp image that is magnified up to 100,000⨯. Thus, EMs can resolve
subcellular structures as well as some molecular structures (e.g., single strands of DNA); however, electron microscopy cannot be
used on living material because of the methods needed to prepare the specimens.
There are two basic types of EM: the transmission electron microscope (TEM) and the scanning electron microscope (SEM)(Figure
3.1.11). The TEM is somewhat analogous to the brightfield light microscope in terms of the way it functions. However, it uses an

electron beam from above the specimen that is focused using a magnetic lens (rather than a glass lens) and projected through the
specimen onto a detector. Electrons pass through the specimen, and then the detector captures the image (Figure 3.1.12).

Figure 3.1.11 : (a) A transmission electron microscope (TEM). (b) A scanning electron microscope (SEM). (credit a: modification
of work by “Deshi”/Wikimedia Commons; credit b: modification of work by “ZEISS Microscopy”/Flickr)

Figure 3.1.12 : Electron microscopes use magnets to focus electron beams similarly to the way that light microscopes use lenses to
focus light.
For electrons to pass through the specimen in a TEM, the specimen must be extremely thin (20–100 nm thick). The image is
produced because of varying opacity in various parts of the specimen. This opacity can be enhanced by staining the specimen with
materials such as heavy metals, which are electron dense. TEM requires that the beam and specimen be in a vacuum and that the
specimen be very thin and dehydrated. The specific steps needed to prepare a specimen for observation under an EM are discussed
in detail in the next section.

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SEMs form images of surfaces of specimens, usually from electrons that are knocked off of specimens by a beam of electrons. This
can create highly detailed images with a three-dimensional appearance that are displayed on a monitor (Figure 3.1.13). Typically,
specimens are dried and prepared with fixatives that reduce artifacts, such as shriveling, that can be produced by drying, before
being sputter-coated with a thin layer of metal such as gold. Whereas transmission electron microscopy requires very thin sections
and allows one to see internal structures such as organelles and the interior of membranes, scanning electron microscopy can be
used to view the surfaces of larger objects (such as a pollen grain) as well as the surfaces of very small samples (Figure 3.1.14).
Some EMs can magnify an image up to 2,000,000⨯.1

Figure 3.1.13 : These schematic illustrations compare the components of transmission electron microscopes and scanning electron
microscopes.

Figure 3.1.14 : (a) This TEM image of cells in a biofilm shows well-defined internal structures of the cells because of varying
levels of opacity in the specimen. (b) This color-enhanced SEM image of the bacterium Staphylococcus aureus illustrates the
ability of scanning electron microscopy to render three-dimensional images of the surface structure of cells. (credit a: modification
of work by American Society for Microbiology; credit b: modification of work by Centers for Disease Control and Prevention)

Exercise 3.1.6

1. What are some advantages and disadvantages of electron microscopy, as opposed to light microscopy, for examining
microbiological specimens?
2. What kinds of specimens are best examined using TEM? SEM?

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Figure 3.1.15 : (credit “Brightfield”: modification of work by American Society for Microbiology; credit “Darkfield”: modification
of work by American Society for Microbiology; credit “Phase contrast”: modification of work by American Society for
Microbiology; credit “DIC”: modification of work by American Society for Microbiology; credit “Fluorescence”: modification of
work by American Society for Microbiology; credit “Confocal”: modification of work by American Society for Microbiology;
credit “Two-photon”: modification of work by Alberto Diaspro, Paolo Bianchini, Giuseppe Vicidomini, Mario Faretta, Paola
Ramoino, Cesare Usai).

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 Figure 3.1.16 : (credit “TEM”: modification of work by American Society for Microbiology; credit “SEM”: modification of work
by American Society for Microbiology)

Key Concepts and Summary

Light waves interacting with materials may be reflected, absorbed, or transmitted, depending on the properties of the material.
Light waves can interact with each other (interference) or be distorted by interactions with small objects or openings
(diffraction).
Refraction occurs when light waves change speed and direction as they pass from one medium to another. Differences in the
refraction indices of two materials determine the magnitude of directional changes when light passes from one to the other.
A lens is a medium with a curved surface that refracts and focuses light to produce an image.
Visible light is part of the electromagnetic spectrum; light waves of different frequencies and wavelengths are distinguished as
colors by the human eye.
A prism can separate the colors of white light (dispersion) because different frequencies of light have different refractive indices
for a given material.
Fluorescent dyes and phosphorescent materials can effectively transform nonvisible electromagnetic radiation into visible light.
The power of a microscope can be described in terms of its magnification and resolution.
Resolution can be increased by shortening wavelength, increasing the numerical aperture of the lens, or using stains that
enhance contrast.
Antonie van Leeuwenhoek is credited with the first observation of microbes, including protists and bacteria, with simple
microscopes that he made.
Robert Hooke was the first to describe what we now call cells.
Simple microscopes have a single lens, while compound microscopes have multiple lenses.
Numerous types of microscopes use various technologies to generate micrographs. Most are useful for a particular type of
specimen or application.
Light microscopy uses lenses to focus light on a specimen to produce an image. Commonly used light microscopes include
brightfield, darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, and two-photon microscopes.
Electron microscopy focuses electrons on the specimen using magnets, producing much greater magnification than light
microscopy. The transmission electron microscope (TEM) and scanning electron microscope (SEM) are two common forms.

Glossary
absorbance
when a molecule captures energy from a photon and vibrates or stretches, using the energy

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amplitude
the height of a wave

brightfield microscope
a compound light microscope with two lenses; it produces a dark image on a bright background
chromophores
pigments that absorb and reflect particular wavelengths of light (giving them a color)
coarse focusing knob
a knob on a microscope that produces relatively large movements to adjust focus
condenser lens
a lens on a microscope that focuses light from the light source onto the specimen
contrast
visible differences between parts of a microscopic specimen

compound microscope
a microscope that uses multiple lenses to focus light from the specimen
diaphragm
a component of a microscope; typically consists of a disk under the stage with holes of various sizes; can be adjusted to allow
more or less light from the light source to reach the specimen
diffraction
the changing of direction (bending or spreading) that occurs when a light wave interacts with an opening or barrier

dispersion
the separation of light of different frequencies due to different degrees of refraction

electron microscope
a type of microscope that uses short-wavelength electron beams rather than light to increase magnification and resolution
fine focusing knob
a knob on a microscope that produces relatively small movements to adjust focus
fluorescent
the ability of certain materials to absorb energy and then immediately release that energy in the form of light

focal length
the distance from the lens to the image point when the object is at a definite distance from the lens (this is also the distance to
the focal point)

focal point
a property of the lens; the image point when light entering the lens is parallel (i.e., the object is an infinite distance from the
lens)

frequency
the rate of vibration for a light wave or other electromagnetic wave

Footnotes

illuminator
the light source on a microscope
image point (focus)
a property of the lens and the distance of the object to the lens; the point at which an image is in focus (the image point is often
called the focus)
interference

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 distortion of a light wave due to interaction with another wave
magnification
the power of a microscope (or lens) to produce an image that appears larger than the actual specimen, expressed as a factor of
the actual size
numerical aperture
a measure of a lens’s ability to gather light
objective lenses
on a light microscope, the lenses closest to the specimen, typically located at the ends of turrets
ocular lens
on a microscope, the lens closest to the eye (also called an eyepiece)
oil immersion lens
a special objective lens on a microscope designed to be used with immersion oil to improve resolution
opacity
the property of absorbing or blocking light
phosphorescence
the ability of certain materials to absorb energy and then release that energy as light after a delay
reflection
when light bounces back from a surface
refraction
bending of light waves, which occurs when a light wave passes from one medium to another
refractive index
a measure of the magnitude of slowing of light waves by a particular medium
resolution
the ability to distinguish between two points in an image
rheostat
a dimmer switch that controls the intensity of the illuminator on a light microscope
scanning electron microscope (SEM)
a type of electron microscope that bounces electrons off of the specimen, forming an image of the surface
simple microscope
a type of microscope with only one lens to focus light from the specimen
stage
the platform of a microscope on which slides are placed
total magnification
in a light microscope is a value calculated by multiplying the magnification of the ocular by the magnification of the objective
lenses
transmission electron microscope (TEM)
a type of electron microscope that uses an electron beam, focused with magnets, that passes through a thin specimen
transmittance
the amount of light that passes through a medium
transparency
the property of allowing light to pass through
wavelength
the distance between one peak of a wave and the next peak
x-y mechanical stage knobs
knobs on a microscope that are used to adjust the position of the specimen on the stage surface, generally to center it directly
above the light

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1 “JEM-ARM200F Transmission Electron Microscope,” JEOL USA Inc,
www.jeolusa.com/PRODUCTS/Tran...specifications. Accessed 8/28/2015.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 3.1: How Microscopes Work is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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3.2: Staining Microscopic Specimens and Descriptions
Learning Objectives
Differentiate between simple and differential stains
Describe the unique features of commonly used stains
Explain the procedures and name clinical applications for Gram, endospore, acid-fast, negative capsule, and flagella
staining
What to describe about cells under the microscope

Clinical Focus: part 2

Wound infections like Cindy’s can be caused by many different types of bacteria, some of which can spread rapidly with
serious complications. Identifying the specific cause is very important to select a medication that can kill or stop the growth of
the bacteria.
After calling a local doctor about Cindy’s case, the camp nurse sends the sample from the wound to the closest medical
laboratory. Unfortunately, since the camp is in a remote area, the nearest lab is small and poorly equipped. A more modern lab
would likely use other methods to culture, grow, and identify the bacteria, but in this case, the technician decides to make a wet
mount from the specimen and view it under a brightfield microscope. In a wet mount, a small drop of water is added to the
slide, and a cover slip is placed over the specimen to keep it in place before it is positioned under the objective lens.
Under the brightfield microscope, the technician can barely see the bacteria cells because they are nearly transparent against
the bright background. To increase contrast, the technician inserts an opaque light stop above the illuminator. The resulting
darkfield image clearly shows that the bacteria cells are spherical and grouped in clusters, like grapes.

Exercise 3.2.1
1. Why is it important to identify the shape and growth patterns of cells in a specimen?
2. What other types of microscopy could be used effectively to view this specimen?

In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. This
makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without
artificially treating specimens. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this
section we will discuss specific techniques for sample preparation in greater detail. Indeed, numerous methods have been
developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the
microscope. Here, we will focus on the most clinically relevant techniques.

Preparing Specimens for Light Microscopy

In clinical settings, light microscopes are the most commonly used microscopes. There are two basic types of preparation used to
view specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed on the slide in a drop of liquid. Some
specimens, such as a drop of urine, are already in a liquid form and can be deposited on the slide using a dropper. Solid specimens,
such as a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid
used is simply water, but often stains are added to enhance contrast. Once the liquid has been added to the slide, a coverslip is
placed on top and the specimen is ready for examination under the microscope.
The second method of preparing specimens for light microscopy is fixation. The “fixing” of a sample refers to the process of
attaching cells to a slide. Fixation is often achieved either by heating (heat fixing) or chemically treating the specimen. In addition
to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism
while preserving the integrity of their cellular components for observation.
To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear), and the slide is then briefly heated over a
heat source (Figure 3.2.1). Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic

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acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and
stabilize cell structures in tissue samples (Figure 3.2.1).

Figure 3.2.1 : (a) A specimen can be heat-fixed by using a slide warmer like this one. (b) Another method for heat-fixing a
specimen is to hold a slide with a smear over a microincinerator. (c) This tissue sample is being fixed in a solution of formalin (also
known as formaldehyde). Chemical fixation kills microorganisms in the specimen, stopping degradation of the tissues and
preserving their structure so that they can be examined later under the microscope. (credit a: modification of work by Nina Parker;
credit b: modification of work by Nina Parker; credit c: modification of work by “University of Bristol”/YouTube)
In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light
microscope. Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive
or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is called the counterion. If the chromophore
is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an
acidic dye.
Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how
the dye will interact with the specimen. In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the cells
or organisms being observed, adding color to objects of interest to make them stand out against the background. However, there are
scenarios in which it is advantageous to use a negative stain, which is absorbed by the background but not by the cells or organisms
in the specimen. Negative staining produces an outline or silhouette of the organisms against a colorful background (Figure 3.2.2).

Figure 3.2.2 : (a) These Bacillus anthracis cells have absorbed crystal violet, a basic positive stain. (b) This specimen of
Spinoloricus, a microscopic marine organism, has been stained with rose bengal, a positive acidic stain. (c) These B. megaterium
appear to be white because they have not absorbed the negative red stain applied to the slide. (credit a: modification of work by
Centers for Disease Control and Prevention; credit b: modification of work by Roberto Danovaro, Antonio Pusceddu, Cristina
Gambi, Iben Heiner, Reinhardt Mobjerg Kristensen; credit c: modification of work by Anh-Hue Tu)
Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls,
making them positive stains. Thus, commonly used basic dyes such as basic fuchsin, crystal violet, malachite green, methylene
blue, and safranin typically serve as positive stains. On the other hand, the negatively charged chromophores in acidic dyes are
repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin, eosin,
and rose bengal.

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Some staining techniques involve the application of only one dye to the sample; others require more than one dye. In simple
staining, a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the
organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. In contrast,
differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a
differentially stained sample may appear to be different colors. Differential staining techniques commonly used in clinical settings
include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.

Exercise 3.2.2

1. Explain why it is important to fix a specimen before viewing it under a light microscope.
2. What types of specimens should be chemically fixed as opposed to heat-fixed?
3. Why might an acidic dye react differently with a given specimen than a basic dye?
4. Explain the difference between a positive stain and a negative stain.
5. Explain the difference between simple and differential staining.

Gram Staining
The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish
microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell
walls, and even today it remains one of the most frequently used staining techniques. The steps of the Gram stain procedure
are illustrated in Figure 3.2.3 and listed below.

Figure 3.2.3 : Gram-staining is a differential staining technique that uses a primary stain and a secondary counterstain to distinguish
between gram-positive and gram-negative bacteria.
1. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color.
2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram’s
iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet–iodine complex clump and
stay contained in thick layers of peptidoglycan in the cell walls.
3. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells that have thick peptidoglycan layers in
their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple.

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 However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again
colorless.
4. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized cells pink and is less noticeable in the
cells that still contain the crystal violet dye.
The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative
(Figure 3.2.4). However, there are several important considerations in interpreting the results of a Gram stain. First, older bacterial
cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive. Thus, it is
best to use fresh bacterial cultures for Gram staining. Second, errors such as leaving on decolorizer too long can affect the results.
In some cases, most cells will appear gram-positive while a few appear gram-negative (as in Figure 3.2.4). This suggests damage
to the individual cells or that decolorizer was left on for too long; the cells should still be classified as gram-positive if they are all
the same species rather than a mixed culture.
Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-
negative cells have other implications with clinical relevance. For example, Gram staining can help clinicians classify bacterial
pathogens in a sample into categories associated with specific properties. Gram-negative bacteria tend to be more resistant to
certain antibiotics than gram-positive bacteria. We will discuss this and other applications of Gram staining in more detail in later
chapters.

Figure 3.2.4 : In this specimen, the gram-positive bacterium Staphylococcus aureus retains crystal violet dye even after the
decolorizing agent is added. Gram-negative Escherichia coli, the most common Gram stain quality-control bacterium, is
decolorized, and is only visible after the addition of the pink counterstain safranin. (credit: modification of work by Nina Parker)

Exercise 3.2.3

1. Explain the role of Gram’s iodine in the Gram stain procedure.

2. Explain the role of alcohol in the Gram stain procedure.
3. What color are gram-positive and gram-negative cells, respectively, after the Gram stain procedure?

Clinical Focus: Part 3

Viewing Cindy’s specimen under the darkfield microscope has provided the technician with some important clues about the
identity of the microbe causing her infection. However, more information is needed to make a conclusive diagnosis. The
technician decides to make a Gram stain of the specimen. This technique is commonly used as an early step in identifying
pathogenic bacteria. After completing the Gram stain procedure, the technician views the slide under the brightfield
microscope and sees purple, grape-like clusters of spherical cells (Figure 3.2.5).

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 Figure 3.2.5: Cindy's specimen (credit: modification of work by American Society for Microbiology)

Exercise 3.2.4

1. Are these bacteria gram-positive or gram-negative?

2. What does this reveal about their cell walls?

Acid-Fast Stains
Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. An acid-fast
stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that
do not. Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique. Both use carbol-
fuchsin as the primary stain. The waxy, acid-fast cells retain the carbol-fuchsin even after a decolorizing agent (an acid-alcohol
solution) is applied. A secondary counterstain, methylene blue, is then applied, which renders non–acid-fast cells blue.
The fundamental difference between the two carbol-fuchsin-based methods is whether heat is used during the primary staining
process. The Ziehl-Neelsen method uses heat to infuse the carbol-fuchsin into the acid-fast cells, whereas the Kinyoun method does
not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria
(AFB). If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the
surrounding tissue cells (Figure 3.2.6).

Exercise 3.2.5

Why are acid-fast stains useful?

Using Microscopy to Diagnose Tuberculosis

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, can be detected in specimens based on the presence of
acid-fast bacilli. Often, a smear is prepared from a sample of the patient’s sputum and then stained using the Ziehl-Neelsen
technique (Figure 3.2.6). If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification.
Variations of this approach can be used as a first step in determining whether M. tuberculosis or other acid-fast bacteria are
present, though samples from elsewhere in the body (such as urine) may contain other Mycobacterium species.
An alternative approach for determining the presence of M. tuberculosis is immunofluorescence. In this technique,
fluorochrome-labeled antibodies bind to M. tuberculosis, if present. Antibody-specific fluorescent dyes can be used to view the
mycobacteria with a fluorescence microscope.

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 Figure 3.2.6 : Ziehl-Neelsen staining has rendered these Mycobacterium tuberculosis cells red and the surrounding growth
indicator medium blue. (credit: modification of work by American Society for Microbiology)

Capsule Staining
Certain bacteria and yeasts have a protective outer structure called a capsule. Since the presence of a capsule is directly related to a
microbe’s virulence (its ability to cause disease), the ability to determine whether cells in a sample have capsules is an important
diagnostic tool. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is
typically used for capsule staining. The dye stains the background but does not penetrate the capsules, which appear like halos
around the borders of the cell. The specimen does not need to be heat-fixed prior to negative staining.
One common negative staining technique for identifying encapsulated yeast and bacteria is to add a few drops of India ink or
nigrosin to a specimen. Other capsular stains can also be used to negatively stain encapsulated cells (Figure 3.2.7). Alternatively,
positive and negative staining techniques can be combined to visualize capsules: The positive stain colors the body of the cell, and
the negative stain colors the background but not the capsule, leaving halo around each cell.

Figure 3.2.7 : (a) India-ink was used to stain the background around these cells of the yeast Cryptococcus neoformans. The halos
surrounding the cells are the polysaccharide capsules. (b) Crystal violet and copper sulfate dyes cannot penetrate the encapsulated
Bacillus cells in this negatively stained sample. Encapsulated cells appear to have a light-blue halo. (credit a: modification of work
by American Society for Microbiology; credit b: modification of work by American Society for Microbiology)

Exercise 3.2.6

How does negative staining help us visualize capsules?

Endospore Staining
Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. Gram staining alone
cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two
stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most commonly used endospore-
staining technique) uses heat to push the primary stain (malachite green) into the endospore. Washing with water decolorizes the
cell, but the endospore retains the green stain. The cell is then counterstained pink with safranin. The resulting image reveals the
shape and location of endospores, if they are present. The green endospores will appear either within the pink vegetative cells or as

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separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible (Figure
3.2.8).

Figure 3.2.8 : A stained preparation of Bacillus subtilis showing endospores as green and the vegetative cells as pink. (credit:
modification of work by American Society for Microbiology)
Endospore-staining techniques are important for identifying Bacillus and Clostridium, two genera of endospore-producing bacteria
that contain clinically significant species. Among others, B. anthracis (which causes anthrax) has been of particular interest
because of concern that its spores could be used as a bioterrorism agent. C. difficile is a particularly important species responsible
for the typically hospital-acquired infection known as “C. diff.”

Exercise 3.2.7

Is endospore staining an example of positive, negative, or differential staining?

Flagella Staining
Flagella (singular: flagellum) are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes.
Because they are so thin, prokaryote flagella typically cannot be seen under a light microscope without a specialized flagella
staining technique. Flagella staining thickens the flagella by first applying mordant (generally tannic acid, but sometimes potassium
alum), which coats the flagella; then the specimen is stained with pararosaniline (most commonly) or basic fuchsin (Figure 3.2.9).

Figure 3.2.9 : A flagella stain of Bacillus cereus, a common cause of foodborne illness, reveals that the cells have numerous
flagella, used for locomotion. (credit: modification of work by Centers for Disease Control and Prevention)
Though flagella staining is uncommon in clinical settings, the technique is commonly used by microbiologists, since the location
and number of flagella can be useful in classifying and identifying bacteria in a sample. When using this technique, it is important
to handle the specimen with great care; flagella are delicate structures that can easily be damaged or pulled off, compromising
attempts to accurately locate and count the number of flagella.

Exercise 3.2.8

Is the flagella stain showing the true size of the flagella?

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Figure 3.2.10 : (credit “basic stains”: modification of work by Centers for Disease Control and Prevention; credit “Acidic stains”:
modification of work by Roberto Danovaro, Antonio Dell’Anno, Antonio Pusceddu, Cristina Gambi, Iben Heiner, Reinhardt
Mobjerg Kristensen; credit “Negative stains”: modification of work by Anh-Hue Tu)

Figure 3.2.11 : (credit “Gram stain”: modification of work by Nina Parker; credit “Acid-fast stain”: modification of work by
American Society for Microbiology; credit “Endospore stain”: modification of work by American Society for Microbiology; credit
“Capsule stain” : modification of work by American Society for Microbiology; credit “Flagella stain”: modification of work by
Centers for Disease Control and Prevention)

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Preparing Specimens for Electron Microscopy
Samples to be analyzed using a TEM must have very thin sections. But cells are too soft to cut thinly, even with diamond knives.
To cut cells without damage, the cells must be embedded in plastic resin and then dehydrated through a series of soaks in ethanol
solutions (50%, 60%, 70%, and so on). The ethanol replaces the water in the cells, and the resin dissolves in ethanol and enters the
cell, where it solidifies. Next, thin sections are cut using a specialized device called an ultramicrotome (Figure 3.2.12). Finally,
samples are fixed to fine copper wire or carbon-fiber grids and stained—not with colored dyes, but with substances like uranyl
acetate or osmium tetroxide, which contain electron-dense heavy metal atoms.

Figure 3.2.12 : (a) An ultramicrotome used to prepare specimens for a TEM. (b) A technician uses an ultramicrotome to slice a
specimen into thin sections. (credit a: modification of work by “Frost Museum”/Flickr; credit b: modification of work by U.S. Fish
and Wildlife Service Northeast Region)
When samples are prepared for viewing using an SEM, they must also be dehydrated using an ethanol series. However, they must
be even drier than is necessary for a TEM. Critical point drying with inert liquid carbon dioxide under pressure is used to displace
the water from the specimen. After drying, the specimens are sputter-coated with metal by knocking atoms off of a palladium
target, with energetic particles. Sputter-coating prevents specimens from becoming charged by the SEM’s electron beam.

Exercise 3.2.9

1. Why is it important to dehydrate cells before examining them under an electron microscope?
2. Name the device that is used to create thin sections of specimens for electron microscopy.

Using Microscopy to Diagnose Syphilis

Figure 3.2.13 : (a) Living, unstained Treponema pallidum spirochetes can be viewed under a darkfield microscope. (b) In this
brightfield image, a modified Steiner silver stain is used to visualized T. pallidum spirochetes. Though the stain kills the cells,
it increases the contrast to make them more visible. (c) While not used for standard diagnostic testing, T. pallidum can also be
examined using scanning electron microscopy. (credit a: modification of work by Centers for Disease Control and Prevention;
credit b: modification of work by Centers for Disease Control and Prevention; credit c: modification of work by Centers for
Disease Control and Prevention)
The causative agent of syphilis is Treponema pallidum, a flexible, spiral cell (spirochete) that can be very thin (<0.15 μm) and
match the refractive index of the medium, making it difficult to view using brightfield microscopy. Additionally, this species
has not been successfully cultured in the laboratory on an artificial medium; therefore, diagnosis depends upon successful
identification using microscopic techniques and serology (analysis of body fluids, often looking for antibodies to a pathogen).
Since fixation and staining would kill the cells, darkfield microscopy is typically used for observing live specimens and
viewing their movements. However, other approaches can also be used. For example, the cells can be thickened with silver

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 particles (in tissue sections) and observed using a light microscope. It is also possible to use fluorescence or electron
microscopy to view Treponema (Figure 3.2.13).
In clinical settings, indirect immunofluorescence is often used to identify Treponema. A primary, unstained antibody attaches
directly to the pathogen surface, and secondary antibodies “tagged” with a fluorescent stain attach to the primary antibody.
Multiple secondary antibodies can attach to each primary antibody, amplifying the amount of stain attached to each Treponema
cell, making them easier to spot (Figure 3.2.14).

Figure 3.2.14 : Indirect immunofluorescence can be used to identify T. pallidum, the causative agent of syphilis, in a specimen.

Preparation and Staining for Other Microscopes

Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are
fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific
proteins on specific types of cells (immunofluorescence); others may attach to DNA molecules in a process called fluorescence in
situ hybridization (FISH), causing cells to be stained based on whether they have a specific DNA sequence. Sample preparation for
two-photon microscopy is similar to fluorescence microscopy, except for the use of infrared dyes.

Exercise 3.2.10

What is the main difference between preparing a sample for fluorescence microscopy versus light microscopy?

Cornell University’s Case Studies in Microscopy offers a series of clinical problems based on real-life events. Each case study
walks you through a clinical problem using appropriate techniques in microscopy at each step.

Microscopy and Antibiotic Resistance

As the use of antibiotics has proliferated in medicine, as well as agriculture, microbes have evolved to become more resistant.
Strains of bacteria such as methicillin-resistant S. aureus (MRSA), which has developed a high level of resistance to many
antibiotics, are an increasingly worrying problem, so much so that research is underway to develop new and more diversified
antibiotics.
Fluorescence microscopy can be useful in testing the effectiveness of new antibiotics against resistant strains like MRSA. In a
test of one new antibiotic derived from a marine bacterium, MC21-A (bromophene), researchers used the fluorescent dye
SYTOX Green to stain samples of MRSA. SYTOX Green is often used to distinguish dead cells from living cells, with
fluorescence microscopy. Live cells will not absorb the dye, but cells killed by an antibiotic will absorb the dye, since the
antibiotic has damaged the bacterial cell membrane. In this particular case, MRSA bacteria that had been exposed to MC21-A
did, indeed, appear green under the fluorescence microscope, leading researchers to conclude that it is an effective antibiotic
against MRSA.
Of course, some argue that developing new antibiotics will only lead to even more antibiotic-resistant microbes, so-called
superbugs that could spawn epidemics before new treatments can be developed. For this reason, many health professionals are
beginning to exercise more discretion in prescribing antibiotics. Whereas antibiotics were once routinely prescribed for
common illnesses without a definite diagnosis, doctors and hospitals are much more likely to conduct additional testing to
determine whether an antibiotic is necessary and appropriate before prescribing.
A sick patient might reasonably object to this stingy approach to prescribing antibiotics. To the patient who simply wants to
feel better as quickly as possible, the potential benefits of taking an antibiotic may seem to outweigh any immediate health
risks that might occur if the antibiotic is ineffective. But at what point do the risks of widespread antibiotic use supersede the
desire to use them in individual cases?

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Description of Cells Under the Microscope
Once you have stained the cells, the next step is to use the microscope to describe them. You will always look for cell shape, the
grouping and the color(s) that appear. These descriptions of cell morphology (shape and structure) are your microscopic
observations. This is most commonly done with prokaryotes and the following tables contain proper names for shapes and
groupings you should use.

Figure 3.2.15: (credit “Coccus” micrograph: modification of work by Janice Haney Carr, Centers for Disease Control and
Prevention; credit “Coccobacillus” micrograph: modification of work by Janice Carr, Centers for Disease Control and Prevention;
credit “Spirochete” micrograph: modification of work by Centers for Disease Control and Prevention)

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 Figure 3.2.16 : Common cell arrangements.
Eukaryotic cells display a wide variety of different cell morphologies. Possible shapes include spheroid, ovoid, cuboidal,
cylindrical, flat, lenticular, fusiform, discoidal, crescent, ring stellate, and polygonal (Figure 3.2.17). Some eukaryotic cells are
irregular in shape, and some are capable of changing shape. The shape of a particular type of eukaryotic cell may be influenced by
factors such as its primary function, the organization of its cytoskeleton, the viscosity of its cytoplasm, the rigidity of its cell
membrane or cell wall (if it has one), and the physical pressure exerted on it by the surrounding environment and/or adjoining cells.

Figure 3.2.17 : Eukaryotic cells come in a variety of cell shapes. (a) Spheroid Chromulina alga. (b) Fusiform shaped Trypanosoma.
(c) Bell-shaped Vorticella. (d) Ovoid Paramecium. (e) Ring-shaped Plasmodium ovale. (credit a: modification of work by NOAA;
credit b, e: modification of work by Centers for Disease Control and Prevention).

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 Exercise 3.2.11

1. What are the proper names for the common shapes of bacterial cells?
2. What are the proper names for the common groupings of bacterial cells?
3. Why do eukaryote cells not always follow the same shapes and groupings as bacterial cells?

Key Concepts and Summary

Samples must be properly prepared for microscopy. This may involve staining, fixation, and/or cutting thin sections.
A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining, capsule
staining, endospore staining, and flagella staining.
Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.
Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.
Cells have several things unique about them when being viewed under the microscope. When recording observations, make
sure to describe cell shape, grouping and color(s).

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 3.2: Staining Microscopic Specimens and Descriptions is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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3.3: Cells as Living Things
Learning Objectives
Identify and describe the properties of life
Identify the major parts of all cells and their functions

Biology is the science that studies life, but what exactly is life? This may sound like a silly question with an obvious response, but
it is not always easy to define life. For example, virology studies viruses, which exhibit some of the characteristics of living entities
but lack others. It turns out that although viruses can attack living organisms, cause diseases, and even reproduce, they do not meet
the criteria that biologists use to define life. Consequently, virologists are not biologists, strictly speaking. Similarly, some
biologists study the early molecular evolution that gave rise to life; since the events that preceded life are not biological events,
these scientists are also excluded from biology in the strict sense of the term.
From its earliest beginnings, biology has wrestled with three questions: What are the shared properties that make something
“alive”? And once we know something is alive, how do we find meaningful levels of organization in its structure? And, finally,
when faced with the remarkable diversity of life, how do we organize the different kinds of organisms so that we can better
understand them? As new organisms are discovered every day, biologists continue to seek answers to these and other questions.

Properties of Life
All living organisms (whether they are bacteria, archaea or eukaryote) share several key characteristics, properties or functions:
order, sensitivity or response to the environment, reproduction, growth and development, regulation (including homeostasis),
energy processing, and evolution with adaptation. When viewed together, these seven characteristics serve to define life.

Order
Organisms are highly organized, coordinated structures that consist of one or more cells. Even very simple, single-celled organisms
are remarkably complex: inside each cell, atoms make up molecules; these in turn make up cell organelles and other cellular parts.
In multicellular organisms (Figure 3.3.1), similar cells come together to form tissues. Tissues, in turn, collaborate to create organs
(body structures with a distinct function). Organs work together to form organ systems.

Figure 3.3.1 : A toad represents a highly organized structure consisting of cells, tissues, organs, and organ systems. (credit:
“Ivengo”/Wikimedia Commons)

Sensitivity or Response to Stimuli

Organisms respond to diverse stimuli. For example, plants can bend toward a source of light, climb on fences and walls, or respond
to touch (Figure 3.3.2). Even tiny bacteria can move toward or away from chemicals (a process called chemotaxis) or light
(phototaxis). Movement toward a stimulus is considered a positive response, while movement away from a stimulus is considered a
negative response.

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 Figure 3.3.2 : The leaves of this sensitive plant (Mimosa pudica) will instantly droop and fold when touched. After a few minutes,
the plant returns to normal. (credit: Alex Lomas)

Video: Watch this video to see how plants respond to a stimulus—from opening to light, to wrapping a tendril
around a branch, to capturing prey.

Reproduction
Single-celled organisms reproduce by first duplicating their DNA, and then dividing it equally as the cell prepares to divide to form
two new cells. Multicellular organisms often produce specialized reproductive germline cells that will form new individuals. When
reproduction occurs, genes containing DNA are passed along to an organism’s offspring. These genes ensure that the offspring will
belong to the same species and will have similar characteristics, such as size and shape.

Growth and Development

Organisms grow and develop following specific instructions coded for by their genes. These genes provide instructions that will
direct cellular growth and development, ensuring that a species’ young (Figure 3.3.3) will grow up to exhibit many of the same
characteristics as its parents.

Figure 3.3.3 : Although no two look alike, these kittens have inherited genes from both parents and share many of the same
characteristics. (credit: Rocky Mountain Feline Rescue)

Regulation
Even the smallest organisms are complex and require multiple regulatory mechanisms to coordinate internal functions, respond to
stimuli, and cope with environmental stresses. Two examples of internal functions regulated in an organism are nutrient transport

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and blood flow. Organs (groups of tissues working together) perform specific functions, such as carrying oxygen throughout the
body, removing wastes, delivering nutrients to every cell, and cooling the body. A special type of internal regulation is homeostasis.
In order to function properly, cells need to have appropriate conditions such as proper temperature, pH, and appropriate
concentration of diverse chemicals. These conditions may, however, change from one moment to the next. Organisms are able to
maintain internal conditions within a narrow range almost constantly, despite environmental changes, through homeostasis
(literally, “steady state”)—the ability of an organism to maintain constant internal conditions. For example, an organism needs to
regulate body temperature through a process known as thermoregulation. Organisms that live in cold climates, such as the polar
bear (Figure 3.3.4), have body structures that help them withstand low temperatures and conserve body heat. Structures that aid in
this type of insulation include fur, feathers, blubber, and fat. In hot climates, organisms have methods (such as perspiration in
humans or panting in dogs) that help them to shed excess body heat.

Figure 3.3.4 : Polar bears (Ursus maritimus) and other mammals living in ice-covered regions maintain their body temperature by
generating heat and reducing heat loss through thick fur and a dense layer of fat under their skin. (credit: “longhorndave”/Flickr)

Energy Processing
All organisms use a source of energy for their metabolic activities. Some organisms capture energy from the sun and convert it into
chemical energy in food; others use chemical energy in molecules they take in as food (Figure 3.3.5).

Figure 3.3.5 : The California condor (Gymnogyps californianus) uses chemical energy derived from food to power flight. California
condors are an endangered species; this bird has a wing tag that helps biologists identify the individual. (credit: Pacific Southwest
Region U.S. Fish and Wildlife Service)

Evolution with Adaptation

Evolution and adaptation rely on the cell's ability to copy its DNA (replication) and give that copy to the next generation
(reproduction). However, this copying is sometimes carried out with mistakes (mutations). Chemicals and other mutagens

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(anything that can cause a mutation) can also cause mistakes that will potentially be passed down. These mutations are often
though of as bad, but it depends on which piece of information was affected and how. Sometimes these mistakes give advantages to
the organism- like antibiotic resistance- that can allow the organism to adapt and overcome obstacles. We will discuss the mutations
and how their changes play out further in later chapters.

Exercise 3.3.1

1. What connects all seven properties or characteristics of life?

2. Do all organisms display the seven properties of life in the same way?

Construction of a cell
Cell theory states that the cell is the fundamental unit of life. However, cells vary significantly in size, shape, structure, and
function. At the simplest level of construction, all cells possess a few fundamental components. These include cytoplasm (a gel-like
substance composed of water and dissolved chemicals needed for growth), which is contained within a plasma membrane (also
called a cell membrane or cytoplasmic membrane); within the cytoplasm are one or more chromosomes, which contain the genetic
blueprints of the cell; and ribosomes, parts used for the production of proteins.

The Cytoplasm
The cytoplasm is the entire region of a cell surrounded by the plasma membrane. It is made up of nutrients, enzymes and other
chemicals suspended in the gel-like cytosol, and the cytoskeleton. Even though the cytoplasm consists of 70 to 80 percent water, it
has a semi-solid consistency, which comes from the proteins within it. However, proteins are not the only organic molecules found
in the cytoplasm. Glucose and other simple sugars, polysaccharides, amino acids, nucleic acids, fatty acids, and derivatives of
glycerol are found there, too. Ions of sodium, potassium, calcium, and many other elements are also dissolved in the cytoplasm.
Many metabolic reactions, including protein synthesis, take place in the cytoplasm.

Plasma Membrane
All cells (prokaryotic and eukaryotic) have a plasma membrane (also called cytoplasmic membrane or cell membrane) that exhibits
selective permeability, allowing some molecules to enter or leave the cell while restricting the passage of others. The structure of
the plasma membrane is often described in terms of the fluid mosaic model, which refers to the ability of membrane components to
move fluidly within the plane of the membrane. It is also a reference to the mosaic-like composition of the components, which
include a diverse array of lipid and protein components (Figure 3.3.6).

Figure 3.3.6 : The bacterial plasma membrane is a phospholipid bilayer with a variety of embedded proteins that perform various
functions for the cell. Note the presence of glycoproteins and glycolipids, whose carbohydrate components extend out from the
surface of the cell. The abundance and arrangement of these proteins and lipids can vary greatly between species.
The plasma membrane structure of most bacterial and eukaryotic cell types is a bilayer composed mainly of phospholipids formed
with ester linkages and proteins. These phospholipids and proteins have the ability to move laterally (side-to-side) within the plane

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of the membranes as well as between the two phospholipid layers (less common). The membrane also has a lot of flexibility warp
and flex into or away from the cell's contents which helps with in cells that lack a cell wall.
The plasma membrane of eukaryotic cells is similar in structure to the prokaryotic plasma membrane in that it is composed mainly
of phospholipids forming a bilayer with embedded peripheral and integral proteins (Figure 3.3.6). These membrane components
move within the plane of the membrane according to the fluid mosaic model. However, unlike the prokaryotic membrane,
eukaryotic membranes contain sterols, including cholesterol, that alter membrane fluidity. Additionally, many eukaryotic cells
contain some specialized lipids, including sphingolipids, which are thought to play a role in maintaining membrane stability as well
as being involved in signal transduction pathways and cell-to-cell communication.
Archaeal membranes are fundamentally different from bacterial and eukaryotic membranes in a few significant ways. First,
archaeal membrane phospholipids are formed with ether linkages, in contrast to the ester linkages found in bacterial or eukaryotic
cell membranes. Second, archaeal phospholipids have branched chains, whereas those of bacterial and eukaryotic cells are straight
chained. Finally, although some archaeal membranes can be formed of bilayers like those found in bacteria and eukaryotes, other
archaeal plasma membranes are lipid monolayers.
Proteins on the cell’s surface are important for a variety of functions, including cell-to-cell communication, and sensing
environmental conditions and pathogenic virulence factors. Membrane proteins and phospholipids may have carbohydrates (sugars)
associated with them and are called glycoproteins or glycolipids, respectively. These glycoprotein and glycolipid complexes extend
out from the surface of the cell, allowing the cell to interact with the external environment (Figure 3.3.10). Glycoproteins and
glycolipids in the plasma membrane can vary considerably in chemical composition among archaea, bacteria, and eukaryotes,
allowing scientists to use them to characterize unique species. Plasma membranes from different cells types also contain unique
phospholipids, which contain fatty acids. More of the membrane will be discussed in chapter 8.

DNA
All cellular life has a double stranded DNA genome organized into one or more chromosomes- the DNA carrying the required
information of life. This DNA carries information used to direct reproduction, intake of food, response to stimuli in its environment
and any other typical functions. Prokaryotic chromosomes are typically circular, haploid (unpaired), and not bound by a complex
nuclear membrane. In eukaryotes, chromosomes are linear structures. Every eukaryotic species has a specific number of
chromosomes in the nuclei of its body’s cells. For example, in humans, the chromosome number is 46, while in fruit flies, it is
eight. Chromosomes are only visible and distinguishable from one another when the cell is getting ready to divide. When the cell is
in the growth and maintenance phases of its life cycle, proteins are attached to chromosomes, and they resemble an unwound,
jumbled bunch of threads. These unwound protein-chromosome complexes are called chromatin.
Cells may also contain extrachromosomal DNA, or DNA that is not part of the chromosome. This extrachromosomal DNA is often
found as plasmids, which are small, circular, double-stranded DNA molecules. Plasmids often carry genes that confer advantageous
traits such as antibiotic resistance; thus, they are important but not required to the survival of the organism on a regular basis. Cells
that have plasmids can have hundreds of them within a single cell. Plasmids are more commonly found in bacteria; however,
plasmids have also been found in archaea and eukaryotic organisms. The DNA in plasmids and other sources of extrachromosomal
DNA functions exactly the same way as regular chromosomal DNA.

Ribosomes
All cellular life synthesizes proteins, and organisms in all three domains of life possess ribosomes, structures responsible protein
synthesis. However, ribosomes in each of the three domains are structurally different. Ribosomes, themselves, are constructed from
proteins, along with ribosomal RNA (rRNA). Prokaryotic ribosomes are found in the cytoplasm. They are called 70S ribosomes
because they have a size of 70S split between a large and small subunit (Figure 3.3.7). (The S stands for Svedberg unit, a measure
of sedimentation in an ultracentrifuge, which is based on size, shape, and surface qualities of the structure being analyzed).
Although they are the same size, bacterial and archaeal ribosomes have different proteins and rRNA molecules, and the archaeal
versions are more similar in construction to their eukaryotic counterparts than to those found in bacteria. Ribosomes in eukaryotic
cells are 80S ribosomes, also composed of a small subunit and a large subunit. In terms of size and composition, this makes them
distinct from the ribosomes of prokaryotic cells.

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 Figure 3.3.7: Ribosomes are made up of a large subunit (top) and a small subunit (bottom). During protein synthesis,
ribosomes assemble amino acids into proteins.

Because proteins synthesis is an essential function of all cells (including enzymes, hormones, antibodies, pigments, structural
components, and surface receptors), ribosomes are found in practically every cell. Ribosomes are particularly abundant in cells that
synthesize large amounts of protein. For example, the pancreas is responsible for creating several digestive enzymes and the cells
that produce these enzymes contain many ribosomes. Thus, we see another example of form following function.
Beyond these basic components, cells can vary greatly between organisms, and even within the same multicellular organism. The
two largest categories of cells—prokaryotic cells and eukaryotic cells—are defined by major differences in several cell structures.
Prokaryotic cells lack a nucleus surrounded by a complex nuclear membrane and generally have a single, circular chromosome
located in a nucleoid. Prokaryotes also lack other membrane-bound interior structures. Eukaryotic cells have a nucleus surrounded
by a complex nuclear membrane that contains multiple, rod-shaped chromosomes in addition to many membrane-bound organelles
of varying functions.1

Exercise 3.3.2
1. What are the important parts that are found in all cells?
2. Do all cells have these parts constructed in the same way? Why or why not?
3. Why are all of these part important to all cells?

Clinical Focus: Resolution

From the results of the Gram stain, the technician now knows that Cindy’s infection is caused by spherical, gram-positive
bacteria that form grape-like clusters, which is typical of staphylococcal bacteria. After some additional testing, the technician
determines that these bacteria are the medically important species known as Staphylococcus aureus, a common culprit in
wound infections. Because some strains of S. aureus are resistant to many antibiotics, skin infections may spread to other areas
of the body and become serious, sometimes even resulting in amputations or death if the correct antibiotics are not used.
After testing several antibiotics, the lab is able to identify one that is effective against this particular strain of S. aureus. Cindy’s
doctor quickly prescribes the medication and emphasizes the importance of taking the entire course of antibiotics, even if the
infection appears to clear up before the last scheduled dose. This reduces the risk that any especially resistant bacteria could
survive, causing a second infection or spreading to another person.

Key Concepts and Summary

All living things have several properties or characteristics that they share.
All organisms have order or organization to them, include the level of cells.

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 All organisms have to be able to respond to stimuli in their environment
All cells and organisms reproduce, including the copying of their DNA.
The DNA directs cell to grow and develop.
All cells regulate themselves and maintain homeostasis.
All cells process energy and nutrients.
All cells use changes in its DNA to adapt to its environment.
All cells contain a cytoplasm surrounded by a plasma membrane, DNA in the form of chromosomes and ribosomes.
The cytoplasm is the main metabolic reaction area.
The plasma membrane of phospholipids which helps with regulation, response and many more interactions for the cell.
The DNA in the cell is mainly the required chromosomal DNA and possibly extrachromosomal plasmid(s). This directs the
cell's activities.
Ribosomes aid in the conversion of the information from the DNA into proteins.

Footnotes
1. Y.-H.M. Chan, W.F. Marshall. “Scaling Properties of Cell and Organelle Size.” Organogenesis 6 no. 2 (2010):88–96.

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Chapter 3 Exercises
Review Questions for Chapter 3
Multiple Choice

1) Which of the following has the highest energy?

A. light with a long wavelength
B. light with an intermediate wavelength
C. light with a short wavelength
D. It is impossible to tell from the information given.

2) You place a specimen under the microscope and notice that parts of the specimen begin to emit light immediately. These
materials can be described as _____________.
A. fluorescent
B. phosphorescent
C. transparent
D. opaque

3) Who was the first to describe “cells” in dead cork tissue?

A. Hans Janssen
B. Zaccharias Janssen
C. Antonie van Leeuwenhoek
D. Robert Hooke

4) Who is the probable inventor of the compound microscope?

A. Girolamo Fracastoro
B. Zaccharias Janssen
C. Antonie van Leeuwenhoek
D. Robert Hooke

5) Which would be the best choice for viewing internal structures of a living protist such as a Paramecium?
a. a brightfield microscope with a stain
b. a brightfield microscope without a stain
c. a darkfield microscope
d. a transmission electron microscope

6) Which type of microscope is especially useful for viewing thick structures such as biofilms?
a. a transmission electron microscope
b. a scanning electron microscopes
c. a phase-contrast microscope
d. a confocal scanning laser microscope
e. an atomic force microscope

7) Which type of microscope would be the best choice for viewing very small surface structures of a cell?

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 a. a transmission electron microscope
b. a scanning electron microscope
c. a brightfield microscope
d. a darkfield microscope
e. a phase-contrast microscope

8) What type of microscope uses an annular stop?

a. a transmission electron microscope
b. a scanning electron microscope
c. a brightfield microscope
d. a darkfield microscope
e. a phase-contrast microscope

9) What type of microscope uses a cone of light so that light only hits the specimen indirectly, producing a darker image on a
brighter background?
a. a transmission electron microscope
b. a scanning electron microscope
c. a brightfield microscope
d. a darkfield microscope
e. a phase-contrast microscope

10) What mordant is used in Gram staining?

A. crystal violet
B. safranin
C. acid-alcohol
D. iodine

11) What is one difference between specimen preparation for a transmission electron microscope (TEM) and preparation for a
scanning electron microscope (SEM)?
A. Only the TEM specimen requires sputter coating.
B. Only the SEM specimen requires sputter-coating.
C. Only the TEM specimen must be dehydrated.
D. Only the SEM specimen must be dehydrated.

12) The smallest unit of biological structure that meets the functional requirements of “living” is the ________.
a. organ
b. organelle
c. cell
d. macromolecule

13) Viruses are not considered living because they ________.

a. are not made of cells
b. lack cell nuclei
c. do not contain DNA or RNA
d. cannot reproduce

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14) The presence of a membrane-enclosed nucleus is a characteristic of ________.
a. prokaryotic cells
b. eukaryotic cells
c. living organisms
d. bacteria

Fill-in-the-Blanks

15) When you see light bend as it moves from air into water, you are observing _________.

16) A microscope that uses multiple lenses is called a _________ microscope.

17) Chromophores that absorb and then emit light are called __________.

18) In a(n) _______ microscope, a probe located just above the specimen moves up and down in response to forces between the
atoms and the tip of the probe.

19) What is the total magnification of a specimen that is being viewed with a standard ocular lens and a 40⨯ objective lens?

20) Ziehl-Neelsen staining, a type of _______ staining, is diagnostic for Mycobacterium tuberculosis.

21) The _______ is used to differentiate bacterial cells based on the components of their cell walls.

22) The four main parts of a cell are collectively responsible for the properties of life. For example, DNA directly influences
evolution and reproduction. The ___________ is directly influential over regulation, including homeostasis.

Short Answer

23) Explain how a prism separates white light into different colors.

24) Why is Antonie van Leeuwenhoek’s work much better known than that of Zaccharias Janssen?

25) Why did the cork cells observed by Robert Hooke appear to be empty, as opposed to being full of other structures?

26) What is the function of the condenser in a brightfield microscope?

27) Art Connection: Label each component of the brightfield microscope.

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28) How could you identify whether a particular bacterial sample contained specimens with mycolic acid-rich cell walls?

29) Select two items that biologists agree are necessary in order to consider an organism “alive.” For each, give an example of a
nonliving object that otherwise fits the definition of “alive.”

Critical Thinking

30) Which of the following has the lowest energy?

A. visible light
B. X-rays
C. ultraviolet rays
D. infrared rays

31) When focusing a light microscope, why is it best to adjust the focus using the coarse focusing knob before using the fine
focusing knob?

32) You need to identify structures within a cell using a microscope. However, the image appears very blurry even though you have
a high magnification. What are some things that you could try to improve the resolution of the image? Describe the most basic
factors that affect resolution when you first put the slide onto the stage; then consider more specific factors that could affect
resolution for 40⨯ and 100⨯ lenses.

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33) You use the Gram staining procedure to stain an L-form bacterium (a bacterium that lacks a cell wall). What color will the
bacterium be after the staining procedure is finished?

34) You go for a long walk on a hot day. Give an example of a way in which homeostasis keeps your body healthy.

35) Using examples, explain how biology can be studied from a microscopic approach to a global approach.

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 CHAPTER OVERVIEW

4: Prokaryotic Diversity
Scientists have studied prokaryotes for centuries, but it wasn’t until 1966 that scientist Thomas Brock (1926–) discovered that
certain bacteria can live in boiling water. This led many to wonder whether prokaryotes may also live in other extreme
environments, such as at the bottom of the ocean, at high altitudes, or inside volcanoes, or even on other planets. Prokaryotes have
an important role in changing, shaping, and sustaining the entire biosphere. They can produce proteins and other substances used
by molecular biologists in basic research and in medicine and industry. For example, the bacterium Shewanella lives in the deep
sea, where oxygen is scarce. It grows long appendages, which have special sensors used to seek the limited oxygen in its
environment. It can also digest toxic waste and generate electricity. Other species of prokaryotes can produce more oxygen than the
entire Amazon rainforest, while still others supply plants, animals, and humans with usable forms of nitrogen; and inhabit our body,
protecting us from harmful microorganisms and producing some vitally important substances. This chapter will examine the
diversity, structure, and function of prokaryotes.
4.1: Unique Characteristics of Prokaryotic Cells
4.2: Classifying Prokaryotes and Examples
Chapter 4 Exercises

Thumbnail: A cladogram linking all major groups of living organisms to the LUCA (the black trunk at the bottom), based on
ribosomal RNA sequence data.

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1
4.1: Unique Characteristics of Prokaryotic Cells
Learning Objectives
Explain the distinguishing characteristics of prokaryotic cells
Describe common cell morphologies and cellular arrangements typical of prokaryotic cells and explain how cells maintain
their morphology
Describe internal and external structures of prokaryotic cells in terms of their physical structure, chemical structure, and
function
Compare the distinguishing characteristics of bacterial and archaeal cells

Cell theory states that the cell is the fundamental unit of life. However, cells vary significantly in size, shape, structure, and
function. The two largest categories of cells—prokaryotic cells and eukaryotic cells—are defined by major differences in several
cell structures. Prokaryotic cells lack a nucleus surrounded by a complex nuclear membrane and generally have a single, circular
chromosome located in a nucleoid. Prokaryotic microorganisms are classified within the domains Archaea and Bacteria.
The structures inside a cell are analogous to the organs inside a human body, with unique structures suited to specific functions.
Some of the structures found in prokaryotic cells are similar to those found in some eukaryotic cells; others are unique to
prokaryotes. Although there are some exceptions, eukaryotic cells tend to be larger than prokaryotic cells. The comparatively larger
size of eukaryotic cells dictates the need to compartmentalize various chemical processes within different areas of the cell, using
complex membrane-bound organelles. In contrast, prokaryotic cells generally lack membrane-bound organelles; however, they
often contain inclusions that compartmentalize their cytoplasm. Figure 4.1.1 illustrates structures typically associated with
prokaryotic cells. These structures are described in more detail in the next section.

Figure 4.1.1 : A typical prokaryotic cell contains a cell membrane, chromosomal DNA that is concentrated in a nucleoid,
ribosomes, and a cell wall. Some prokaryotic cells may also possess flagella, pili, fimbriae, and capsules.

Common Cell Morphologies and Arrangements

Individual cells of a particular prokaryotic organism are typically similar in shape, or cell morphology. Although thousands of
prokaryotic organisms have been identified, only a handful of cell morphologies are commonly seen microscopically. Recall that

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cells are generally coccus (round/spherical), bacillus (rod/elongated), or spiral in shape (rigid spirillia or flexible spirochetes).
However vibrio (curved rods) or coccobacillus (short rods) are also possible. In addition to cellular shape, prokaryotic cells of the
same species may group together in certain distinctive arrangements depending on the plane of cell division. Some common
arrangements are singles, diplo- (pairs), tetrads (set of four in a square), strepto- (chains) or staphylo- (clusters). (Check out
Chapter 3, section 2 for reminders).

Clinical Focus - part 1

Marsha, a 20-year-old university student, recently returned to the United States from a trip to Nigeria, where she had interned
as a medical assistant for an organization working to improve access to laboratory services for tuberculosis testing. When she
returned, Marsha began to feel fatigue, which she initially attributed to jet lag. However, the fatigue persisted, and Marsha soon
began to experience other bothersome symptoms, such as occasional coughing, night sweats, loss of appetite, and a low-grade
fever of 37.4 °C (99.3 °F).
Marsha expected her symptoms would subside in a few days, but instead, they gradually became more severe. About two
weeks after returning home, she coughed up some sputum and noticed that it contained blood and small whitish clumps
resembling cottage cheese. Her fever spiked to 38.2 °C (100.8 °F), and she began feeling sharp pains in her chest when
breathing deeply. Concerned that she seemed to be getting worse, Marsha scheduled an appointment with her physician.

Exercise 4.1.1

Could Marsha’s symptoms be related to her overseas travel, even several weeks after returning home?

The Cellular Envelope: Between Outside and In

Cell Wall
In most prokaryotic cells, morphology is maintained by the cell wall in combination with cytoskeletal elements. The cell wall is a
structure found in most prokaryotes and some eukaryotes; it envelopes the cell membrane, protecting the cell from changes in
osmotic pressure. Osmotic pressure occurs because of differences in the concentration of solutes on opposing sides of a selectively
or semi-permeable membrane. Water is able to pass through a membrane, but solutes (dissolved molecules like salts, sugars, and
other compounds) cannot. When the concentration of solutes is greater on one side of the membrane, water diffuses across the
membrane from the side with the lower concentration (more water) to the side with the higher concentration (less water) until the
concentrations on both sides become equal. This diffusion of water is called osmosis, and it can cause extreme osmotic pressure on
a cell when its external environment changes. This will be discussed further in chapter 8. When present, there are notable
similarities and differences among the cell walls of archaea, bacteria, and eukaryotes.
The major component of bacterial cell walls is called peptidoglycan (or murein); it is only found in bacteria. Structurally,
peptidoglycan resembles a layer of meshwork or fabric (Figure 4.1.2). Each layer is composed of long chains of alternating
molecules of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). The structure of the long chains has significant two-
dimensional tensile strength due to the formation of peptide bridges that connect NAG and NAM within each peptidoglycan layer.
In gram-negative bacteria, tetrapeptide chains extending from each NAM unit are directly cross-linked, whereas in gram-positive
bacteria, these tetrapeptide chains are linked by pentaglycine cross-bridges. Peptidoglycan subunits are made inside of the bacterial
cell and then exported and assembled in layers, giving the cell its shape.
Since peptidoglycan is unique to bacteria, many antibiotic drugs are designed to interfere with peptidoglycan synthesis, weakening
the cell wall and making bacterial cells more susceptible to the effects of osmotic pressure. In addition, certain cells of the human
immune system are able “recognize” bacterial pathogens by detecting peptidoglycan on the surface of a bacterial cell; these cells
then engulf and destroy the bacterial cell, using enzymes such as lysozyme, which breaks down and digests the peptidoglycan in
their cell walls.

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 Figure 4.1.2 : Peptidoglycan is composed of polymers of alternating NAM and NAG subunits, which are cross-linked by peptide
bridges linking NAM subunits from various glycan chains. This provides the cell wall with tensile strength in two dimensions.
The Gram staining protocol is used to differentiate two most common types of cell wall structures (Figure 4.1.3). Gram-positive
cells have a cell wall consisting of many layers of peptidoglycan totaling 30–100 nm in thickness. These peptidoglycan layers are
commonly embedded with teichoic acids (TAs), carbohydrate chains that extend through and beyond the peptidoglycan layer.4 TA
is thought to stabilize peptidoglycan by increasing its rigidity. TA also plays a role in the ability of pathogenic gram-positive
bacteria such as Streptococcus to bind to certain proteins on the surface of host cells, enhancing their ability to cause infection. In
addition to peptidoglycan and TAs, bacteria of the family Mycobacteriaceae have an external layer of waxy mycolic acids in their
cell wall; these bacteria are referred to as acid-fast, since acid-fast stains must be used to penetrate the mycolic acid layer for
purposes of microscopy (Figure 4.1.4).

Figure 4.1.3 : Bacteria contain two common cell wall structural types. Gram-positive cell walls are structurally simple, containing a
thick layer of peptidoglycan with embedded teichoic acid external to the plasma membrane.5 Gram-negative cell walls are
structurally more complex, containing three layers: the inner membrane, a thin layer of peptidoglycan, and an outer membrane
containing lipopolysaccharide. (credit: modification of work by “Franciscosp2”/Wikimedia Commons)

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 Figure 4.1.4 : (a) Some gram-positive bacteria, including members of the Mycobacteriaceae, produce waxy mycolic acids found
exterior to their structurally-distinct peptidoglycan. (b) The acid-fast staining protocol detects the presence of cell walls that are
rich in mycolic acid. Acid-fast cells are stained red by carbolfuschin. (credit a: modification of work by “Franciscosp2”/Wikimedia
Commons; credit b: modification of work by Centers for Disease Control and Prevention)
Gram-negative cells have a much thinner layer of peptidoglycan (no more than about 4 nm thick6) than gram-positive cells, and the
overall structure of their cell envelope is more complex. In gram-negative cells, a gel-like matrix occupies the periplasmic space
between the cell wall and the plasma membrane, and there is a second lipid bilayer called the outer membrane, which is external to
the peptidoglycan layer (Figure 4.1.3). This outer membrane is attached to the peptidoglycan by murein lipoprotein. The outer
leaflet of the outer membrane contains the molecule lipopolysaccharide (LPS), which functions as an endotoxin in infections
involving gram-negative bacteria, contributing to symptoms such as fever, hemorrhaging, and septic shock. Each LPS molecule is
composed of Lipid A, a core polysaccharide, and an O side chain that is composed of sugar-like molecules that comprise the
external face of the LPS (Figure 4.1.5). The composition of the O side chain varies between different species and strains of
bacteria. Parts of the O side chain called antigens can be detected using serological or immunological tests to identify specific
pathogenic strains like Escherichia coli O157:H7, a deadly strain of bacteria that causes bloody diarrhea and kidney failure.

Figure 4.1.5 : The outer membrane of a gram-negative bacterial cell contains lipopolysaccharide (LPS), a toxin composed of Lipid
A embedded in the outer membrane, a core polysaccharide, and the O side chain.
Archaeal cell wall structure differs from that of bacteria in several significant ways. First, archaeal cell walls do not contain
peptidoglycan; instead, they contain a similar polymer called pseudopeptidoglycan (pseudomurein) in which NAM is replaced with

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a different subunit. Other archaea may have a layer of glycoproteins or polysaccharides that serves as the cell wall instead of
pseudopeptidoglycan. Last, as is the case with some bacterial species, there are a few archaea that appear to lack cell walls entirely.

Plasma Membrane
The plasma membrane structure of most bacterial and eukaryotic cell types is a bilayer composed mainly of phospholipids formed
with ester linkages and proteins. Proteins on the cell’s surface are important for a variety of functions. (review chapter 3 section 3
for more detail, and this will be described more in chapter 8)
Photosynthetic Membrane Structures
Some prokaryotic cells, namely cyanobacteria and photosynthetic bacteria, have membrane structures that enable them to perform
photosynthesis. These structures consist of an infolding of the plasma membrane that encloses photosynthetic pigments such as
green chlorophylls and bacteriochlorophylls. In cyanobacteria, these membrane structures are called thylakoids; in photosynthetic
bacteria, they are called chromatophores, lamellae, or chlorosomes. These membranes are still contiguous with the plasma
membrane.

Exercise 4.1.1

1. Explain the difference between cell morphology and arrangement.

2. What advantages do cell walls provide prokaryotic cells?
3. What parts are included in the cellular envelope?

On the Inside
The Nucleoid
All cellular life has a DNA genome organized into one or more chromosomes. Prokaryotic chromosomes are typically circular,
haploid (unpaired), and not bound by a complex nuclear membrane. Because the chromosome contains only one copy of each gene,
prokaryotes are haploid. Prokaryotic DNA and DNA-associated proteins are concentrated within the nucleoid region of the cell
(Figure 4.1.6). In general, prokaryotic DNA interacts with nucleoid-associated proteins (NAPs) that assist in the organization and
packaging of the chromosome, as it would be many times longer than the cell. In bacteria, NAPs function similar to histones, which
are the DNA-organizing proteins found in eukaryotic cells. In archaea, the nucleoid is organized by either NAPs or histone-like
DNA organizing proteins.

Figure 4.1.6 : The nucleoid region (the area enclosed by the green dashed line) is a condensed area of DNA found within
prokaryotic cells. Because of the density of the area, it does not readily stain and appears lighter in color when viewed with a
transmission electron microscope.

Plasmids
Prokaryotic cells may also contain extrachromosomal DNA, or DNA that is not part of the chromosome. This extrachromosomal
DNA is found in plasmids, which are small, circular, double-stranded DNA molecules. Cells that have plasmids often have
hundreds of them within a single cell. Plasmids are more commonly found in bacteria; however, plasmids have been found in
archaea and eukaryotic organisms. Plasmids often carry genes that confer advantageous traits such as antibiotic resistance; thus,
they are important to the survival of the organism.

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 Lethal Plasmids
Maria, a 20-year-old anthropology student from Texas, recently became ill in the African nation of Botswana, where she was
conducting research as part of a study-abroad program. Maria’s research was focused on traditional African methods of tanning
hides for the production of leather. Over a period of three weeks, she visited a tannery daily for several hours to observe and
participate in the tanning process. One day, after returning from the tannery, Maria developed a fever, chills, and a headache,
along with chest pain, muscle aches, nausea, and other flu-like symptoms. Initially, she was not concerned, but when her fever
spiked and she began to cough up blood, her African host family became alarmed and rushed her to the hospital, where her
condition continued to worsen.
After learning about her recent work at the tannery, the physician suspected that Maria had been exposed to anthrax. He
ordered a chest X-ray, a blood sample, and a spinal tap, and immediately started her on a course of intravenous penicillin.
Unfortunately, lab tests confirmed the physician’s presumptive diagnosis. Maria’s chest X-ray exhibited pleural effusion, the
accumulation of fluid in the space between the pleural membranes, and a Gram stain of her blood revealed the presence of
gram-positive, rod-shaped bacteria in short chains, consistent with Bacillus anthracis. Blood and bacteria were also shown to
be present in her cerebrospinal fluid, indicating that the infection had progressed to meningitis. Despite supportive treatment
and aggressive antibiotic therapy, Maria slipped into an unresponsive state and died three days later.
Anthrax is a disease caused by the introduction of endospores from the gram-positive bacterium B. anthracis into the body.
Once infected, patients typically develop meningitis, often with fatal results. In Maria’s case, she inhaled the endospores while
handling the hides of animals that had been infected.
The genome of B. anthracis illustrates how small structural differences can lead to major differences in virulence. In 2003, the
genomes of B. anthracis and Bacillus cereus, a similar but less pathogenic bacterium of the same genus, were sequenced and
compared.4 Researchers discovered that the 16S rRNA gene sequences of these bacteria are more than 99% identical, meaning
that they are actually members of the same species despite their traditional classification as separate species. Although their
chromosomal sequences also revealed a great deal of similarity, several virulence factors of B. anthracis were found to be
encoded on two large plasmids not found in B. cereus. The plasmid pX01 encodes a three-part toxin that suppresses the host
immune system, whereas the plasmid pX02 encodes a capsular polysaccharide that further protects the bacterium from the host
immune system (Figure 4.1.7). Since B. cereus lacks these plasmids, it does not produce these virulence factors, and although
it is still pathogenic, it is typically associated with mild cases of diarrhea from which the body can quickly recover.
Unfortunately for Maria, the presence of these toxin-encoding plasmids in B. anthracis gives it its lethal virulence.

Figure 4.1.7 : Genome sequencing of Bacillus anthracis and its close relative B. cereus reveals that the pathogenicity of B.
anthracis is due to the maintenance of two plasmids, pX01 and pX02, which encode virulence factors.

Exercise 4.1.2

What do you think would happen to the pathogenicity of B. anthracis if it lost one or both of its plasmids?

Ribosomes
All cellular life synthesizes proteins, and organisms in all three domains of life possess ribosomes, structures responsible protein
synthesis. However, ribosomes in each of the three domains are structurally different. Ribosomes, themselves, are constructed from
proteins, along with ribosomal RNA (rRNA). Prokaryotic ribosomes are found in the cytoplasm. They are called 70S ribosomes
because they have a total size of 70S (Figure 4.1.8). This is built up from two subunits, a large 50S subunit and a small 30S

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subunit. The S stands for Svedberg unit, a measure of sedimentation in an ultracentrifuge, which is based on size, shape, and
surface qualities of the structure being analyzed. Although they are the same size, bacterial and archaeal ribosomes have different
proteins and rRNA molecules, and the archaeal versions are more similar to their eukaryotic counterparts than to those found in
bacteria.

Figure 4.1.8 : Prokaryotic ribosomes (70S) are composed of two subunits: the 30S (small subunit) and the 50S (large subunit), each
of which are composed of protein and rRNA components.

Inclusions
As single-celled organisms living in unstable environments, some prokaryotic cells have the ability to store excess nutrients within
cytoplasmic areas called inclusions. Storing nutrients in a polymerized form is advantageous because it reduces the buildup of
osmotic pressure that occurs as a cell accumulates solutes. Various types of inclusions store glycogen and starches, which contain
carbon that cells can access for energy. Volutin granules, also called metachromatic granules because of their staining
characteristics, are inclusions that store polymerized inorganic phosphate that can be used in metabolism and assist in the formation
of biofilms. Microbes known to contain volutin granules include the archaea Methanosarcina, the bacterium Corynebacterium
diphtheriae, and the unicellular eukaryotic alga Chlamydomonas. Sulfur granules, another type of inclusion, are found in sulfur
bacteria of the genus Thiobacillus; these granules store elemental sulfur, which the bacteria use for metabolism.
Occasionally, certain types of inclusions are surrounded by a phospholipid monolayer embedded with protein. Polyhydroxybutyrate
(PHB), which can be produced by species of Bacillus and Pseudomonas, is an example of an inclusion that displays this type of
monolayer structure. Industrially, PHB has also been used as a source of biodegradable polymers for bioplastics. Several different
types of inclusions are shown in Figure 4.1.9.
Some prokaryotic cells have other types of inclusions that serve purposes other than nutrient storage. For example, some
prokaryotic cells produce gas globules, accumulations of small, protein-lined globs of gas. These gas globs allow the prokaryotic
cells that synthesize them to alter their buoyancy so that they can adjust their location in the water column. Magnetotactic bacteria,
such as Magnetospirillum magnetotacticum, contain magnetosomes, which are inclusions of magnetic iron oxide or iron sulfide
surrounded by a lipid layer. These allow cells to align along a magnetic field, aiding their movement (Figure 4.1.9). Cyanobacteria
such as Anabaena cylindrica and bacteria such as Halothiobacillus neapolitanus produce carboxysome inclusions. Carboxysomes
are composed of outer shells of thousands of protein subunits. Their interior is filled with ribulose-1,5-bisphosphate
carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Both of these compounds are used for carbon metabolism. Some
prokaryotic cells also possess carboxysomes that sequester functionally related enzymes in one location. These structures are
considered proto-organelles because they compartmentalize important compounds or chemical reactions, much like many
eukaryotic organelles.

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 Figure 4.1.9 : Prokaryotic cells may have various types of inclusions. (a) A transmission electron micrograph of
polyhydroxybutryrate lipid droplets. (b) A light micrograph of volutin granules. (c) A phase-contrast micrograph of sulfur granules.
(d) A transmission electron micrograph of magnetosomes. (e) A transmission electron micrograph of gas vacuoles. (credit b, c, d:
modification of work by American Society for Microbiology)

Endospores
Bacterial cells are generally observed as vegetative cells, but some genera of bacteria have the ability to form endospores,
structures that essentially protect the bacterial genome in a dormant state when environmental conditions are unfavorable.
Endospores (not to be confused with the reproductive spores formed by fungi) allow some bacterial cells to survive long periods
without food or water, as well as exposure to chemicals, extreme temperatures, and even radiation. Table 4.1.1 compares the
characteristics of vegetative cells and endospores.
Table 4.1.1 : Characteristics of Vegetative Cells versus Endospores
Vegetative Cells Endospores

Sensitive to extreme temperatures and radiation Resistant to extreme temperatures and radiation

Gram-positive Do not absorb Gram stain, only special endospore stains

Normal water content and enzymatic activity Dehydrated; no metabolic activity

Capable of active growth and metabolism Dormant; no growth or metabolic activity

The process by which vegetative cells transform into endospores is called sporulation, and it generally begins when nutrients
become depleted or environmental conditions become otherwise unfavorable (Figure 4.1.10). The process begins with the
formation of a septum in the vegetative bacterial cell. The septum divides the cell asymmetrically, separating a DNA forespore
from the mother cell. The forespore, which will form the core of the endospore, is essentially a copy of the cell’s chromosomes, and
is separated from the mother cell by a second membrane. A cortex gradually forms around the forespore by laying down layers of
calcium and dipicolinic acid between membranes. A protein spore coat then forms around the cortex while the DNA of the mother
cell disintegrates. Further maturation of the endospore occurs with the formation of an outermost exosporium. The endospore is
released upon disintegration of the mother cell, completing sporulation.

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 Figure 4.1.10: (a) Sporulation begins following asymmetric cell division. The forespore becomes surrounded by a double layer of
membrane, a cortex, and a protein spore coat, before being released as a mature endospore upon disintegration of the mother cell.
(b) An electron micrograph of a Carboxydothermus hydrogenoformans endospore. (c) These Bacillus spp. cells are undergoing
sporulation. The endospores have been visualized using Malachite Green spore stain. (credit b: modification of work by Jonathan
Eisen)
Endospores of certain species have been shown to persist in a dormant state for extended periods of time, up to thousands of years.2
However, when living conditions improve, endospores undergo germination, reentering a vegetative state. After germination, the
cell becomes metabolically active again and is able to carry out all of its normal functions, including growth and cell division.
Not all bacteria have the ability to form endospores; however, there are a number of clinically significant endospore-forming gram-
positive bacteria of the genera Bacillus and Clostridium. These include B. anthracis, the causative agent of anthrax, which produces
endospores capable of survive for many decades3; C. tetani (causes tetanus); C. difficile (causes pseudomembranous colitis); C.
perfringens (causes gas gangrene); and C. botulinum (causes botulism). Pathogens such as these are particularly difficult to combat
because their endospores are so hard to kill. Special sterilization methods for endospore-forming bacteria are discussed in later
chapters.

Exercise 4.1.3

1. What is an inclusion?
2. What is the function of an endospore?

On the Outside
Glycocalyces and S-Layers
Although most prokaryotic cells have cell walls, some may have additional cell envelope structures exterior to the cell wall, such as
glycocalyces and S-layers. A glycocalyx is a sugar coat, of which there are two important types: capsules and slime layers. A
capsule is an organized layer located outside of the cell wall and usually composed of polysaccharides or proteins (Figure 4.1.11).
A slime layer is a less tightly organized layer that is only loosely attached to the cell wall and can be more easily washed off. Slime
layers may be composed of polysaccharides, glycoproteins, or glycolipids.

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Glycocalyces allows cells to adhere to surfaces, aiding in the formation of biofilms (colonies of microbes that form in layers on
surfaces). In nature, most microbes live in mixed communities within biofilms, partly because the biofilm affords them some level
of protection. Biofilms are abundant and frequently occupy complex niches within ecosystems. In medicine, biofilms can coat
medical devices and exist within the body. Because they possess unique characteristics, such as increased resistance against the
immune system, disinfectants, predation and to antimicrobial drugs, biofilms are of particular interest to microbiologists and
clinicians alike. Biofilms generally hold water like a sponge, preventing desiccation.
All of these properties are advantageous to the microbes living in a biofilm, but they present challenges in a clinical setting, where
the goal is often to eliminate microbes. The ability to produce a capsule can contribute to a microbe’s pathogenicity (ability to
cause disease) because the capsule can make it more difficult for phagocytic cells (such as white blood cells) to engulf and kill the
microorganism. Streptococcus pneumoniae, for example, produces a capsule that is well known to aid in this bacterium’s
pathogenicity. Capsules are difficult to stain for microscopy; negative staining techniques are typically used.

Figure 4.1.11: (a) Capsules are a type of glycocalyx composed of an organized layer of polysaccharides. (b) A capsule stain of
Pseudomonas aeruginosa, a bacterial pathogen capable of causing many different types of infections in humans. (credit b:
modification of work by American Society for Microbiology)

Figure 4.1.12: A biofilm forms when planktonic (free-floating) bacteria of one or more species adhere to a surface, produce slime,
and form a colony. This diagram shows the five stages of biofilm development of Pseudomonas aeruginosa. All photomicrographs
are shown to the same scale. (credit: Public Library of Science).
An S-layer is another type of cell envelope structure; it is composed of a mixture of structural proteins and glycoproteins. In
bacteria, S-layers are found outside the cell wall, but in some archaea, the S-layer serves as the cell wall. The exact function of S-

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layers is not entirely understood, and they are difficult to study; but available evidence suggests that they may play a variety of
functions in different prokaryotic cells, such as helping the cell withstand osmotic pressure and, for certain pathogens, interacting
with the host immune system.

Filamentous Appendages
Many bacterial cells have protein appendages embedded within their cell envelopes that extend outward, allowing interaction with
the environment. These appendages can attach to other surfaces, transfer DNA, or provide movement. Filamentous appendages
include fimbriae, pili, and flagella.
Fimbriae

Fimbriae and pili are structurally similar and, because differentiation between the two is problematic, these terms are often used
interchangeably.7 8 However, common use it that the term fimbriae refers to short bristle-like proteins projecting from the cell
surface by the hundreds. Fimbriae enable a cell to attach to surfaces and to other cells. For pathogenic bacteria, adherence to host
cells is important for colonization, infectivity, and virulence. Adherence to surfaces is also important in biofilm formation.
Pili
The term pili (singular: pilus) commonly refers to longer, hollow, less numerous protein appendages (Figure 4.1.13). More
specifically called the F pilus or sex pilus, it is important in the transfer of DNA between bacterial cells, which occurs between
members of the same generation when two cells physically transfer or exchange parts of their respective genomes.

Figure 4.1.13 : Bacteria may produce two different types of protein appendages that aid in surface attachment. Fimbriae typically
are more numerous and shorter, whereas pili (shown here) are longer and less numerous per cell. (credit: modification of work by
American Society for Microbiology)
Flagella

Flagella are structures used by cells to move in aqueous environments. Bacterial flagella act like propellers. They are stiff spiral
filaments composed of flagellin protein subunits that extend outward from the cell and spin in solution. The basal body is the motor
for the flagellum and is embedded in the plasma membrane (Figure 4.1.14). A hook region connects the basal body to the filament.
Gram-positive and gram-negative bacteria have different basal body configurations due to differences in cell wall structure.
Different types of motile bacteria exhibit different arrangements of flagella (Figure 4.1.14). A bacterium with a singular flagellum,
typically located at one end of the cell (polar), is said to have a monotrichous flagellum. An example of a monotrichously
flagellated bacterial pathogen is Vibrio cholerae, the gram-negative bacterium that causes cholera. Cells with amphitrichous
flagella have a flagellum or tufts of flagella at each end. An example is Spirillum minor, the cause of spirillary (Asian) rat-bite
fever or sodoku. Cells with lophotrichous flagella have a tuft at one end of the cell. The gram-negative bacillus Pseudomonas
aeruginosa, an opportunistic pathogen known for causing many infections, including “swimmer’s ear” and burn wound infections,
has lophotrichous flagella. Flagella that cover the entire surface of a bacterial cell are called peritrichous flagella. The gram-
negative bacterium E. coli shows a peritrichous arrangement of flagella.

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 Figure 4.1.14 : The basic structure of a bacterial flagellum consists of a basal body, hook, and filament. The basal body composition
and arrangement differ between gram-positive and gram-negative bacteria. (credit: modification of work by “LadyofHats”/Mariana
Ruiz Villareal)

Figure 4.1.15 : Flagellated bacteria may exhibit multiple arrangements of their flagella. Common arrangements include
monotrichous, amphitrichous, lophotrichous, or peritrichous.

Directional movement depends on the configuration of the flagella. Bacteria can move in response to a variety of environmental
signals, including light (phototaxis), magnetic fields (magnetotaxis) using magnetosomes, and, most commonly, chemical gradients
(chemotaxis). Purposeful movement toward a chemical attractant, like a food source, or away from a repellent, like a poisonous
chemical, is achieved by increasing the length of runs and decreasing the length of tumbles. When running, flagella rotate in a
counterclockwise direction, allowing the bacterial cell to move forward. In a peritrichous bacterium, the flagella are all bundled
together in a very streamlined way (Figure 4.1.16), allowing for efficient movement. When tumbling, flagella are splayed out while
rotating in a clockwise direction, creating a looping motion and preventing meaningful forward movement but reorienting the cell
toward the direction of the attractant. When an attractant exists, runs and tumbles still occur; however, the length of runs is longer,
while the length of the tumbles is reduced, allowing overall movement toward the higher concentration of the attractant. When no
chemical gradient exists, the lengths of runs and tumbles are more equal, and overall movement is more random (Figure 4.1.17).

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 Figure 4.1.16 : Bacteria achieve directional movement by changing the rotation of their flagella. In a cell with peritrichous flagella,
the flagella bundle when they rotate in a counterclockwise direction, resulting in a run. However, when the flagella rotate in a
clockwise direction, the flagella are no longer bundled, resulting in tumbles.

Figure 4.1.17 : Without a chemical gradient, flagellar rotation cycles between counterclockwise (run) and clockwise (tumble) with
no overall directional movement. However, when a chemical gradient of an attractant exists, the length of runs is extended, while
the length of tumbles is decreased. This leads to chemotaxis: an overall directional movement toward the higher concentration of
the attractant.

Exercise 4.1.4

1. What is the peptidoglycan layer and how does it differ between gram-positive and gram-negative bacteria?
2. Compare and contrast monotrichous, amphitrichous, lophotrichous, and peritrichous flagella.

Summary
Prokaryotic cells differ from eukaryotic cells in that their genetic material is contained in a nucleoid rather than a membrane-
bound nucleus. In addition, prokaryotic cells generally lack membrane-bound organelles.
Prokaryotic cells of the same species typically share a similar cell morphology and cellular arrangement.
Most prokaryotic cells have a cell wall that helps the organism maintain cellular morphology and protects it against changes in
osmotic pressure.
In prokaryotic cells, the cell envelope always includes a plasma membrane and usually includes a cell wall. The proteins in
the membrane serve a variety of functions, including transport, cell-to-cell communication, and sensing environmental

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 conditions. Archaeal membranes are distinct in that they are composed of fatty acids that are ether-linked to phospholipids.
Prokaryotic cell walls may be composed of peptidoglycan (bacteria) or pseudopeptidoglycan (archaea).
Gram-positive bacterial cells are characterized by a thick peptidoglycan layer, whereas gram-negative bacterial cells are
characterized by a thin peptidoglycan layer surrounded by an outer membrane.
Prokaryote DNA is generally found in the nucleoid. Outside of the nucleoid, prokaryotic cells may contain extrachromosomal
DNA in plasmids.
Prokaryotic ribosomes that are found in the cytoplasm have a size of 70S.
Some prokaryotic cells have inclusions that store nutrients or chemicals for other uses.
Some prokaryotic cells are able to form endospores through sporulation to survive in a dormant state when conditions are
unfavorable. Endospores can germinate, transforming back into vegetative cells when conditions improve.
Some prokaryotic cells produce glycocalyx coatings, such as capsules and slime layers, that aid in attachment to surfaces
and/or evasion of the host immune system.
Some prokaryotic cells have fimbriae or pili, filamentous appendages. Fimbriae aid in attachment. Pili are used in the transfer
of genetic material between cells.
Some prokaryotic cells use one or more flagella to move through water. Peritrichous bacteria, which have numerous flagella,
use runs and tumbles to move purposefully in the direction of a chemical attractant.

Footnotes
1.
2. F. Rothfuss, M Bender, R Conrad. “Survival and Activity of Bacteria in a Deep, Aged Lake Sediment (Lake Constance).”
Microbial Ecology 33 no. 1 (1997):69–77.
3. R. Sinclair et al. “Persistence of Category A Select Agents in the Environment.” Applied and Environmental Microbiology 74
no. 3 (2008):555–563.
4. T.J. Silhavy, D. Kahne, S. Walker. “The Bacterial Cell Envelope.” Cold Spring Harbor Perspectives in Biology 2 no. 5
(2010):a000414.
5. B. Zuber et al. “Granular Layer in the Periplasmic Space of Gram-Positive Bacteria and Fine Structures of Enterococcus
gallinarum and Streptococcus gordonii Septa Revealed by Cryo-Electron Microscopy of Vitreous Sections.” Journal of
Bacteriology 188 no. 18 (2006):6652–6660
6. L. Gana, S. Chena, G.J. Jensena. “Molecular Organization of Gram-Negative Peptidoglycan.” Proceedings of the National
Academy of Sciences of the United States of America 105 no. 48 (2008):18953–18957.
7. J.A. Garnetta et al. “Structural Insights Into the Biogenesis and Biofilm Formation by the Escherichia coli Common Pilus.”
Proceedings of the National Academy of Sciences of the United States of America 109 no. 10 (2012):3950–3955.
8. T. Proft, E.N. Baker. “Pili in Gram-Negative and Gram-Positive Bacteria—Structure, Assembly and Their Role in Disease.”
Cellular and Molecular Life Sciences 66 (2009):613.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 4.1: Unique Characteristics of Prokaryotic Cells is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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4.2: Classifying Prokaryotes and Examples
Learning Objectives
Explain how prokaryotes are classified what criteria are used to help group them into phylum
Highlight some interesting and diverse prokaryotes

Taxonomy and Systematics

Assigning prokaryotes to a certain species is challenging. They do not reproduce sexually, so it is not possible to classify them
according to the presence or absence of interbreeding. Also, they do not have many morphological features. Traditionally, the
classification of prokaryotes was based on their shape, staining patterns, and biochemical or physiological differences. More
recently, as technology has improved, the nucleotide sequences in genes (particularly rRNA) have become an important criterion of
microbial classification.
In 1923, American microbiologist David Hendricks Bergey (1860–1937) published A Manual in Determinative Bacteriology. With
this manual, he attempted to summarize the information about the kinds of bacteria known at that time, using Latin binomial
classification. Bergey also included the morphological, physiological, and biochemical properties of these organisms. His manual
has been updated multiple times to include newer bacteria and their properties. It is a great aid in bacterial taxonomy and methods
of characterization of bacteria. A more recent sister publication, the five-volume Bergey’s Manual of Systematic Bacteriology,
expands on Bergey’s original manual. It includes a large number of additional species, along with up-to-date descriptions of the
taxonomy and biological properties of all named prokaryotic taxa. This publication incorporates the approved names of bacteria as
determined by the List of Prokaryotic Names with Standing in Nomenclature (LPSN). It can also be found as a living document,
available through subscription, to try and keep it as up-to-date as possible.

Classification by Staining Patterns

According to their staining patterns, which depend on the properties of their cell walls, bacteria have traditionally been classified
into gram-positive, gram-negative, and “atypical,” meaning neither gram-positive nor gram-negative. As explained in earlier, gram-
positive bacteria possess a thick peptidoglycan cell wall that retains the primary stain (crystal violet) during the decolorizing step;
they remain purple after the gram-stain procedure because the crystal violet dominates the light red/pink color of the secondary
counterstain, safranin. In contrast, gram-negative bacteria possess a thin peptidoglycan cell wall that does not prevent the crystal
violet from washing away during the decolorizing step; therefore, they appear light red/pink after staining with the safranin.
Bacteria that cannot be stained by the standard Gram stain procedure are called atypical bacteria. Included in the atypical category
are species of Mycoplasma and Chlamydia, which lack a cell wall and therefore cannot retain the gram-stain reagents. Rickettsia
are also considered atypical because they are too small to be evaluated by the Gram stain.
More recently, scientists have begun to further classify gram-negative and gram-positive bacteria. They have added a special group
of deeply branching bacteria based on a combination of physiological, biochemical, and genetic features. They also now further
classify gram-negative bacteria into Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB), and spirochetes.
The deeply branching bacteria are thought to be a very early evolutionary form of bacteria. They live in hot, acidic, ultraviolet-
light-exposed, and anaerobic (deprived of oxygen) conditions. Proteobacteria is a phylum of very diverse groups of gram-negative
bacteria; it includes some important human pathogens (e.g., E. coli and Bordetella pertussis). The CFB group of bacteria includes
components of the normal human gut microbiota, like Bacteroides. The spirochetes are spiral-shaped bacteria and include the
pathogen Treponema pallidum, which causes syphilis. We will characterize these groups of bacteria in more detail later in this
section.
Based on their prevalence of guanine and cytosine nucleotides, gram-positive bacteria are also classified into low G+C and high
G+C gram-positive bacteria. The low G+C gram-positive bacteria have less than 50% of guanine and cytosine nucleotides in their
DNA. They include human pathogens, such as those that cause anthrax (Bacillus anthracis), tetanus (Clostridium tetani), and
listeriosis (Listeria monocytogenes). High G+C gram-positive bacteria, which have more than 50% guanine and cytosine
nucleotides in their DNA, include the bacteria that cause diphtheria (Corynebacterium diphtheriae), tuberculosis (Mycobacterium
tuberculosis), and other diseases.
Using these two descriptions however, will only get small amounts of separation, so microbiologist turn to two other descriptions:
the microbes interaction with or reliance on oxygen gas, and their main mode of gaining of nutrients (both more fully described in

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chapters 7 and 8). The classifications of prokaryotes are constantly changing as new species are being discovered and new
information comes to light. Included in the next section are some highlights from the different major groups and phylum of the
Bacterial and Archaeal domains.

Exercise 4.2.1

What characteristics do scientists use to classify prokaryotes?

Proteobacteria
In 1987, the American microbiologist Carl Woese (1928–2012) suggested that a large and diverse group of bacteria that he called
“purple bacteria and their relatives” should be defined as a separate phylum within the domain Bacteria based on the similarity of
the nucleotide sequences in their genome.1 This phylum of gram-negative bacteria subsequently received the name Proteobacteria.
It includes many bacteria that are part of the normal human microbiota as well as many pathogens. The Proteobacteria are further
divided into five classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and
Epsilonproteobacteria.

Alphaproteobacteria include intracellular parasites

Among the Alphaproteobacteria are two taxa, chlamydias and rickettsias, that are obligate intracellular pathogens, meaning that
part of their life cycle must occur inside other cells called host cells. When not growing inside a host cell, Chlamydia and Rickettsia
are metabolically inactive outside of the host cell. They cannot synthesize their own adenosine triphosphate (ATP), and, therefore,
rely on cells for their energy needs.
Rickettsia spp. include a number of serious human pathogens. For example, R. rickettsii causes Rocky Mountain spotted fever, a
life-threatening form of meningoencephalitis (inflammation of the membranes that wrap the brain). R. rickettsii infects ticks and
can be transmitted to humans via a bite from an infected tick (Figure 4.2.1). Another species of Rickettsia, R. prowazekii, is spread
by lice. It causes epidemic typhus, a severe infectious disease common during warfare and mass migrations of people. R.
prowazekii infects human endothelium cells, causing inflammation of the inner lining of blood vessels, high fever, abdominal pain,
and sometimes delirium. A relative, R. typhi, causes a less severe disease known as murine or endemic typhus, which is still
observed in the southwestern United States during warm seasons.

Figure 4.2.1 : Rickettsias require special staining methods to see them under a microscope. Here, R. rickettsii, which causes Rocky
Mountain spotted fever, is shown infecting the cells of a tick. (credit: modification of work by Centers for Disease Control and
Prevention)
Chlamydia is another taxon of the Alphaproteobacteria. Members of this genus are extremely resistant to the cellular defenses,
giving them the ability to spread from host to host rapidly via elementary bodies. The metabolically and reproductively inactive
elementary bodies are the endospore-like form of intracellular bacteria that enter an epithelial cell, where they become active.
Figure 4.2.2 illustrates the life cycle of Chlamydia. C. trachomatis is a human pathogen that causes trachoma, a disease of the eyes,
often leading to blindness. C. trachomatis also causes the sexually transmitted disease lymphogranuloma venereum (LGV). This
disease is often mildly symptomatic, manifesting as regional lymph node swelling, or it may be asymptomatic, but it is extremely
contagious and is common on college campuses.

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 Figure 4.2.2 : Chlamydia begins infection of a host when the metabolically inactive elementary bodies enter an epithelial cell. Once
inside the host cell, the elementary bodies turn into active reticulate bodies. The reticulate bodies multiply and release more
elementary bodies when the cell dies after the Chlamydia uses all of the host cell’s ATP. (credit: modification of work by Centers
for Disease Control and Prevention)

Betaproteobacteria include other familiar infectious bacteria

Unlike Alphaproteobacteria, which survive on a minimal amount of nutrients, the class Betaproteobacteria are eutrophs (or
copiotrophs), meaning that they require a copious amount of organic nutrients. Betaproteobacteria often grow between aerobic and
anaerobic areas (e.g., in mammalian intestines). Some genera include species that are human pathogens, able to cause severe,
sometimes life-threatening disease. The pathogen responsible for pertussis (whooping cough) is also a member of
Betaproteobacteria. The bacterium Bordetella pertussis, from the order Burkholderiales, produces several toxins that paralyze the
movement of cilia in the human respiratory tract and directly damage cells of the respiratory tract, causing a severe cough.
The genus Neisseria, for example, includes the bacteria N. gonorrhoeae, the causative agent of the STI gonorrhea, and N.
meningitides, the causative agent of bacterial meningitis. Neisseria are cocci that live on mucosal surfaces of the human body. They
are fastidious, or difficult to culture, and they require high levels of moisture, nutrient supplements, and carbon dioxide. Also,
Neisseria are microaerophilic, meaning that they require low levels of oxygen. For optimal growth and for the purposes of
identification, Neisseria spp. are grown on chocolate agar (i.e., agar supplemented by partially hemolyzed red blood cells). Their
characteristic pattern of growth in culture is diplococcal: pairs of cells resembling coffee beans (Figure 4.2.3).

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 Figure 4.2.3 : Neisseria meningitidis growing in colonies on a chocolate agar plate. (credit: Centers for Disease Control and
Prevention)

Gammaproteobacteria
The most diverse class of gram-negative bacteria is Gammaproteobacteria, and it includes a number of human pathogens. For
example, a large and diverse family, Pseudomonaceae, includes the genus Pseudomonas. Within this genus is the species P.
aeruginosa, a pathogen responsible for diverse infections in various regions of the body. It often infects wounds and burns, can be
the cause of chronic urinary tract infections, and can be an important cause of respiratory infections in patients with cystic fibrosis
or patients on mechanical ventilators. Infections by P. aeruginosa are often difficult to treat because the bacterium is resistant to
many antibiotics and has a remarkable ability to form biofilms. Other representatives of Pseudomonas include the fluorescent
(glowing) bacterium P. fluorescens and the soil bacteria P. putida, which is known for its ability to degrade xenobiotics (substances
not naturally produced or found in living organisms).
Another famous member is E. coli has been perhaps the most studied bacterium since it was first described in 1886 by Theodor
Escherich (1857–1911). Many strains of E. coli are in mutualistic relationships with humans. However, some strains produce a
potentially deadly toxin called Shiga toxin, which perforates cellular membranes in the large intestine, causing bloody diarrhea and
peritonitis (inflammation of the inner linings of the abdominal cavity). Other E. coli strains may cause traveler’s diarrhea, a less
severe but very widespread disease. The genera Pasteruella, Legionella, Haemophilus and Salmonella.
The order Vibrionales includes the human pathogen Vibrio cholerae. This comma-shaped aquatic bacterium thrives in highly
alkaline environments like shallow lagoons and sea ports. A toxin produced by V. cholerae causes hypersecretion of electrolytes
and water in the large intestine, leading to profuse watery diarrhea and dehydration. V. parahaemolyticus is also a cause of
gastrointestinal disease in humans, whereas V. vulnificus causes serious and potentially life-threatening cellulitis (infection of the
skin and deeper tissues) and blood-borne infections. Another representative of Vibrionales, Aliivibrio fischeri, engages in a
symbiotic relationship with squid. The squid provides nutrients for the bacteria to grow and the bacteria produce bioluminescence
that protects the squid from predators (Figure 4.2.4).

Figure 4.2.4 : (a) Aliivibrio fischeri is a bioluminescent bacterium. (b) A. fischeri colonizes and lives in a mutualistic relationship
with the Hawaiian bobtail squid (Euprymna scolopes). (credit a: modification of work by American Society for Microbiology;
credit b: modification of work by Margaret McFall-Ngai)

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Deltaproteobacteria include potentially helpful a scattering of helpful bacteria
The Deltaproteobacteria is a small class of gram-negative Proteobacteria that includes sulfate-reducing bacteria(SRBs), so named
because they use sulfate as the final electron acceptor in the electron transport chain. Few SRBs are pathogenic. However, the SRB
Desulfovibrio orale is associated with periodontal disease (disease of the gums).
Deltaproteobacteria also includes the genus Bdellovibrio, species of which are parasites of other gram-negative bacteria.
Bdellovibrio invades the cells of the host bacterium, positioning itself in the periplasm, the space between the plasma membrane
and the cell wall, feeding on the host’s proteins and polysaccharides. The infection is lethal for the host cells.
Another type of Deltaproteobacteria, myxobacteria, lives in the soil, scavenging inorganic compounds. Motile and highly social,
they interact with other bacteria within and outside their own group. They can form multicellular, macroscopic “fruiting bodies”
(Figure 4.2.5), structures that are still being studied by biologists and bacterial ecologists.2 These bacteria can also form
metabolically inactive myxospores.

Figure 4.2.5 : Myxobacteria form fruiting bodies. (credit: modification of work by Michiel Vos)

Epsilonproteobacteria includes familiar GI pathogens

The smallest class of Proteobacteria is Epsilonproteobacteria, which are gram-negative microaerophilic bacteria (meaning they
only require small amounts of oxygen in their environment). Two clinically relevant genera of Epsilonproteobacteria are
Campylobacter and Helicobacter, both of which include human pathogens. Campylobacter can cause food poisoning that manifests
as severe enteritis (inflammation in the small intestine). This condition, caused by the species C. jejuni, is rather common in
developed countries, usually because of eating contaminated poultry products. Chickens often harbor C. jejuni in their
gastrointestinal tract and feces, and their meat can become contaminated during processing.
Within the genus Helicobacter, the helical, flagellated bacterium H. pylori has been identified as a beneficial member of the
stomach microbiota, but it is also the most common cause of chronic gastritis and ulcers of the stomach and duodenum (Figure
3
4.2.6). Studies have also shown that H. pylori is linked to stomach cancer. H. pylori is somewhat unusual in its ability to survive

in the highly acidic environment of the stomach. It produces urease and other enzymes that modify its environment to make it less
acidic.

Figure 4.2.6 : Helicobacter pylori can cause chronic gastritis, which can lead to ulcers and stomach cancer.

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Nonproteobacteria Gram-negative Bacteria and Phototrophic Bacteria
The majority of the gram-negative bacteria belong to the phylum Proteobacteria, discussed in the previous section. Those that do
not are called the nonproteobacteria. In this section, we will describe three classes of gram-negative nonproteobacteria: the
spirochetes, the CFB group, and the Planctomycetes. A diverse group of phototrophic bacteria that includes Proteobacteria and
nonproteobacteria will be discussed at the end of this section.

Spirochetes
Several genera of spirochetes include human pathogens. For example, the genus Treponema includes a species T. pallidum, which
is further classified into four subspecies: T. pallidum pallidum, T. pallidum pertenue, T. pallidum carateum, and T. pallidum
endemicum. The subspecies T. pallidum pallidum causes the sexually transmitted infection known as syphilis, the third most
prevalent sexually transmitted bacterial infection in the United States, after chlamydia and gonorrhea. The other subspecies of T.
pallidum cause tropical infectious diseases of the skin, bones, and joints.
Another genus of spirochete, Borrelia, contains a number of pathogenic species. B. burgdorferi causes Lyme disease, which is
transmitted by several genera of ticks (notably Ixodes and Amblyomma) and often produces a “bull’s eye” rash, fever, fatigue, and,
sometimes, debilitating arthritis. B. recurrens causes a condition known as relapsing fever.
Spirochetes are characterized by their long (up to 250 μm), spiral-shaped bodies. Most spirochetes are also very thin, which makes
it difficult to examine gram-stained preparations under a conventional brightfield microscope. Darkfield fluorescent microscopy is
typically used instead. Spirochetes are also difficult or even impossible to culture. They are highly motile, using their axial filament
to propel themselves. The axial filament is similar to a flagellum, but it wraps around the cell and runs inside the cell body of a
spirochete in the periplasmic space between the outer membrane and the plasma membrane (Figure 4.2.7).

Figure 4.2.7 : Spirochetes are typically observed using darkfield microscopy (left). However, electron microscopy (top center,
bottom center) provides a more detailed view of their cellular morphology. The flagella found between the inner and outer
membranes of spirochetes wrap around the bacterium, causing a twisting motion used for locomotion. (credit “spirochetes”
micrograph: modification of work by Centers for Disease Control and Prevention; credit “SEM/TEM”: modification of work by
Guyard C, Raffel SJ, Schrumpf ME, Dahlstrom E, Sturdevant D, Ricklefs SM, Martens C, Hayes SF, Fischer ER, Hansen BT,
Porcella SF, Schwan TG)

Phototrophic Bacteria
The phototrophic bacteria are a large and diverse category of bacteria that do not represent a taxon but, rather, a group of bacteria
that use sunlight as their primary source of energy. This group contains both Proteobacteria and nonproteobacteria. They use solar
energy to synthesize ATP through photosynthesis. When they produce oxygen, they perform oxygenic photosynthesis. When they

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do not produce oxygen, they perform anoxygenic photosynthesis. With the exception of some cyanobacteria, the majority of
phototrophic bacteria perform anoxygenic photosynthesis.
One large group of phototrophic bacteria includes the purple or green bacteria that perform photosynthesis with the help of
bacteriochlorophylls, which are green, purple, or blue pigments similar to chlorophyll in plants. Some of these bacteria have a
varying amount of red or orange pigments called carotenoids. Their color varies from orange to red to purple to green (Figure
4.2.8), and they are able to absorb light of various wavelengths. Traditionally, these bacteria are classified into sulfur and nonsulfur

bacteria; they are further differentiated by color. The sulfur bacteria perform anoxygenic photosynthesis, using sulfites as electron
donors and releasing free elemental sulfur. Nonsulfur bacteria use organic substrates, such as succinate and malate, as donors of
electrons.

Figure 4.2.8 : Purple and green sulfur bacteria use bacteriochlorophylls to perform photosynthesis.
Another large, diverse group of phototrophic bacteria compose the phylum Cyanobacteria; they get their blue-green color from the
chlorophyll contained in their cells (Figure 4.2.9). Species of this group perform oxygenic photosynthesis, producing megatons of
gaseous oxygen. Scientists hypothesize that cyanobacteria played a critical role in the change of our planet’s anoxic atmosphere 1–
2 billion years ago to the oxygen-rich environment we have today.4 Cyanobacteria have other remarkable properties. Amazingly
adaptable, they thrive in many habitats, including marine and freshwater environments, soil, and even rocks. They can live at a
wide range of temperatures, even in the extreme temperatures of the Antarctic. They can live as unicellular organisms or in
colonies, and they can be filamentous, forming sheaths or biofilms. Many of them fix nitrogen, converting molecular nitrogen into
nitrites and nitrates that other bacteria, plants, and animals can use. The reactions of nitrogen fixation occur in specialized cells
called heterocysts.

Figure 4.2.9 : (a) Microcystis aeruginosa is a type of cyanobacteria commonly found in freshwater environments. (b) In warm
temperatures, M. aeruginosa and other cyanobacteria can multiply rapidly and produce neurotoxins, resulting in blooms that are
harmful to fish and other aquatic animals. (credit a: modification of work by Dr. Barry H. Rosen/U.S. Geological Survey; credit b:
modification of work by NOAA)

Actinobacteria: High G+C Gram-Positive Bacteria

The name Actinobacteria comes from the Greek words for rays and small rod, but Actinobacteria are very diverse. Their
microscopic appearance can range from thin filamentous branching rods to coccobacilli. Some Actinobacteria are very large and
complex, whereas others are among the smallest independently living organisms. Most Actinobacteria live in the soil, but some are

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aquatic. The vast majority are aerobic. One distinctive feature of this group is the presence of several different peptidoglycans in
the cell wall.
The genus Actinomyces is a much studied representative of Actinobacteria. Actinomyces spp. play an important role in soil ecology,
and some species are human pathogens. A number of Actinomyces spp. inhabit the human mouth and are opportunistic pathogens,
causing infectious diseases like periodontitis (inflammation of the gums) and oral abscesses. The species A. israelii is an anaerobe
notorious for causing endocarditis (inflammation of the inner lining of the heart).The genus Mycobacterium is represented by
bacilli covered with a mycolic acid coat. This waxy coat protects the bacteria from some antibiotics, prevents them from drying
out, and blocks penetration by Gram stain reagents. Because of this, a special acid-fast staining procedure is used to visualize these
bacteria. The genus Mycobacterium is an important cause of a diverse group of infectious diseases. M. tuberculosis is the causative
agent of tuberculosis, a disease that primarily impacts the lungs but can infect other parts of the body as well. It has been estimated
that one-third of the world’s population has been infected with M. tuberculosis and millions of new infections occur each year.
Treatment of M. tuberculosis is challenging and requires patients to take a combination of drugs for an extended time.
Complicating treatment even further is the development and spread of multidrug-resistant strains of this pathogen. Another
pathogenic species, M. leprae, is the cause of Hansen’s disease (leprosy), a chronic disease that impacts peripheral nerves and the
integrity of the skin and mucosal surface of the respiratory tract. Loss of pain sensation and the presence of skin lesions increase
susceptibility to secondary injuries and infections with other pathogens.

Low G+C Gram-positive Bacteria Groups

The low G+C gram-positive bacteria have less than 50% guanine and cytosine in their DNA, and this group of bacteria includes a
number of genera of bacteria that are pathogenic.

Clostridia
One large and diverse class of low G+C gram-positive bacteria is Clostridia. The best studied genus of this class is Clostridium.
These rod-shaped bacteria are generally obligate anaerobes that produce endospores and can be found in anaerobic habitats like soil
and aquatic sediments rich in organic nutrients. The endospores may survive for many years.
Clostridium spp. produce more kinds of protein toxins than any other bacterial genus, and several species are human pathogens. C.
perfringens is the third most common cause of food poisoning in the United States and is the causative agent of an even more
serious disease called gas gangrene. Gas gangrene occurs when C. perfringens endospores enter a wound and germinate, becoming
viable bacterial cells and producing a toxin that can cause the necrosis (death) of tissue. C. tetani, which causes tetanus, produces a
neurotoxin that is able to enter neurons, travel to regions of the central nervous system where it blocks the inhibition of nerve
impulses involved in muscle contractions, and cause a life-threatening spastic paralysis. C. botulinum produces botulinum
neurotoxin, the most lethal biological toxin known. Botulinum toxin is responsible for rare but frequently fatal cases of botulism.
The toxin blocks the release of acetylcholine in neuromuscular junctions, causing flaccid paralysis. In very small concentrations,
botulinum toxin has been used to treat muscle pathologies in humans and in a cosmetic procedure to eliminate wrinkles. C. difficile
is a common source of hospital-acquired infections (Figure 4.2.10) that can result in serious and even fatal cases of colitis
(inflammation of the large intestine). Infections often occur in patients who are immunosuppressed or undergoing antibiotic therapy
that alters the normal microbiota of the gastrointestinal tract.

Figure 4.2.10 : Clostridium difficile, a gram-positive, rod-shaped bacterium, causes severe colitis and diarrhea, often after the
normal gut microbiota is eradicated by antibiotics. (credit: modification of work by Centers for Disease Control and Prevention)

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Lactobacillales
The order Lactobacillales comprises low G+C gram-positive bacteria that include both bacilli and cocci in the genera
Lactobacillus, Leuconostoc, Enterococcus, and Streptococcus. Bacteria of the latter three genera typically are spherical or ovoid
and often form chains.
Streptococcus, the name of which comes from the Greek word for twisted chain, is responsible for many types of infectious
diseases in humans. Species from this genus, often referred to as streptococci, are usually classified by serotypes called Lancefield
groups, and by their ability to lyse red blood cells when grown on blood agar.
S. pyogenes belongs to the Lancefield group A, β-hemolytic Streptococcus. This species is considered a pyogenic pathogen because
of the associated pus production observed with infections it causes (Figure 4.2.11). S. pyogenes is the most common cause of
bacterial pharyngitis (strep throat); it is also an important cause of various skin infections that can be relatively mild (e.g.,
impetigo) or life threatening (e.g., necrotizing fasciitis, also known as flesh eating disease), life threatening.

Figure 4.2.11 : (a) A gram-stained specimen of Streptococcus pyogenes shows the chains of cocci characteristic of this organism’s
morphology. (b) S. pyogenes on blood agar shows characteristic lysis of red blood cells, indicated by the halo of clearing around
colonies. (credit a, b: modification of work by American Society for Microbiology)

Bacilli
The name of the class Bacilli suggests that it is made up of bacteria that are bacillus in shape, but it is a morphologically diverse
class that includes bacillus-shaped and cocccus-shaped genera. Among the many genera in this class are two that are very important
clinically: Bacillus and Staphylococcus. Bacteria in the genus Bacillus are bacillus in shape and can produce endospores. They
include aerobes or facultative anaerobes. A number of Bacillus spp. are used in various industries, including the production of
antibiotics (e.g., barnase), enzymes (e.g., alpha-amylase, BamH1 restriction endonuclease), and detergents (e.g., subtilisin).
Two notable pathogens belong to the genus Bacillus. B. anthracis is the pathogen that causes anthrax, a severe disease that affects
wild and domesticated animals and can spread from infected animals to humans. Anthrax manifests in humans as charcoal-black
ulcers on the skin, severe enterocolitis, pneumonia, and brain damage due to swelling. If untreated, anthrax is lethal. B. cereus, a
closely related species, is a pathogen that may cause food poisoning. It is a rod-shaped species that forms chains. Colonies appear
milky white with irregular shapes when cultured on blood agar (Figure 4.2.12). One other important species is B. thuringiensis.
This bacterium produces a number of substances used as insecticides because they are toxic for insects.
The genus Staphylococcus also belongs to the class Bacilli, even though its shape is coccus rather than a bacillus. The name
Staphylococcus comes from a Greek word for bunches of grapes, which describes their microscopic appearance in culture.
Staphylococcus spp. are facultative anaerobic, halophilic, and nonmotile. The two best-studied species of this genus are S.
epidermidis and S. aureus.
S. epidermidis, whose main habitat is the human skin, is thought to be nonpathogenic for humans with healthy immune systems,
but in patients with immunodeficiency, it may cause infections in skin wounds and prostheses (e.g., artificial joints, heart valves). S.
epidermidis is also an important cause of infections associated with intravenous catheters. This makes it a dangerous pathogen in
hospital settings, where many patients may be immunocompromised.

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 Figure 4.2.12 : (a) In this gram-stained specimen, the violet rod-shaped cells forming chains are the gram-positive bacteria Bacillus
cereus. The small, pink cells are the gram-negative bacteria Escherichia coli. (b) In this culture, white colonies of B. cereus have
been grown on sheep blood agar. (credit a: modification of work by “Bibliomaniac 15”/Wikimedia Commons; credit b:
modification of work by Centers for Disease Control and Prevention)

Strains of S. aureus cause a wide variety of infections in humans, including skin infections that produce boils, carbuncles, cellulitis,
or impetigo. Certain strains of S. aureus produce a substance called enterotoxin, which can cause severe enteritis, often called staph
food poisoning. Some strains of S. aureus produce the toxin responsible for toxic shock syndrome, which can result in
cardiovascular collapse and death.

Mycoplasmas
Although Mycoplasma spp. do not possess a cell wall and, therefore, are not stained by Gram-stain reagents, this genus is still
included with the low G+C gram-positive bacteria. The genus Mycoplasma includes more than 100 species, which share several
unique characteristics. They are very small cells, some with a diameter of about 0.2 μm, which is smaller than some large viruses.
They have no cell walls and, therefore, are pleomorphic, meaning that they may take on a variety of shapes and can even resemble
very small animal cells. Because they lack a characteristic shape, they can be difficult to identify. One species, M. pneumoniae,
causes the mild form of pneumonia known as “walking pneumonia” or “atypical pneumonia.” This form of pneumonia is typically
less severe than forms caused by other bacteria or viruses.

Clinical Focus - Resolution

Marsha’s sputum sample was sent to the microbiology lab to confirm the identity of the microorganism causing her infection.
The lab also performed antimicrobial susceptibility testing (AST) on the sample to confirm that the physician has prescribed
the correct antimicrobial drugs.
Direct microscopic examination of the sputum revealed acid-fast bacteria (AFB) present in Marsha’s sputum. When placed in
culture, there were no signs of growth for the first 8 days, suggesting that microorganism was either dead or growing very
slowly. Slow growth is a distinctive characteristic of M. tuberculosis.
After four weeks, the lab microbiologist observed distinctive colorless granulated colonies (Figure 4.2.13). The colonies
contained AFB showing the same microscopic characteristics as those revealed during the direct microscopic examination of
Marsha’s sputum. To confirm the identification of the AFB, samples of the colonies were analyzed using nucleic acid
hybridization, or direct nucleic acid amplification (NAA) testing. When a bacterium is acid-fast, it is classified in the family
Mycobacteriaceae. DNA sequencing of variable genomic regions of the DNA extracted from these bacteria revealed that it was
high G+C. This fact served to finalize Marsha’s diagnosis as infection with M. tuberculosis. After nine months of treatment
with the drugs prescribed by her doctor, Marsha made a full recovery.

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 Figure 4.2.13 : M. tuberculosis grows on Löwenstein-Jensen (LJ) agar in distinct colonies. (credit: Centers for Disease Control
and Prevention)

Biopiracy and Bioprospecting

In 1969, an employee of a Swiss pharmaceutical company was vacationing in Norway and decided to collect some soil
samples. He took them back to his lab, and the Swiss company subsequently used the fungus Tolypocladium inflatum in those
samples to develop cyclosporine A, a drug widely used in patients who undergo tissue or organ transplantation. The Swiss
company earns more than $1 billion a year for production of cyclosporine A, yet Norway receives nothing in return—no
payment to the government or benefit for the Norwegian people. Despite the fact the cyclosporine A saves numerous lives,
many consider the means by which the soil samples were obtained to be an act of “biopiracy,” essentially a form of theft. Do
the ends justify the means in a case like this?
Nature is full of as-yet-undiscovered bacteria and other microorganisms that could one day be used to develop new life-saving
drugs or treatments.5 Pharmaceutical and biotechnology companies stand to reap huge profits from such discoveries, but
ethical questions remain. To whom do biological resources belong? Should companies who invest (and risk) millions of dollars
in research and development be required to share revenue or royalties for the right to access biological resources?
Compensation is not the only issue when it comes to bioprospecting. Some communities and cultures are philosophically
opposed to bioprospecting, fearing unforeseen consequences of collecting genetic or biological material. Native Hawaiians, for
example, are very protective of their unique biological resources.
For many years, it was unclear what rights government agencies, private corporations, and citizens had when it came to
collecting samples of microorganisms from public land. Then, in 1993, the Convention on Biological Diversity granted each
nation the rights to any genetic and biological material found on their own land. Scientists can no longer collect samples
without a prior arrangement with the land owner for compensation. This convention now ensures that companies act ethically
in obtaining the samples they use to create their products.

Deeply Branching Bacteria

On a phylogenetic tree, the trunk or root of the tree represents a common ancient evolutionary ancestor, often called the last
universal common ancestor (LUCA), and the branches are its evolutionary descendants. Scientists consider the deeply branching
bacteria, such as the genus Acetothermus, to be the first of these non-LUCA forms of life produced by evolution some 3.5 billion
years ago. When placed on the phylogenetic tree, they stem from the common root of life, deep and close to the LUCA root—
hence the name “deeply branching.” (see chapter 1, section 4 for review)
The deeply branching bacteria may provide clues regarding the structure and function of ancient and now extinct forms of life. We
can hypothesize that ancient bacteria, like the deeply branching bacteria that still exist, were thermophiles or hyperthermophiles,
meaning that they thrived at very high temperatures. Acetothermus paucivorans, a gram-negative anaerobic bacterium discovered
in 1988 in sewage sludge, is a thermophile growing at an optimal temperature of 58 °C.6 Scientists have determined it to be the
deepest branching bacterium, or the closest evolutionary relative of the LUCA.

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The class Thermotogae is represented mostly by hyperthermophilic, as well as some mesophilic (preferring moderate
temperatures), anaerobic gram-negative bacteria whose cells are wrapped in a peculiar sheath-like outer membrane called a toga.
The thin layer of peptidoglycan in their cell wall has an unusual structure; it contains diaminopimelic acid and D-lysine. These
bacteria are able to use a variety of organic substrates and produce molecular hydrogen, which can be used in industry. The class
contains several genera, of which the best known is the genus Thermotoga. One species of this genus, T. maritima, lives near the
thermal ocean vents and thrives in temperatures of 90 °C; another species, T. subterranea, lives in underground oil reservoirs.
Finally, the deeply branching bacterium Deinococcus radiodurans belongs to a genus whose name is derived from a Greek word
meaning terrible berry. Nicknamed “Conan the Bacterium,” D. radiodurans is considered a polyextremophile because of its ability
to survive under the many different kinds of extreme conditions—extreme heat, drought, vacuum, acidity, and radiation. It owes its
name to its ability to withstand doses of ionizing radiation that kill all other known bacteria; this special ability is attributed to some
unique mechanisms of DNA repair.

Figure 4.2.14 : Deinococcus radiodurans, or “Conan the Bacterium,” survives in the harshest conditions on earth.

Archaea
Like organisms in the domain Bacteria, organisms of the domain Archaea are all unicellular organisms. However, archaea differ
structurally from bacteria in several significant ways. To summarize:
The archaeal cell membrane is composed of ether linkages with branched isoprene chains (as opposed to the bacterial cell
membrane, which has ester linkages with unbranched fatty acids).
Archaeal cell walls lack peptidoglycan, but some contain a structurally similar substance called pseudopeptidoglycan or
pseudomurein.
The genomes of Archaea are larger and more complex than those of bacteria.
Domain Archaea is as diverse as domain Bacteria, and its representatives can be found in any habitat. Some archaea are
mesophiles, and many are extremophiles, preferring extreme hot or cold, extreme salinity, or other conditions that are hostile to
most other forms of life on earth. Their metabolism is adapted to the harsh environments, and they can perform methanogenesis,
for example, which bacteria and eukaryotes cannot. With few exceptions, archaea are not present in the human microbiota, and
none are currently known to be associated with infectious diseases in humans, animals, plants, or microorganisms. However, many
play important roles in the environment and may thus have an indirect impact on human health.
The size and complexity of the archaeal genome makes it difficult to classify. Most taxonomists agree that within the Archaea,
there are currently five major phyla: Crenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota, and Thaumarchaeota. There are
likely many other archaeal groups that have not yet been systematically studied and classified.

Crenarchaeota
Crenarchaeota is a class of Archaea that is extremely diverse, containing genera and species that differ vastly in their morphology
and requirements for growth. All Crenarchaeota are aquatic organisms, and they are thought to be the most abundant
microorganisms in the oceans. Most, but not all, Crenarchaeota are hyperthermophiles; some of them (notably, the genus
Pyrolobus) are able to grow at temperatures up to 113 °C.7

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Euryarchaeota
The phylum Euryarchaeota includes several distinct classes. Species in the classes Methanobacteria, Methanococci, and
Methanomicrobia represent Archaea that can be generally described as methanogens. Methanogens are unique in that they can
reduce carbon dioxide in the presence of hydrogen, producing methane. They can live in the most extreme environments and can
reproduce at temperatures varying from below freezing to boiling. Methanogens have been found in hot springs as well as deep
under ice in Greenland. Some scientists have even hypothesized that methanogens may inhabit the planet Mars because the mixture
of gases produced by methanogens resembles the makeup of the Martian atmosphere.8
Methanogens are thought to contribute to the formation of anoxic sediments by producing hydrogen sulfide, making “marsh gas.”
They also produce gases in ruminants and humans. Some genera of methanogens, notably Methanosarcina, can grow and produce
methane in the presence of oxygen, although the vast majority are strict anaerobes.
The class Halobacteria (which was named before scientists recognized the distinction between Archaea and Bacteria) includes
halophilic (“salt-loving”) archaea. Halobacteria require a very high concentrations of sodium chloride in their aquatic environment.
The required concentration is close to saturation, at 36%; such environments include the Dead Sea as well as some salty lakes in
Antarctica and south-central Asia. One remarkable feature of these organisms is that they perform photosynthesis using the protein
bacteriorhodopsin, which gives them, and the bodies of water they inhabit, a beautiful purple color (Figure 4.2.15).

Figure 4.2.15 : Halobacteria growing in these salt ponds gives them a distinct purple color. (credit: modification of work by Tony
Hisgett)
Notable species of Halobacteria include Halobacterium salinarum, which may be the oldest living organism on earth; scientists
have isolated its DNA from fossils that are 250 million years old.9 Another species, Haloferax volcanii, shows a very sophisticated
system of ion exchange, which enables it to balance the concentration of salts at high temperatures

Clinical Focus: Resolution

When Marsha finally went to the doctor’s office, the physician listened to her breathing through a stethoscope. He heard some
crepitation (a crackling sound) in her lungs, so he ordered a chest radiograph and asked the nurse to collect a sputum sample
for microbiological evaluation and cytology. The radiologic evaluation found cavities, opacities, and a particular pattern of
distribution of abnormal material (Figure 4.2.16).
Based on her symptoms, Marsha’s doctor suspected that she had a case of tuberculosis. Although less common in the United
States, tuberculosis is still extremely common in many parts of the world, including Nigeria. Marsha’s work there in a medical
lab likely exposed her to Mycobacterium tuberculosis, the bacterium that causes tuberculosis.
Marsha’s doctor ordered her to stay at home, wear a respiratory mask, and confine herself to one room as much as possible. He
also said that Marsha had to take one semester off school. He prescribed isoniazid and rifampin, antibiotics used in a drug
cocktail to treat tuberculosis, which Marsha was to take three times a day for at least three months.

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 Figure 4.2.16 : This anteroposterior radiograph shows the presence of bilateral pulmonary infiltrate (white triangles) and
“caving formation” (black arrows) present in the right apical region. (credit: Centers for Disease Control and Prevention)

Exercise 4.2.1
What are some possible diseases that could be responsible for Marsha’s radiograph results?
Why did the doctor order Marsha to stay home for three months?

Summary
In recent years, the traditional approaches to classification of prokaryotes have been supplemented by approaches based on
molecular genetics.
We separate the Phylum in the Bacterial and Archaeal domains by wall composition (Gram positive, Gram negative or other),
their G+C percentage, how they get their energy, and how they react to oxygen gas.
Proteobacteria is a phylum of gram-negative bacteria discovered by Carl Woese in the 1980s based on nucleotide sequence
homology. Proteobacteria are further classified into the classes alpha-, beta-, gamma-, delta- and epsilonproteobacteria, each
class having separate orders, families, genera, and species.
Non-proteobacteria that are Gram negative include groups like the spirochetes and photosynthetic bacteria.
Actinobacteria are a group of high G+C Gram positive bacteria including Actinomyces and Mycobacterium.
Low G+C Gram positive bacteria include many smaller groups such as Clostridia, Lactobacillales, Bacilli and the highly
variable Mycoplasma.
Deeply branching bacteria are phylogenetically the most ancient forms of life, being the closest to the last universal common
ancestor.
Archaea are unicellular, prokaryotic microorganisms that differ from bacteria in their genetics, biochemistry, and ecology.
Some archaea are extremophiles, living in environments with extremely high or low temperatures, or extreme salinity.
Only archaea are known to produce methane. Methane-producing archaea are called methanogens.

Footnotes
1. C.R. Woese. “Bacterial Evolution.” Microbiological Review 51 no. 2 (1987):221–271.
2. H. Reichenbach. “Myxobacteria, Producers of Novel Bioactive Substances.” Journal of Industrial Microbiology &
Biotechnology 27 no. 3 (2001):149–156.
3. S. Suerbaum, P. Michetti. “Helicobacter pylori infection.” New England Journal of Medicine 347 no. 15 (2002):1175–1186.
4. A. De los Rios et al. “Ultrastructural and Genetic Characteristics of Endolithic Cyanobacterial Biofilms Colonizing Antarctic
Granite Rocks.” FEMS Microbiology Ecology 59 no. 2 (2007):386–395.
5. J. Andre. Bioethics as Practice. Chapel Hill, NC: University of North Carolina Press, 2002

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 6. G. Dietrich et al. “Acetothermus paucivorans, gen. nov., sp. Nov., a Strictly Anaerobic, Thermophilic Bacterium From Sewage
Sludge, Fermenting Hexoses to Acetate, CO2, and H2.” Systematic and Applied Microbiology 10 no. 2 (1988):174–179.
7. E. Blochl et al.“Pyrolobus fumani, gen. and sp. nov., represents a novel group of Archaea, extending the upper temperature limit
for life to 113°C.” Extremophiles 1 (1997):14–21.
8. R.R. Britt “Crater Critters: Where Mars Microbes Might Lurk.” www.space.com/1880-crater-cri...obes-lurk.html. Accessed
April 7, 2015.
9. H. Vreeland et al. “Fatty acid and DA Analyses of Permian Bacterium Isolated From Ancient Salt Crystals Reveal Differences
With Their Modern Relatives.” Extremophiles 10 (2006):71–78.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 4.2: Classifying Prokaryotes and Examples is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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Chapter 4 Exercises
Review Questions for Chapter 4:

Multiple Choice

1) Which of the following terms refers to a prokaryotic cell that is comma shaped?
A. coccus
B. coccobacilli
C. vibrio
D. spirillum

2) Which bacterial structures are important for adherence to surfaces? (Select all that apply.)
A. endospores
B. cell walls
C. fimbriae
D. capsules
E. flagella

3) Which of the following cell wall components is unique to gram-negative cells?

A. lipopolysaccharide
B. teichoic acid
C. mycolic acid
D. peptidoglycan

4) Which of the following terms refers to a bacterial cell having a single tuft of flagella at one end?
A. monotrichous
B. amphitrichous
C. peritrichous
D. lophotrichous

5) Bacterial cell walls are primarily composed of which of the following?

A. phospholipid
B. protein
C. carbohydrate
D. peptidoglycan

6) The term prokaryotes refers to which of the following?

A. very small organisms
B. unicellular organisms that have no nucleus
C. multicellular organisms
D. cells that resemble animal cells more than plant cells

7) The term microbiota refers to which of the following?

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A. all microorganisms of the same species
B. all of the microorganisms involved in a symbiotic relationship
C. all microorganisms in a certain region of the human body
D. all microorganisms in a certain geographic region

8) Which of the following describes Proteobacteria in domain Bacteria?

A. phylum
B. class
C. species
D. genus

9) Which of the following Alphaproteobacteria is the cause of Rocky Mountain spotted fever and typhus?
A. Bartonella
B. Coxiella
C. Rickettsia
D. Ehrlichia
E. Brucella

10) Class Betaproteobacteria includes all but which of the following genera?
A. Neisseria.
B. Bordetella.
C. Leptothrix.
D. Campylobacter.

11) Haemophilus influenzae is a common cause of which of the following?

A. influenza
B. dysentery
C. upper respiratory tract infections
D. hemophilia

12) Which of the following is the organelle that spirochetes use to propel themselves?
A. plasma membrane
B. axial filament
C. pilum
D. fimbria

13) Which of the following bacteria are the most prevalent in the human gut?
A. cyanobacteria
B. staphylococci
C. Borrelia
D. Bacteroides

14) Which of the following refers to photosynthesis performed by bacteria with the use of water as the donor of electrons?
A. oxygenic
B. anoxygenic

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C. heterotrophic
D. phototrophic

15) Which of the following bacterial species is classified as high G+C gram-positive?
A. Corynebacterium diphtheriae
B. Staphylococcus aureus
C. Bacillus anthracis
D. Streptococcus pneumonia

16) The term “deeply branching” refers to which of the following?

A. the cellular shape of deeply branching bacteria
B. the position in the evolutionary tree of deeply branching bacteria
C. the ability of deeply branching bacteria to live in deep ocean waters
D. the pattern of growth in culture of deeply branching bacteria

17) Which of these deeply branching bacteria is considered a polyextremophile?

A. Aquifex pyrophilus
B. Deinococcus radiodurans
C. Staphylococcus aureus
D. Mycobacterium tuberculosis

18) Archaea and Bacteria are most similar in terms of their ________.
A. genetics
B. cell wall structure
C. ecology
D. unicellular structure

19) Which of the following is true of archaea that produce methane?

A. They reduce carbon dioxide in the presence of nitrogen.
B. They live in the most extreme environments.
C. They are always anaerobes.
D. They have been discovered on Mars.

Fill-in-the-Blanks

20) Prokaryotic cells that are rod-shaped are called _____________.

21) The type of inclusion containing polymerized inorganic phosphate is called _____________.

22) The domain ________ does not include prokaryotes.

23) Pathogenic bacteria that are part of the transient microbiota can sometimes be eliminated by ________ therapy.

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24) Nitrogen-fixing bacteria provide other organisms with usable nitrogen in the form of ________.

25) Rickettsias are ________ intracellular bacteria.

26) The species ________, which belongs to Epsilonproteobacteria, causes peptic ulcers of the stomach and duodenum.

27) The genus Salmonella belongs to the class ________ and includes pathogens that cause salmonellosis and typhoid fever.

28) The bacterium that causes syphilis is called ________.

29) Bacteria in the genus Rhodospirillum that use hydrogen for oxidation and fix nitrogen are ________ bacteria.

30) Streptococcus is the ________ of bacteria that is responsible for many human diseases.

31) One species of Streptococcus, S. pyogenes, is a classified as a ________ pathogen due to the characteristic production of pus in
infections it causes.

32) Propionibacterium belongs to ________ G+C gram-positive bacteria. One of its species is used in the food industry and
another causes acne.

33) The length of the branches of the evolutionary tree characterizes the evolutionary ________ between organisms.

34) The deeply branching bacteria are thought to be the form of life closest to the last universal ________ ________.

35) Many of the deeply branching bacteria are aquatic and hyperthermophilic, found near underwater volcanoes and thermal ocean
________.

36) The deeply branching bacterium Deinococcus radiodurans is able to survive exposure to high doses of ________.

37) ________ is a genus of Archaea. Its optimal environmental temperature ranges from 70 °C to 80 °C, and its optimal pH is 2–3.
It oxidizes sulfur and produces sulfuric acid.

38) ________ was once thought to be the cause of periodontal disease, but, more recently, the causal relationship between this
archaean and the disease was not confirmed.

Short Answer

39) How do bacterial flagella respond to a chemical gradient of an attractant to move toward a higher concentration of the
chemical?

40) Label the parts of the prokaryotic cell.

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41) What is the metabolic difference between coliforms and noncoliforms? Which category contains several species of intestinal
pathogens?

42) Why are Mycoplasma and Chlamydia classified as obligate intracellular pathogens?

43) Explain the term CFB group and name the genera that this group includes.

44) Name and briefly describe the bacterium that causes Lyme disease.

46) Characterize the phylum Cyanobacteria.

47) Name and describe two types of S. aureus that show multiple antibiotic resistance.

48) Briefly describe the significance of deeply branching bacteria for basic science and for industry.

49) What is thought to account for the unique radiation resistance of D. radiodurans?

50) What accounts for the purple color in salt ponds inhabited by halophilic archaea?

51) What evidence supports the hypothesis that some archaea live on Mars?

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Critical Thinking

52) Which of the following slides is a good example of staphylococci?

(credit a: modification of work by U.S. Department of Agriculture; credit b: modification of work by Centers for Disease Control
and Prevention; credit c: modification of work by NIAID)

53) Provide some examples of bacterial structures that might be used as antibiotic targets and explain why.

54) The causative agent of botulism, a deadly form of food poisoning, is an endospore-forming bacterium called Clostridium
botulinim. Why might it be difficult to kill this bacterium in contaminated food?

55) The cell shown is found in the human stomach and is now known to cause peptic ulcers. What is the name of this bacterium?

(credit: American Society for Microbiology)

56) The microscopic growth pattern shown is characteristic of which genus of bacteria?

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(credit: modification of work by Janice Haney Carr/Centers for Disease Control and Prevention)

57) What is the connection between this methane bog and archaea?

(credit: Chad Skeers)

Chapter 4 Exercises is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

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 CHAPTER OVERVIEW

5: The Eukaryotes of Microbiology

5.1: Characteristics of Eukaryotic Cells
5.2: Classifying Eukaryotic Microbes and Examples
Chapter 5 Exercises

Thumbnail: Mold growing on a clementine. (CC SA-BY 3.0; NotFromUtrecht).

This page titled 5: The Eukaryotes of Microbiology is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

1
5.1: Characteristics of Eukaryotic Cells
Learning Objectives
Identify and describe structures and organelles unique to eukaryotic cells
Compare and contrast similar structures found in prokaryotic and eukaryotic cells

Eukaryotic organisms include protozoans, algae, fungi, plants, and animals. Some eukaryotic cells are independent, single-celled
microorganisms, whereas others are part of multicellular organisms. The cells of eukaryotic organisms have several distinguishing
characteristics. Above all, eukaryotic cells are defined by the presence of a nucleus surrounded by a complex nuclear membrane.
Also, eukaryotic cells are characterized by the presence of membrane-bound organelles in the cytoplasm. Organelles such as
mitochondria, the endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and peroxisomes are held in place by the cytoskeleton,
an internal network that supports transport of intracellular components and helps maintain cell shape (Figure 5.1.1). The genome of
eukaryotic cells is packaged in multiple, rod-shaped chromosomes as opposed to the single, circular-shaped chromosome that
characterizes most prokaryotic cells. Table 5.1.1 compares the characteristics of eukaryotic cell structures with those of bacteria
and archaea.

Figure 5.1.1 : An illustration of a generalized, single-celled eukaryotic organism. Note that cells of eukaryotic organisms vary
greatly in terms of structure and function, and a particular cell may not have all of the structures shown here.

Table 5.1.1 : Summary of Cell Structures.

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 Prokaryotes
Cell Structure Eukaryotes
Bacteria Archaea

Size ~0.5–1 μM ~0.5–1 μM ~5–20 μM

Surface area-to-volume ratio High High Low

Nucleus No No Yes

Single chromosome Single chromosome Multiple chromosomes

Circular Circular Linear
Genome characteristics
Haploid Haploid Haploid or diploid
Lacks histones Contains histones-like proteins Contains histones

Cell division Binary fission Binary fission Mitosis, meiosis

Ester-linked
Ester-linked Ether-linked
Straight-chain fatty acids
Membrane lipid composition Straight-chain fatty acids Branched isoprenoids
Sterols
Bilayer Bilayer or monolayer
Bilayer

Pseudopeptidoglycan, or Cellulose (plants, some algae)

Glycopeptide, or Chitin (molluscs, insects,
Peptidoglycan, or
Cell wall composition Polysaccharide, or crustaceans, and fungi)
None
Protein (S-layer), or Silica (some algae)
None Most others lack cell walls

Flexible flagella and cilia

Rigid spiral flagella composed of Rigid spiral flagella composed of
Motility structures composed of microtubules,
flagellin archaeal flagellins
membrane-bound

Membrane-bound organelles No No Yes

Endomembrane system No No Yes (ER, Golgi, lysosomes)

80S in cytoplasm and rough

ER
Ribosomes 70S 70S
70S in mitochondria,
chloroplasts

Exercise 5.1.1

Identify two differences between eukaryotic and prokaryotic cell.

Clinical Focus: part 1

Upon arriving home from school, 7-year-old Sarah complains that a large spot on her arm will not stop itching. She keeps
scratching at it, drawing the attention of her parents. Looking more closely, they see that it is a red circular spot with a raised
red edge (Figure 5.1.2).

Figure 5.1.2: Ringworm presents as a raised, red ring on the skin. (credit: Centers for Disease Control and Prevention)
The next day, Sarah’s parents take her to their doctor, who examines the spot using a Wood’s lamp. A Wood’s lamp produces
ultraviolet light that causes the spot on Sarah’s arm to fluoresce, which confirms what the doctor already suspected: Sarah has a

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 case of ringworm. Sarah’s mother is mortified to hear that her daughter has a “worm.” How could this happen?

Exercise 5.1.2

What are some likely ways that Sarah might have contracted ringworm?

Inside the cell:

Nucleus
Unlike prokaryotic cells, in which DNA is loosely contained in the nucleoid region, eukaryotic cells possess a nucleus (plural =
nuclei), which is surrounded by a complex nuclear membrane that houses the DNA genome (Figure 5.1.3). By containing the cell’s
DNA, the nucleus ultimately controls all activities of the cell and also serves an essential role in reproduction and heredity.
Eukaryotic cells typically have their DNA organized into multiple linear chromosomes. The DNA within the nucleus is highly
organized and condensed to fit inside the nucleus, which is accomplished by wrapping the DNA around proteins called histones.

Figure 5.1.3 : Eukaryotic cells have a well-defined nucleus. The nucleus of this mammalian lung cell is the large, dark, oval-shaped
structure in the lower half of the image.
Although most eukaryotic cells have only one nucleus, exceptions exist. For example, protozoans of the genus Paramecium
typically have two complete nuclei: a small nucleus that is used for reproduction (micronucleus) and a large nucleus that directs
cellular metabolism (macronucleus). Additionally, some fungi transiently form cells with two nuclei, called heterokaryotic cells,
during sexual reproduction. Cells whose nuclei divide, but whose cytoplasm does not, are called coenocytes.
The nucleus is bound by a complex nuclear membrane, often called the nuclear envelope, that consists of two distinct lipid bilayers
that are contiguous with each other (Figure 5.1.4). Despite these connections between the inner and outer membranes, each
membrane contains unique lipids and proteins on its inner and outer surfaces. The nuclear envelope contains nuclear pores, which
are large, rosette-shaped protein complexes that control the movement of materials into and out of the nucleus. The overall shape of
the nucleus is determined by the nuclear lamina, a meshwork of intermediate filaments found just inside the nuclear envelope
membranes. Outside the nucleus, additional intermediate filaments form a looser mesh and serve to anchor the nucleus in position
within the cell.

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 Figure 5.1.4 : In this fluorescent microscope image, all the intermediate filaments have been stained with a bright green fluorescent
stain. The nuclear lamina is the intense bright green ring around the faint red nuclei.

Nucleolus
The nucleolus is a dense region within the nucleus where ribosomal RNA (rRNA) biosynthesis occurs. In addition, the nucleolus is
also the site where assembly of ribosomes begins. How does it do this? Some chromosomes have sections of DNA that encode
ribosomal RNA. Pre-ribosomal complexes are assembled from rRNA and proteins into the ribosomal subunits in the nucleolus;
these are then transported out to the cytoplasm, where ribosome assembly is completed (Figure 5.1.5).

Figure 5.1.5 : (a) The nucleolus is the dark, dense area within the nucleus. It is the site of rRNA synthesis and preribosomal
assembly. (b) Electron micrograph showing the nucleolus.

Chromatin and Chromosomes

Between the nucleolus and the nuclear envelope is the chromatin. To understand chromatin, it is helpful to first consider
chromosomes. Chromosomes are structures within the nucleus that are made up of DNA, the hereditary material, and carry
information (genes) that are required. You may remember that in prokaryotes, DNA is organized into a single circular chromosome.
Eukaryotic chromosomes are typically linear, and eukaryotic cells contain multiple distinct chromosomes. Many eukaryotic cells
contain two copies of each chromosome and, therefore, are diploid. The length of a even a single chromosome greatly exceeds the
length of the cell, so a chromosome needs to be packaged into a very small space to fit within the cell. For example, the combined
length of all of the DNA of the human genome would measure approximately 2 meters if completely stretched out, and some
eukaryotic genomes are many times larger than the human genome. Every eukaryotic species has a specific number of

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chromosomes in the nuclei of its body’s cells. For example, in humans, the chromosome number is 46, while in fruit flies, it is
eight. Chromosomes are only visible and distinguishable from one another when the cell is getting ready to divide. When the cell is
in the growth and maintenance phases of its life cycle, proteins are attached to chromosomes, and they resemble an unwound,
jumbled bunch of threads. These unwound protein-chromosome complexes are called chromatin (Figure 5.1.6); chromatin
describes the material that makes up the chromosomes both when condensed and decondensed.

Figure 5.1.6: (a) This image shows various levels of the organization of chromatin (DNA and protein). (b) This image shows
paired chromosomes. (credit b: modification of work by NIH; scale-bar data from Matt Russell)

Ribosomes
Cytoplasmic ribosomes in eukaryotic cells are 80S ribosomes, composed of a 40S small subunit and a 60S large subunit. In terms
of size and composition, this makes them distinct from the ribosomes of prokaryotic cells. The two types of cytoplasmic eukaryotic
ribosomes are defined by their location in the cell: free ribosomes and membrane-bound ribosomes. Free ribosomes are found
floating in the cytoplasm and serve to synthesize water-soluble proteins; membrane-bound ribosomes are found attached to the
cytoplasmic side of the rough endoplasmic reticulum and make proteins for insertion into the cell membrane or proteins destined
for export from the cell.
In contrast, ribosomes found in eukaryotic organelles such as mitochondria or chloroplasts have 70S ribosomes—the same size as
prokaryotic ribosomes. The differences between eukaryotic and prokaryotic ribosomes are clinically relevant because certain
antibiotic drugs are designed to target one or the other. For example, cycloheximide targets eukaryotic action, whereas
chloramphenicol targets prokaryotic ribosomes.1 Since human cells are eukaryotic, they generally are not harmed by antibiotics
that destroy the prokaryotic ribosomes in bacteria. However, sometimes negative side effects may occur because mitochondria in
human cells contain prokaryotic ribosomes.

Endomembrane System
The endomembrane system, unique to eukaryotic cells, is a series of membranous tubules, sacs, and flattened disks that synthesize
many cell components and move materials around within the cell (Figure 5.1.7). Because of their larger cell size, eukaryotic cells
require this system to transport materials that cannot be dispersed by diffusion alone. The endomembrane system comprises several
organelles and connections between them, including the endoplasmic reticulum, Golgi apparatus, lysosomes, and vesicles.

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 Figure 5.1.7 : The endomembrane system is composed of a series of membranous intracellular structures that facilitate movement
of materials throughout the cell and to the cell membrane.

Endoplasmic Reticulum
The endoplasmic reticulum (ER) is an interconnected array of tubules and cisternae (flattened sacs) with a single lipid bilayer
(Figure 5.1.7). The spaces inside of the cisternae are called lumen of the ER. There are two types of ER, rough endoplasmic
reticulum (RER) and smooth endoplasmic reticulum (SER). These two different types of ER are sites for the synthesis of distinctly
different types of molecules. RER is studded with ribosomes bound on the cytoplasmic side of the membrane. These ribosomes
make proteins destined for the plasma membrane (Figure 5.1.8). Following synthesis, these proteins are inserted into the membrane
of the RER. Small sacs of the RER containing these newly synthesized proteins then bud off and move either to the Golgi
apparatus for further processing, directly to the plasma membrane, to the membrane of another organelle, or out of the cell. SER
does not have ribosomes and, therefore, appears “smooth.” It is involved in biosynthesis of lipids, carbohydrate synthesis and
degradation, and detoxification of medications and poisons; and storage of calcium ions.

Figure 5.1.8 : The rough endoplasmic reticulum is studded with ribosomes for the synthesis of membrane proteins (which give it its
rough appearance).

Golgi Apparatus
The Golgi apparatus was discovered within the endomembrane system in 1898 by Italian scientist Camillo Golgi (1843–1926),
who developed a novel staining technique that showed stacked membrane structures within the cells of Plasmodium, the causative
agent of malaria. The Golgi apparatus is composed of a series of membranous disks called dictyosomes, each having a single lipid
bilayer, that are stacked together (Figure 5.1.9).
Modification, sorting, tagging, packaging, and distribution of lipids and proteins takes place in the Golgi apparatus. Enzymes in the
Golgi apparatus modify lipids and proteins transported from the ER to the Golgi, often adding carbohydrate components to them,
producing glycolipids, glycoproteins, or proteoglycans. Glycolipids and glycoproteins are often inserted into the plasma membrane

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and are important for signal recognition by other cells or infectious particles. Different types of cells can be distinguished from one
another by the structure and arrangement of the glycolipids and glycoproteins contained in their plasma membranes. These
glycolipids and glycoproteins commonly also serve as cell surface receptors.

Vesicles and Vacuoles

Vesicles and vacuoles are single-lipid, bilayer, membranous spheres with hollow interiors that function in storage and transport.
Other than the fact that vacuoles are somewhat larger than vesicles, there is a very subtle distinction between them: The membranes
of vesicles can fuse with either the plasma membrane or other membrane systems within the cell. Additionally, some agents such as
enzymes within plant vacuoles break down macromolecules. The membrane of a vacuole does not fuse with the membranes of
other cellular components making them great storage units.
Transport vesicles leaving the ER with proteins, carbohydrates and other goods or waste fuse with a Golgi apparatus on its
receiving, or cis, face. The proteins are processed within the Golgi apparatus, and then additional transport vesicles containing the
modified proteins and lipids pinch off from the Golgi apparatus on its outgoing, or trans, face. These outgoing vesicles move to and
fuse with the plasma membrane (through exocytosis) or the membrane of other organelles (Figure 5.1.9).

Figure 5.1.9 : A transmission electron micrograph (left) of a Golgi apparatus in a white blood cell. The illustration (right) shows the
cup-shaped, stacked disks and several transport vesicles. The Golgi apparatus modifies lipids and proteins, producing glycolipids
and glycoproteins, respectively, which are commonly inserted into the plasma membrane.

Lysosomes
In the 1960s, Belgian scientist Christian de Duve (1917–2013) discovered lysosomes, membrane-bound organelles of the
endomembrane system that contain digestive enzymes. Certain types of eukaryotic cells use lysosomes to break down various
particles, such as food, damaged organelles or cellular debris, microorganisms, or immune complexes. Compartmentalization of the
digestive enzymes within the lysosome allows the cell to efficiently digest matter without harming the cytoplasmic components of
the cell.

Exercise 5.1.3

Name the components of the endomembrane system and describe the function of each component.

Peroxisomes
Christian de Duve is also credited with the discovery of peroxisomes, membrane-bound organelles that are not part of the
endomembrane system (Figure 5.1.10). Peroxisomes form independently in the cytoplasm from the synthesis of peroxin proteins
by free ribosomes and the incorporation of these peroxin proteins into existing peroxisomes. Growing peroxisomes then divide by a
process similar to binary fission.
Peroxisomes were first named for their ability to produce hydrogen peroxide, a highly reactive molecule that helps to break down
molecules such as uric acid, amino acids, and fatty acids. Peroxisomes also possess the enzyme catalase, which can degrade

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hydrogen peroxide. Along with the SER, peroxisomes also play a role in lipid biosynthesis. Like lysosomes, the
compartmentalization of these degradative molecules within an organelle helps protect the cytoplasmic contents from unwanted
damage.
The peroxisomes of certain organisms are specialized to meet their particular functional needs. For example, glyoxysomes are
modified peroxisomes of yeasts and plant cells that perform several metabolic functions, including the production of sugar
molecules. Similarly, glycosomes are modified peroxisomes made by certain trypanosomes, the pathogenic protozoans that cause
Chagas disease and African sleeping sickness.

Figure 5.1.10 : A transmission electron micrograph (left) of a cell containing a peroxisome. The illustration (right) shows the
location of peroxisomes in a cell. These eukaryotic structures play a role in lipid biosynthesis and breaking down various
molecules. They may also have other specialized functions depending on the cell type. (credit “micrograph”: modification of work
by American Society for Microbiology).

Cytoskeleton
Eukaryotic cells have an internal cytoskeleton made of microfilaments, intermediate filaments, and microtubules. This matrix of
fibers and tubes provides structural support as well as a network over which materials can be transported within the cell and on
which organelles can be anchored (Figure 5.1.11). For example, the process of exocytosis involves the movement of a vesicle via
the cytoskeletal network to the plasma membrane, where it can release its contents.

Figure 5.1.11 : The cytoskeleton is a network of microtubules, microfilaments, and intermediate filaments found throughout the
cytoplasm of a eukaryotic cell. In these fluorescently labeled animal cells, the microtubules are green, the actin microfilaments are
red, the nucleus is blue, and keratin (a type of intermediate filament) is yellow.

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Microfilaments are composed of two intertwined strands of actin, each composed of actin monomers forming filamentous cables 6
nm in diameter2 (Figure 5.1.11). The actin filaments work together with motor proteins, like myosin, to effect muscle contraction
in animals or the amoeboid movement of some eukaryotic microbes. In ameboid organisms, actin can be found in two forms: a
stiffer, polymerized, gel form and a more fluid, unpolymerized soluble form. Actin in the gel form creates stability in the
ectoplasm, the gel-like area of cytoplasm just inside the plasma membrane of ameboid protozoans. Temporary extensions of the
cytoplasmic membrane called pseudopodia (meaning “false feet”) are produced through the forward flow of soluble actin filaments
into the pseudopodia, followed by the gel-sol cycling of the actin filaments, resulting in cell motility. Once the cytoplasm extends
outward, forming a pseudopodium, the remaining cytoplasm flows up to join the leading edge, thereby creating forward
locomotion. Beyond amoeboid movement, microfilaments are also involved in a variety of other processes in eukaryotic cells,
including cytoplasmic streaming (the movement or circulation of cytoplasm within the cell), cleavage furrow formation during cell
division, and muscle movement in animals (Figure 5.1.12). These functions are the result of the dynamic nature of microfilaments,
which can polymerize and depolymerize relatively easily in response to cellular signals, and their interactions with molecular
motors in different types of eukaryotic cells.

Figure 5.1.12 : (a) A microfilament is composed of a pair of actin filaments. (b) Each actin filament is a string of polymerized actin
monomers. (c) The dynamic nature of actin, due to its polymerization and depolymerization and its association with myosin, allows
microfilaments to be involved in a variety of cellular processes, including ameboid movement, cytoplasmic streaming, contractile
ring formation during cell division, and muscle contraction in animals.
Intermediate filaments (Figure 5.1.13) are a diverse group of cytoskeletal filaments that act as cables within the cell. They are
termed “intermediate” because their 10-nm diameter is thicker than that of actin but thinner than that of microtubules.3 They are
composed of several strands of polymerized subunits that, in turn, are made up of a wide variety of monomers. Intermediate
filaments tend to be more permanent in the cell and maintain the position of the nucleus. They also form the nuclear lamina (lining
or layer) just inside the nuclear envelope. Additionally, intermediate filaments play a role in anchoring cells together in animal
tissues. The intermediate filament protein desmin is found in desmosomes, the protein structures that join muscle cells together and

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help them resist external physical forces. The intermediate filament protein keratin is a structural protein found in hair, skin, and
nails.

Figure 5.1.13 : (a) Intermediate filaments are composed of multiple strands of polymerized subunits. They are more permanent than
other cytoskeletal structures and serve a variety of functions. (b) Intermediate filaments form much of the nuclear lamina. (c)
Intermediate filaments form the desmosomes between cells in some animal tissues. (credit c “illustration”: modification of work by
Mariana Ruiz Villareal)

Microtubules (Figure 5.1.14) are a third type of cytoskeletal fiber composed of tubulin dimers (α tubulin and β tubulin). These
form hollow tubes 23 nm in diameter that are used as girders within the cytoskeleton.4 Like microfilaments, microtubules are
dynamic and have the ability to rapidly assemble and disassemble. Microtubules also work with motor proteins (such as dynein and
kinesin) to move organelles and vesicles around within the cytoplasm. Additionally, microtubules are the main components of
eukaryotic flagella and cilia, composing both the filament and the basal body components (Figure 5.1.21). Microtubules are also
involved in cell division, forming the mitotic spindle that serves to separate chromosomes during mitosis and meiosis. The mitotic
spindle is produced by two centrosomes, which are essentially microtubule-organizing centers, at opposite ends of the cell.

Figure 5.1.14 : (a) Microtubules are hollow structures composed of polymerized tubulin dimers. (b) They are involved in several
cellular processes, including the movement of organelles throughout the cytoplasm. Motor proteins carry organelles along
microtubule tracks that crisscross the entire cell. (credit b: modification of work by National Institute on Aging)

Exercise 5.1.4

Compare and contrast the three types of cytoskeletal structures described in this section.

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Endosymbiotic Theory
As scientists were making progress toward understanding the role of cells in plant and animal tissues, others were examining the
structures within the cells themselves. In 1831, Scottish botanist Robert Brown (1773–1858) was the first to describe observations
of nuclei, which he observed in plant cells. Then, in the early 1880s, German botanist Andreas Schimper (1856–1901) was the first
to describe the chloroplasts of plant cells, identifying their role in starch formation during photosynthesis and noting that they
divided independent of the nucleus.
Based upon the chloroplasts’ ability to reproduce independently, Russian botanist Konstantin Mereschkowski (1855–1921)
suggested in 1905 that chloroplasts may have originated from ancestral photosynthetic bacteria living symbiotically inside a
eukaryotic cell. He proposed a similar origin for the nucleus of plant cells. This was the first articulation of the endosymbiotic
hypothesis, and would explain how eukaryotic cells evolved from ancestral bacteria.
Mereschkowski’s endosymbiotic hypothesis was furthered by American anatomist Ivan Wallin (1883–1969), who began to
experimentally examine the similarities between mitochondria, chloroplasts, and bacteria—in other words, to put the
endosymbiotic hypothesis to the test using objective investigation. Wallin published a series of papers in the 1920s supporting the
endosymbiotic hypothesis, including a 1926 publication co-authored with Mereschkowski. Wallin claimed he could culture
mitochondria outside of their eukaryotic host cells. Many scientists dismissed his cultures of mitochondria as resulting from
bacterial contamination. Modern genome sequencing work supports the dissenting scientists by showing that much of the genome
of mitochondria had been transferred to the host cell’s nucleus, preventing the mitochondria from being able to live on their own.6 7
Wallin’s ideas regarding the endosymbiotic hypothesis were largely ignored for the next 50 years because scientists were unaware
that these organelles contained their own DNA. However, with the discovery of mitochondrial and chloroplast DNA in the 1960s,
the endosymbiotic hypothesis was resurrected. Lynn Margulis (1938–2011), an American geneticist, published her ideas regarding
the endosymbiotic hypothesis of the origins of mitochondria and chloroplasts in 1967.8 In the decade leading up to her publication,
advances in microscopy had allowed scientists to differentiate prokaryotic cells from eukaryotic cells. In her publication, Margulis
reviewed the literature and argued that the eukaryotic organelles such as mitochondria and chloroplasts are of prokaryotic origin.
She presented a growing body of microscopic, genetic, molecular biology, fossil, and geological data to support her claims.

Figure 5.1.15 : According to the endosymbiotic theory, mitochondria and chloroplasts are each derived from the uptake of bacteria.
These bacteria established a symbiotic relationship with their host cell that eventually led to the bacteria evolving into mitochondria
and chloroplasts.
Again, this hypothesis was not initially popular, but mounting genetic evidence due to the advent of DNA sequencing supported the
endosymbiotic theory, which is now defined as the theory that mitochondria and chloroplasts arose as a result of prokaryotic cells
establishing a symbiotic relationship within a eukaryotic host (Figure 5.1.15). With Margulis’ initial endosymbiotic theory gaining

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wide acceptance, she expanded on the theory in her 1981 book Symbiosis in Cell Evolution. In it, she explains how endosymbiosis
is a major driving factor in the evolution of organisms. More recent genetic sequencing and phylogenetic analysis show that
mitochondrial DNA and chloroplast DNA are highly related to their bacterial counterparts, both in DNA sequence and
chromosome structure. However, mitochondrial DNA and chloroplast DNA are reduced compared with nuclear DNA because
many of the genes have moved from the organelles into the host cell’s nucleus. Additionally, mitochondrial and chloroplast
ribosomes are structurally similar to bacterial ribosomes, rather than to the eukaryotic ribosomes of their hosts. Last, the binary
fission of these organelles strongly resembles the binary fission of bacteria, as compared with mitosis performed by eukaryotic
cells. Since Margulis’ original proposal, scientists have observed several examples of bacterial endosymbionts in modern-day
eukaryotic cells. Examples include the endosymbiotic bacteria found within the guts of certain insects, such as cockroaches,9 and
photosynthetic bacteria-like organelles found in protists.10

Exercise 5.1.5

1. What does the modern endosymbiotic theory state?

2. What evidence supports the endosymbiotic theory?

Mitochondria
Mitochondria (singular = mitochondrion) are often called the “powerhouses” or “energy factories” of a cell because they are
responsible for making adenosine triphosphate (ATP), the cell’s main energy-carrying molecule. ATP represents the short-term
stored energy of the cell. Cellular respiration is the process of making ATP using the chemical energy found in glucose and other
nutrients. In mitochondria, this process uses oxygen and produces carbon dioxide as a waste product. In fact, the carbon dioxide
that you exhale with every breath comes from the cellular reactions that produce carbon dioxide as a byproduct (Figure 5.1.16).
The term “mitochondrion” was first coined by German microbiologist Carl Benda in 1898 and was later connected with the process
of respiration by Otto Warburg in 1913.
Each mitochondrion has two lipid membranes. The outer membrane is a remnant of the original host cell’s membrane structures.
The inner membrane was derived from the bacterial plasma membrane. The electron transport chain for aerobic respiration uses
integral proteins embedded in the inner membrane. The mitochondrial matrix, corresponding to the location of the original
bacterium’s cytoplasm, is the current location of many metabolic enzymes. It also contains mitochondrial DNA and 70S ribosomes.
Invaginations of the inner membrane, called cristae, evolved to increase surface area for the location of biochemical reactions. The
folding patterns of the cristae differ among various types of eukaryotic cells and are used to distinguish different eukaryotic
organisms from each other.

Figure 5.1.16: Each mitochondrion is surrounded by two membranes, the inner of which is extensively folded into cristae and is the
site of the intermembrane space. The mitochondrial matrix contains the mitochondrial DNA, ribosomes, and metabolic enzymes.
The transmission electron micrograph of a mitochondrion, on the right, shows both membranes, including cristae and the
mitochondrial matrix. (credit “micrograph”: modification of work by Matthew Britton; scale-bar data from Matt Russell)

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Chloroplasts
Plant cells and algal cells contain chloroplasts, the organelles in which photosynthesis occurs (Figure 5.1.17). Photosynthesis is the
series of reactions that use carbon dioxide, water, and light energy to make glucose and oxygen. All chloroplasts have at least three
membrane systems: the outer membrane, the inner membrane, and the thylakoid membrane system. Inside the outer and inner
membranes is the chloroplast stroma, a gel-like fluid that makes up much of a chloroplast’s volume, and in which the thylakoid
system floats. The thylakoid system is a highly dynamic collection of folded membrane sacs. It is where the green photosynthetic
pigment chlorophyll is found and the light reactions of photosynthesis occur. In most plant chloroplasts, the thylakoids are arranged
in stacks called grana (singular: granum), whereas in some algal chloroplasts, the thylakoids are free floating.

Figure 5.1.17: Photosynthesis takes place in chloroplasts, which have an outer membrane and an inner membrane. Stacks of
thylakoids called grana form a third membrane layer.
Other organelles similar to mitochondria have arisen in other types of eukaryotes, but their roles differ. Hydrogenosomes are found
in some anaerobic eukaryotes and serve as the location of anaerobic hydrogen production. Hydrogenosomes typically lack their
own DNA and ribosomes. Kinetoplasts are a variation of the mitochondria found in some eukaryotic pathogens. In these
organisms, each cell has a single, long, branched mitochondrion in which kinetoplast DNA, organized as multiple circular pieces of
DNA, is found concentrated at one pole of the cell.

Mitochondria-related Organelles in Protozoan Parasites

Many protozoans, including several protozoan parasites that cause infections in humans, can be identified by their unusual
appearance. Distinguishing features may include complex cell morphologies, the presence of unique organelles, or the absence
of common organelles. The protozoan parasites Giardia lamblia and Trichomonas vaginalis are two examples.
G. lamblia, a frequent cause of diarrhea in humans and many other animals, is an anaerobic parasite that possesses two nuclei
and several flagella. Its Golgi apparatus and endoplasmic reticulum are greatly reduced, and it lacks mitochondria completely.
However, it does have organelles known as mitosomes, double-membrane-bound organelles that appear to be severely reduced
mitochondria. This has led scientists to believe that G. lamblia’s ancestors once possessed mitochondria that evolved to
become mitosomes. T. vaginalis, which causes the sexually transmitted infection vaginitis, is another protozoan parasite that
lacks conventional mitochondria. Instead, it possesses hydrogenosomes, mitochondrial-related, double-membrane-bound
organelles that produce molecular hydrogen used in cellular metabolism. Scientists believe that hydrogenosomes, like
mitosomes, also evolved from mitochondria.5

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The Boundary Layer: the Cellular Envelope
Plasma Membrane
The plasma membrane structure of most bacterial and eukaryotic cell types is a bilayer composed mainly of phospholipids formed
with ester linkages and proteins. Proteins on the cell’s surface are important for a variety of functions. (review chapter 3 section 3
for more detail, will be described more in chapter 8) Unlike the prokaryotic membrane, eukaryotic membranes contain sterols,
including cholesterol, that alter membrane fluidity. Additionally, many eukaryotic cells contain some specialized lipids, including
sphingolipids, which are thought to play a role in maintaining membrane stability as well as being involved in signal transduction
pathways and cell-to-cell communication.

Cell Wall
In addition to a plasma membrane, some eukaryotic cells have a cell wall. Cells of fungi, algae, plants, and even some protists have
cell walls. Depending upon the type of eukaryotic cell, cell walls can be made of a wide range of materials, including cellulose
(fungi and plants); biogenic silica, calcium carbonate, agar, and carrageenan (protists and algae); or chitin (fungi). In general, all
cell walls provide structural stability for the cell and protection from environmental stresses such as desiccation, changes in
osmotic pressure, and traumatic injury.6

Outside the Cell

Extracellular Matrix
Cells of animals and some protozoans do not have cell walls to help maintain shape and provide structural stability. Instead, many
of these types of eukaryotic cells produce an extracellular matrix for this purpose. They secrete a sticky mass of carbohydrates and
proteins into the spaces between adjacent cells (Figure 5.1.18). Some protein components assemble into a basement membrane to
which the remaining extracellular matrix components adhere. Proteoglycans typically form the bulky mass of the extracellular
matrix while fibrous proteins, like collagen, provide strength. Both proteoglycans and collagen are attached to fibronectin proteins,
which, in turn, are attached to integrin proteins. These integrin proteins interact with transmembrane proteins in the plasma
membranes of eukaryotic cells that lack cell walls. This makes them both similar and different to the glycocalyx found in
prokaryote cells.

Figure 5.1.18 : The extracellular matrix is composed of protein and carbohydrate components. It protects cells from physical
stresses and transmits signals arriving at the outside edges of the tissue to cells deeper within the tissue.

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In animal cells, the extracellular matrix allows cells within tissues to withstand external stresses and transmits signals from the
outside of the cell to the inside. The amount of extracellular matrix is quite extensive in various types of connective tissues, and
variations in the extracellular matrix can give different types of tissues their distinct properties. In addition, a host cell’s
extracellular matrix is often the site where microbial pathogens attach themselves to establish infection. For example,
Streptococcus pyogenes, the bacterium that causes strep throat and various other infections, binds to fibronectin in the extracellular
matrix of the cells lining the oropharynx (upper region of the throat).

Flagella and Cilia

Some eukaryotic cells use flagella for locomotion; however, eukaryotic flagella are structurally distinct from those found in
prokaryotic cells. Whereas the prokaryotic flagellum is a stiff, rotating structure, a eukaryotic flagellum is more like a flexible whip
composed of nine parallel pairs of microtubules surrounding a central pair of microtubules. This arrangement is referred to as a 9+2
array (Figure 5.1.19). The parallel microtubules use dynein motor proteins to move relative to each other, causing the flagellum to
bend.
Cilia (singular: cilium) are a similar external structure found in some eukaryotic cells. Unique to eukaryotes, cilia are shorter than
flagella and often cover the entire surface of a cell; however, they are structurally similar to flagella (a 9+2 array of microtubules)
and use the same mechanism for movement. A structure called a basal body is found at the base of each cilium and flagellum. The
basal body, which helps connect the cilium or flagellum to the cell, is composed of an array of triplet microtubules similar to that of
a centriole but embedded in the plasma membrane. Because of their shorter length, cilia use a rapid, flexible, waving motion. In
addition to motility, cilia may have other functions such as sweeping particles past or into cells. For example, ciliated protozoans
use the sweeping of cilia to move food particles into their mouthparts, and ciliated cells in the mammalian respiratory tract beat in
synchrony to sweep mucus and debris up and out of the lungs (Figure 5.1.19).

Figure 5.1.19 : (a) Eukaryotic flagella and cilia are composed of a 9+2 array of microtubules, as seen in this transmission electron
micrograph cross-section. (b) The sliding of these microtubules relative to each other causes a flagellum to bend. (c) An illustration
of Trichomonas vaginalis, a flagellated protozoan parasite that causes vaginitis. (d) Many protozoans, like this Paramecium, have
numerous cilia that aid in locomotion as well as in feeding. Note the mouth opening shown here. (credit d: modification of work by
University of Vermont/National Institutes of Health)

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Exercise 5.1.6

1. Explain how the cellular envelope of eukaryotic cells compares to that of prokaryotic cells.
2. Explain the difference between eukaryotic and prokaryotic flagella.

Key Concepts and Summary

Eukaryotic cells are defined by the presence of a nucleus containing the DNA genome and bound by a nuclear membrane (or
nuclear envelope) composed of two lipid bilayers that regulate transport of materials into and out of the nucleus through
nuclear pores.
Eukaryotic cell morphologies vary greatly and may be maintained by various structures, including the cytoskeleton, the cell
membrane, and/or the cell wall
The nucleolus, located in the nucleus of eukaryotic cells, is the site of ribosomal synthesis and the first stages of ribosome
assembly.
Eukaryotic cells contain 80S ribosomes in the rough endoplasmic reticulum (membrane bound-ribosomes) and cytoplasm
(free ribosomes). They contain 70s ribosomes in mitochondria and chloroplasts.
Eukaryotic cells have evolved an endomembrane system, containing membrane-bound organelles involved in transport. These
include vesicles, the endoplasmic reticulum, and the Golgi apparatus.
The smooth endoplasmic reticulum plays a role in lipid biosynthesis, carbohydrate metabolism, and detoxification of toxic
compounds. The rough endoplasmic reticulum contains membrane-bound 80S ribosomes that synthesize proteins destined for
the cell membrane
The Golgi apparatus processes proteins and lipids, typically through the addition of sugar molecules, producing glycoproteins
or glycolipids, components of the plasma membrane that are used in cell-to-cell communication.
Lysosomes contain digestive enzymes that break down small particles ingested by endocytosis, large particles or cells ingested
by phagocytosis, and damaged intracellular components.
The cytoskeleton, composed of microfilaments, intermediate filaments, and microtubules, provides structural support in
eukaryotic cells and serves as a network for transport of intracellular materials.
Centrosomes are microtubule-organizing centers important in the formation of the mitotic spindle in mitosis.
Endosymbiotic theory states that mitochondria and chloroplasts, organelles found in many types of organisms, have their
origins in bacteria. Significant structural and genetic information support this theory.
Mitochondria are the site of cellular respiration. They have two membranes: an outer membrane and an inner membrane with
cristae. The mitochondrial matrix, within the inner membrane, contains the mitochondrial DNA, 70S ribosomes, and metabolic
enzymes.
Chloroplasts are used by plants and some protists to help collect solar energy and transform it into sugars for food, structural
components or other uses.
The plasma membrane of eukaryotic cells is structurally similar to that found in prokaryotic cells, and membrane components
move according to the fluid mosaic model. However, eukaryotic membranes contain sterols, which alter membrane fluidity, as
well as glycoproteins and glycolipids, which help the cell recognize other cells and infectious particles
Cells of fungi, algae, plants, and some protists have a cell wall, whereas cells of animals and some protozoans have a sticky
extracellular matrix that provides structural support and mediates cellular signaling.
Eukaryotic flagella are structurally distinct from prokaryotic flagella but serve a similar purpose (locomotion). Cilia are
structurally similar to eukaryotic flagella, but shorter; they may be used for locomotion, feeding, or movement of extracellular
particles.

Footnotes
1. A.E. Barnhill, M.T. Brewer, S.A. Carlson. “Adverse Effects of Antimicrobials via Predictable or Idiosyncratic Inhibition of
Host Mitochondrial Components.” Antimicrobial Agents and Chemotherapy 56 no. 8 (2012):4046–4051.
2. Fuchs E, Cleveland DW. “A Structural Scaffolding of Intermediate Filaments in Health and Disease.” Science 279 no. 5350
(1998):514–519.
3. E. Fuchs, D.W. Cleveland. “A Structural Scaffolding of Intermediate Filaments in Health and Disease.” Science 279 no. 5350
(1998):514–519.
4. E. Fuchs, D.W. Cleveland. “A Structural Scaffolding of Intermediate Filaments in Health and Disease.” Science 279 no. 5350
(1998):514–519.

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 5. N. Yarlett, J.H.P. Hackstein. “Hydrogenosomes: One Organelle, Multiple Origins.” BioScience 55 no. 8 (2005):657–658.
6. M. Dudzick. “Protists.” OpenStax CNX. November 27, 2013. http://cnx.org/contents/f7048bb6-e46...ef291cf7049c@1
7. J.M. Jaynes, L.P. Vernon. “The Cyanelle of Cyanophora paradoxa: Almost a Cyanobacterial Chloroplast.” Trends in
Biochemical Sciences 7 no. 1 (1982):22–24.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 5.1: Characteristics of Eukaryotic Cells is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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5.2: Classifying Eukaryotic Microbes and Examples
Learning Objectives
Identify challenges associated with classifying microbial eukaryotes
Explain the taxonomic scheme used for microbial eukaryotes
Give examples of infections caused by microbial eukaryotes

Eukaryotic microbes are an extraordinarily diverse group, including species with a wide range of life cycles, morphological
specializations, and nutritional needs. Although more diseases are caused by viruses and bacteria than by microscopic eukaryotes,
these eukaryotes are responsible for some diseases of great public health importance. Although it may seem surprising, parasitic
worms are included within the study of microbiology because identification depends on observation of microscopic adult worms or
eggs. Even in developed countries, these worms are important parasites of humans and of domestic animals. There are fewer fungal
pathogens, but these are important causes of illness, as well. On the other hand, fungi have been important in producing
antimicrobial substances such as penicillin. In this chapter, we will examine characteristics of protists, worms, and fungi while
considering their roles in causing disease.

Characteristics of Protists
The word protist is a historical term that is now used informally to refer to a diverse group of microscopic eukaryotic organisms. It
is not considered a formal taxonomic term because the organisms it describes do not have a shared evolutionary origin, termed
polyphyletic. Since the current taxonomy is based on evolutionary history (as determined by biochemistry, morphology, and
genetics), protists are scattered across many different taxonomic groups within the domain. Historically, the protists were
informally grouped into the “animal-like” protozoans, the “plant-like” algae, and the “fungus-like” protists such as water molds.
These three groups of protists differ greatly in terms of their basic characteristics. For example, algae are photosynthetic organisms
that can be unicellular or multicellular. Protozoa, on the other hand, are nonphotosynthetic, motile organisms that are always
unicellular. Other informal terms may also be used to describe various groups of protists. For example, microorganisms that drift or
float in water, moved by currents, are referred to as plankton. Types of plankton include zooplankton, which are motile and
nonphotosynthetic, and phytoplankton, which are photosynthetic.

Protozoans
Protozoans inhabit a wide variety of habitats, both aquatic and terrestrial. Protozoans are heterotrophic. Many are free-living, while
others are parasitic, carrying out a life cycle within a host or hosts and potentially causing illness. There are also beneficial
symbionts that provide metabolic services to their hosts. During the feeding and growth part of their life cycle, they are called
trophozoites; these feed on small particulate food sources such as bacteria. For example, the protozoal disease malaria was
responsible for 584,000 deaths worldwide (primarily children in Africa) in 2013, according to the World Health Organization
(WHO). The protist parasite Giardia causes a diarrheal illness (giardiasis) that is easily transmitted through contaminated water
supplies. In the United States, Giardia is the most common human intestinal parasite (Figure 5.2.2).
The genus Entamoeba includes commensal or parasitic species, including the medically important E. histolytica, which is
transmitted by cysts in feces and is the primary cause of amoebic dysentery. The notorious “brain-eating amoeba,” Naegleria
fowleri, is a deadly parasite is found in warm, fresh water and causes primary amoebic meningoencephalitis (PAM). Another
member of this group is Acanthamoeba, which can cause keratitis (corneal inflammation) and blindness.
While some types of protozoa exist exclusively in the trophozoite form, others can develop from trophozoite to an encapsulated
cyst stage when environmental conditions are too harsh for the trophozoite. A cyst is a cell with a protective wall, and the process
by which a trophozoite becomes a cyst is called encystment. When conditions become more favorable, these cysts are triggered by
environmental cues to become active again through excystment. One protozoan genus capable of encystment is Eimeria, which
includes some human and animal pathogens. Figure 5.2.3 illustrates the life cycle of Eimeria.

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Figure 5.2.2 : (a) A scanning electron micrograph shows many Giardia parasites in the trophozoite, or feeding stage, in a gerbil
intestine. (b) An individual trophozoite of G. lamblia, visualized here in a scanning electron micrograph. This waterborne protist
causes severe diarrhea when ingested. (credit a, b: modification of work by Centers for Disease Control and Prevention)

Figure 5.2.3 : In the sexual/asexual life cycle of Eimeria, oocysts (inset) are shed in feces and may cause disease when ingested by
a new host. (credit “life cycle,” “micrograph”: modification of work by USDA)

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The apicomplexans are intra- or extracellular parasites that have an apical complex at one end of the cell. The apical complex is a
concentration of organelles, vacuoles, and microtubules that allows the parasite to enter host cells (Figure 5.2.4). Apicomplexans
have complex life cycles that include an infective sporozoite that undergoes schizogony to make many merozoites. Many are
capable of infecting a variety of animal cells, from insects to livestock to humans, and their life cycles often depend on
transmission between multiple hosts. The genus Plasmodium is an example of this group.
Another example is Toxoplasma gondii causes toxoplasmosis and can be transmitted from cat feces, unwashed fruit and vegetables,
or from undercooked meat. Because toxoplasmosis can be associated with serious birth defects, pregnant women need to be aware
of this risk and use caution if they are exposed to the feces of potentially infected cats. A national survey found the frequency of
individuals with antibodies for toxoplasmosis (and thus who presumably have a current latent infection) in the United States to be
11%. Rates are much higher in other countries, including some developed countries.1 There is also evidence and a good deal of
theorizing that the parasite may be responsible for altering infected humans’ behavior and personality traits.2

Figure 5.2.4 : (a) Apicomplexans are parasitic protists. They have a characteristic apical complex that enables them to infect host
cells. (b) A colorized electron microscope image of a Plasmodium sporozoite. (credit b: modification of work by Ute Frevert)

Fungi-like protists
The Eumycetozoa are an unusual group of organisms called slime molds, which have previously been classified as animals, fungi,
and plants (Figure 5.2.5). Slime molds can be divided into two types: cellular slime molds and plasmodial slime molds. The
cellular slime molds exist as individual amoeboid cells that periodically aggregate into a mobile slug. The aggregate then forms a
fruiting body that produces haploid spores. Plasmodial slime molds exist as large, multinucleate amoeboid cells that form
reproductive stalks to produce spores that divide into gametes. One cellular slime mold, Dictyostelium discoideum, has been an
important study organism for understanding cell differentiation, because it has both single-celled and multicelled life stages, with
the cells showing some degree of differentiation in the multicelled form.

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 Figure 5.2.5 : (a) The cellular slime mold Dictyostelium discoideum can be grown on agar in a Petri dish. In this image, individual
amoeboid cells (visible as small spheres) are streaming together to form an aggregation that is beginning to rise in the upper right
corner of the image. The primitively multicellular aggregation consists of individual cells that each have their own nucleus. (b)
Fuligo septica is a plasmodial slime mold. This brightly colored organism consists of a large cell with many nuclei.

Öomycetes have similarities to fungi and were once classified with them. They are also called water molds. However, they differ
from fungi in several important ways. Öomycetes have cell walls of cellulose (unlike the chitinous cell walls of fungi) and they are
generally diploid, whereas the dominant life forms of fungi are typically haploid. Phytophthora, the plant pathogen found in the
soil that caused the Irish potato famine, is classified within this group (Figure 5.2.6).

Figure 5.2.6 : A saprobic oomycete, or water mold, engulfs a dead insect. (credit: modification of work by Thomas Bresson)
The Euglenozoa are common in the environment and include photosynthetic and nonphotosynthetic species. While some are
photosynthesic these are not considered algae because they feed and are motile. Members of the genus Euglena are typically not
pathogenic. Their cells have two flagella, a pellicle, a stigma (eyespot) to sense light, and chloroplasts for photosynthesis (Figure
5.2.7). The pellicle of Euglena is made of a series of protein bands surrounding the cell; it supports the cell membrane and gives

the cell shape.

The Euglenozoa also include the trypanosomes, which are parasitic pathogens. The genus Trypanosoma includes T. brucei, which
causes African trypanosomiasis (African sleeping sickness and T. cruzi, which causes American trypanosomiasis (Chagas disease).
These tropical diseases are spread by insect bites. In African sleeping sickness, T. brucei colonizes the blood and the brain after
being transmitted via the bite of a tsetse fly (Glossina spp.) (Figure 5.2.8). The early symptoms include confusion, difficulty
sleeping, and lack of coordination. Left untreated, it is fatal. Chagas’ disease originated and is most common in Latin America. The

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disease is transmitted by Triatoma spp., insects often called “kissing bugs,” and affects either the heart tissue or tissues of the
digestive system. Untreated cases can eventually lead to heart failure or significant digestive or neurological disorders.

Figure 5.2.7 : (a) This illustration of a Euglena shows the characteristic structures, such as the stigma and flagellum. (b) The
pellicle, under the cell membrane, gives the cell its distinctive shape and is visible in this image as delicate parallel striations over
the surface of the entire cell (especially visible over the grey contractile vacuole). (credit a: modification of work by Claudio
Miklos; credit b: modification of work by David Shykind)

Figure 5.2.8 : Trypanosoma brucei, the causative agent of African trypanosomiasis, spends part of its life cycle in the tsetse fly and
part in humans. (credit “illustration”: modification of work by Centers for Disease Control and Prevention; credit “photo”:
DPDx/Centers for Disease Control and Prevention)

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 Neglected Parasites
The Centers for Disease Control and Prevention (CDC) is responsible for identifying public health priorities in the United
States and developing strategies to address areas of concern. As part of this mandate, the CDC has officially identified five
parasitic diseases it considers to have been neglected (i.e., not adequately studied). These neglected parasitic infections (NPIs)
include toxoplasmosis, Chagas disease, toxocariasis (a nematode infection transmitted primarily by infected dogs),
cysticercosis (a disease caused by a tissue infection of the tapeworm Taenia solium), and trichomoniasis (a sexually transmitted
disease caused by the parabasalid Trichomonas vaginalis).
The decision to name these specific diseases as NPIs means that the CDC will devote resources toward improving awareness
and developing better diagnostic testing and treatment through studies of available data. The CDC may also advise on
treatment of these diseases and assist in the distribution of medications that might otherwise be difficult to obtain.3
Of course, the CDC does not have unlimited resources, so by prioritizing these five diseases, it is effectively deprioritizing
others. Given that many Americans have never heard of many of these NPIs, it is fair to ask what criteria the CDC used in
prioritizing diseases. According to the CDC, the factors considered were the number of people infected, the severity of the
illness, and whether the illness can be treated or prevented. Although several of these NPIs may seem to be more common
outside the United States, the CDC argues that many cases in the United States likely go undiagnosed and untreated because so
little is known about these diseases.4
What criteria should be considered when prioritizing diseases for purposes of funding or research? Are those identified by the
CDC reasonable? What other factors could be considered? Should government agencies like the CDC have the same criteria as
private pharmaceutical research labs? What are the ethical implications of deprioritizing other potentially neglected parasitic
diseases such as leishmaniasis?

Algae
The algae are autotrophic protists that can be unicellular or multicellular. They are important ecologically and environmentally
because they are responsible for the production of approximately 70% of the oxygen and organic matter in aquatic environments.
Some types of algae, even those that are microscopic, are regularly eaten by humans and other animals. Additionally, algae are the
source for agar, agarose, and carrageenan, solidifying agents used in laboratories and in food production. Although algae are
typically not pathogenic, some produce toxins. Harmful algal blooms, which occur when algae grow quickly and produce dense
populations, can produce high concentrations of toxins that impair liver and nervous-system function in aquatic animals and
humans.
Like protozoans, algae often have complex cell structures. For instance, algal cells can have one or more chloroplasts that contain
structures called pyrenoids to synthesize and store starch. The chloroplasts themselves differ in their number of membranes,
indicative of secondary or rare tertiary endosymbiotic events. Primary chloroplasts have two membranes—one from the original
cyanobacteria that the ancestral eukaryotic cell engulfed, and one from the plasma membrane of the engulfing cell. Chloroplasts in
some lineages appear to have resulted from secondary endosymbiosis, in which another cell engulfed a green or red algal cell that
already had a primary chloroplast within it. The engulfing cell destroyed everything except the chloroplast and possibly the cell
membrane of its original cell, leaving three or four membranes around the chloroplast. Different algal groups have different
pigments, which are reflected in common names such as red algae, brown algae, and green algae. Algae may also have a variety of
life cycles. Reproduction may be asexual by mitosis or sexual using gametes.
Some algae, the seaweeds, are macroscopic and may be confused with plants. Seaweeds can be red, brown, or green, depending on
their photosynthetic pigments. Green algae, in particular, share some important similarities with land plants; however, there are also
important distinctions. For example, seaweeds do not have true tissues or organs like plants do. Additionally, seaweeds do not have
a waxy cuticle to prevent desiccation. Algae can also be confused with cyanobacteria, photosynthetic bacteria that bear a
resemblance to algae; however, cyanobacteria are prokaryotes.
Algal Diversity
The dinoflagellates are mostly marine organisms and are an important component of plankton. They have a variety of nutritional
types and may be phototrophic, heterotrophic, or mixotrophic. Those that are photosynthetic use chlorophyll a, chlorophyll c2, and
other photosynthetic pigments (Figure 5.2.9). They generally have two flagella, causing them to whirl (in fact, the name
dinoflagellate comes from the Greek word for “whirl”: dini). Some have cellulose plates forming a hard outer covering, or theca, as
armor. Additionally, some dinoflagellates produce neurotoxins that can cause paralysis in humans or fish. Exposure can occur

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through contact with water containing the dinoflagellate toxins or by feeding on organisms that have eaten dinoflagellates.When a
population of dinoflagellates becomes particularly dense, a red tide (a type of harmful algal bloom) can occur. Red tides cause harm
to marine life and to humans who consume contaminated marine life. Major toxin producers include the genera Gonyaulax and
Alexandrium, both of which cause paralytic shellfish poisoning. Another species, Pfiesteria piscicida, is known as a fish killer
because, at certain parts of its life cycle, it can produce toxins harmful to fish and it appears to be responsible for a suite of
symptoms, including memory loss and confusion, in humans exposed to water containing the species.

Figure 5.2.9 : (a) These large multicellular kelps are members of the brown algae. Note the “leaves” and “stems” that make them
appear similar to green plants. (b) This is a species of red algae that is also multicellular. (c) The green alga Halimeda incrassata,
shown here growing on the sea floor in shallow water, appears to have plant-like structures, but is not a true plant. (d)
Bioluminesence, visible in the cresting wave in this picture, is a phenomenon of certain dinoflagellates. (e) Diatoms (pictured in
this micrograph) produce silicaceous tests (skeletons) that form diatomaceous earths. (f) Colonial green algae, like volvox in these
three micrographs, exhibit simple cooperative associations of cells. (credit a, e: modification of work by NOAA; credit b:
modification of work by Ed Bierman; credit c: modification of work by James St. John; credit d: modification of work by
“catalano82”/Flickr; credit f: modification of work by Dr. Ralf Wagner)

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 Figure 5.2.10 : The dinoflagellates exhibit great diversity in shape. Many are encased in cellulose armor and have two flagella that
fit in grooves between the plates. Movement of these two perpendicular flagella causes a spinning motion. (credit: modification of
work by CSIRO)

Helminths
Parasitic helminths are animals that are often included within the study of microbiology because many species of these worms are
identified by their microscopic eggs and larvae. There are two major groups of parasitic helminths: the roundworms (Nematoda)
and flatworms (Platyhelminthes). Of the many species that exist in these groups, about half are parasitic and some are important
human pathogens. As animals, they are multicellular and have organ systems. However, the parasitic species often have limited
digestive tracts, nervous systems, and locomotor abilities. Parasitic forms may have complex reproductive cycles with several
different life stages and more than one type of host. Some are monoecious, having both male and female reproductive organs in a
single individual, while others are dioecious, each having either male or female reproductive organs.
Nematoda (Roundworms)
Phylum Nematoda (the roundworms) is a diverse group containing more than 15,000 species, of which several are important
human parasites (Figure 5.2.11). These unsegmented worms have a full digestive system even when parasitic. Some are common
intestinal parasites, and their eggs can sometimes be identified in feces or around the anus of infected individuals. Ascaris
lumbricoides is the largest nematode intestinal parasite found in humans; females may reach lengths greater than 1 meter. A.
lumbricoides is also very widespread, even in developed nations, although it is now a relatively uncommon problem in the United
States. It may cause symptoms ranging from relatively mild (such as a cough and mild abdominal pain) to severe (such as intestinal
blockage and impaired growth).
Trichinellosis, also called trichinosis, caused by Trichinella spiralis, is contracted by consuming undercooked meat, which releases
the larvae and allows them to encyst in muscles. Infection can cause fever, muscle pains, and digestive system problems; severe
infections can lead to lack of coordination, breathing and heart problems, and even death. Finally, heartworm in dogs and other
animals is caused by the nematode Dirofilaria immitis, which is transmitted by mosquitoes. Symptoms include fatigue and cough;
when left untreated, death may result.
Of all nematode infections in the United States, pinworm (caused by Enterobius vermicularis) is the most common. Pinworm
causes sleeplessness and itching around the anus, where the female worms lay their eggs during the night. Toxocara canis and T.
cati are nematodes found in dogs and cats, respectively, that can be transmitted to humans, causing toxocariasis. Antibodies to
these parasites have been found in approximately 13.9% of the U.S. population, suggesting that exposure is common.5 Infection
can cause larval migrans, which can result in vision loss and eye inflammation, or fever, fatigue, coughing, and abdominal pain,
depending on whether the organism infects the eye or the viscera. Another common nematode infection is hookworm, which is
caused by Necator americanus (the New World or North American hookworm) and Ancylostoma duodenale (the Old World
hookworm). Symptoms of hookworm infection can include abdominal pain, diarrhea, loss of appetite, weight loss, fatigue, and
anemia.

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 Figure 5.2.11 : A micrograph of the nematode Enterobius vermicularis, also known as the pinworm. (credit: modification of work
by Centers for Disease Control and Prevention)

Clinical Focus: part 2

The physician explains to Sarah’s mother that ringworm can be transferred between people through touch. “It’s common in
school children, because they often come in close contact with each other, but anyone can become infected,” he adds. “Because
you can transfer it through objects, locker rooms and public pools are also a potential source of infection. It’s very common
among wrestlers and athletes in other contact sports.”
Looking very uncomfortable, Sarah says to her mother “I want this worm out of me.”
The doctor laughs and says, “Sarah, you’re in luck because ringworm is just a name; it is not an actual worm. You have
nothing wriggling around under your skin.”
“Then what is it?” asks Sarah.

Exercise 5.2.2

1. What type of pathogen causes ringworm?

2. What is the most common nematode infection in the United States?

Platyhelminths (Flatworms)
Phylum Platyhelminthes (the platyhelminths) are flatworms. This group includes the flukes, tapeworms, and the turbellarians,
which include planarians. The flukes and tapeworms are medically important parasites (Figure 5.2.12).
The flukes (trematodes) are nonsegmented flatworms that have an oral sucker (Figure 5.2.13) (and sometimes a second ventral
sucker) and attach to the inner walls of intestines, lungs, large blood vessels, or the liver. Trematodes have complex life cycles,
often with multiple hosts. Several important examples are the liver flukes (Clonorchis and Opisthorchis), the intestinal fluke
(Fasciolopsis buski), and the oriental lung fluke (Paragonimus westermani). Schistosomiasis is a serious parasitic disease,
considered second in the scale of its impact on human populations only to malaria. The parasites Schistosoma mansoni, S.
haematobium, and S. japonicum, which are found in freshwater snails, are responsible for schistosomiasis. Immature forms burrow
through the skin into the blood. They migrate to the lungs, then to the liver and, later, other organs. Symptoms include anemia,
malnutrition, fever, abdominal pain, fluid buildup, and sometimes death.

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 Figure 5.2.12 : Phylum Platyhelminthes is divided into four classes. (a) Class Turbellaria includes the Bedford’s flatworm
(Pseudobiceros bedfordi), which is about 8–10 cm long. (b) The parasitic class Monogenea includes Dactylogyrus spp. Worms in
this genus are commonly called gill flukes. The specimen pictured here is about 0.2 mm long and has two anchors, indicated by
arrows, that it uses to latch onto the gills of host fish. (c) The Trematoda class includes the common liver fluke Fasciola hepatica
and the giant liver fluke Fascioloides magna (right). The F. magna specimen shown here is about 7 cm long. (d) Class Cestoda
includes tapeworms such as Taenia saginata, which infects both cattle and humans and can reach lengths of 4–10 meters; the
specimen shown here is about 4 meters long. (credit c: modification of work by “Flukeman”/Wikimedia Commons)

Figure 5.2.13 : (a) The oral sucker is visible on the anterior end of this liver fluke, Opisthorchis viverrini. (b) This micrograph
shows the scolex of the cestode Taenia solium, also known as the pork tapeworm. The visible suckers and hooks allow the worm to
attach itself to the inner wall of the intestine. (credit a: modification of work by Sripa B, Kaewkes S, Sithithaworn P, Mairiang E,
Laha T, and Smout M; credit b: modification of work by Centers for Disease Control and Prevention)
The other medically important group of platyhelminths are commonly known as tapeworms (cestodes) and are segmented
flatworms that may have suckers or hooks at the scolex (head region) (Figure 5.2.13). Tapeworms use these suckers or hooks to

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attach to the wall of the small intestine. The body of the worm is made up of segments called proglottids that contain reproductive
structures; these detach when the gametes are fertilized, releasing gravid proglottids with eggs. Tapeworms often have an
intermediate host that consumes the eggs, which then hatch into a larval form called an oncosphere. The oncosphere migrates to a
particular tissue or organ in the intermediate host, where it forms cysticerci. After being eaten by the definitive host, the cysticerci
develop into adult tapeworms in the host's digestive system. Taenia saginata (the beef tapeworm) and T. solium (the pork
tapeworm) enter humans through ingestion of undercooked, contaminated meat. The adult worms develop and reside in the
intestine, but the larval stage may migrate and be found in other body locations such as skeletal and smooth muscle. The beef
tapeworm is relatively benign, although it can cause digestive problems and, occasionally, allergic reactions. The pork tapeworm
can cause more serious problems when the larvae leave the intestine and colonize other tissues, including those of the central
nervous system. Diphylobothrium latum is the largest human tapeworm and can be ingested in undercooked fish. It can grow to a
length of 15 meters. Echinococcus granulosus, the dog tapeworm, can parasitize humans and uses dogs as an important host.

Food for Worms?

For residents of temperate, developed countries, it may be difficult to imagine just how common helminth infections are in the
human population. In fact, they are quite common and even occur frequently in the United States. Worldwide, approximately
807–1,221 million people are infected with Ascaris lumbricoides (perhaps one-sixth of the human population) and far more are
infected if all nematode species are considered.6 Rates of infection are relatively high even in industrialized nations.
Approximately 604–795 million people are infected with whipworm (Trichuris) worldwide (Trichuris can also infect dogs),
and 576–740 million people are infected with hookworm (Necator americanus and Ancylostoma duodenale).7 Toxocara, a
nematode parasite of dogs and cats, is also able to infect humans. It is widespread in the United States, with about 10,000
symptomatic cases annually. However, one study found 14% of the population (more than 40 million Americans) was
seropositive, meaning they had been exposed to the parasite at one time. More than 200 million people have schistosomiasis
worldwide. Most of the World Health Organization (WHO) neglected tropical diseases are helminths. In some cases, helminths
may cause subclinical illnesses, meaning the symptoms are so mild that that they go unnoticed. In other cases, the effects may
be more severe or chronic, leading to fluid accumulation and organ damage. With so many people affected, these parasites
constitute a major global public health concern.

Eradicating the Guinea Worm

Dracunculiasis, or Guinea worm disease, is caused by a nematode called Dracunculus medinensis. When people consume
contaminated water, water fleas (small crustaceans) containing the nematode larvae may be ingested. These larvae migrate out
of the intestine, mate, and move through the body until females eventually emerge (generally through the feet or lower limbs).
While Guinea worm disease is rarely fatal, it is extremely painful and can be accompanied by secondary infections and edema
(Figure 5.2.14).

Figure 5.2.14 : The Guinea worm can be removed from a leg vein of an infected person by gradually winding it around a stick,
like this matchstick. (credit: Centers for Disease Control and Prevention)
An eradication campaign led by WHO, the CDC, the United Nations Children’s Fund (UNICEF), and the Carter Center
(founded by former U.S. president Jimmy Carter) has been extremely successful in reducing cases of dracunculiasis. This has
been possible because diagnosis is straightforward, there is an inexpensive method of control, there is no animal reservoir, the
water fleas are not airborne (they are restricted to still water), the disease is geographically limited, and there has been a
commitment from the governments involved. Additionally, no vaccines or medication are required for treatment and
prevention. In 1986, 3.5 million people were estimated to be affected. After the eradication campaign, which included helping
people in affected areas learn to filter water with cloth, only four countries continue to report the disease (Chad, Mali, South
Sudan, and Ethiopia) with a total of 126 cases reported to WHO in 2014.

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Fungi
The fungi comprise a diverse group of organisms that are heterotrophic and typically saprozoic. In addition to the well-known
macroscopic fungi (such as mushrooms and molds), many unicellular yeasts and spores of macroscopic fungi are microscopic. For
this reason, fungi are included within the field of microbiology.
Fungi are important to humans in a variety of ways. Both microscopic and macroscopic fungi have medical relevance, with some
pathogenic species that can cause mycoses (illnesses caused by fungi). Some pathogenic fungi are opportunistic, meaning that they
mainly cause infections when the host’s immune defenses are compromised and do not normally cause illness in healthy
individuals. Fungi are important in other ways. They act as decomposers in the environment, and they are critical for the production
of certain foods such as cheeses. Fungi are also major sources of antibiotics, such as penicillin from the fungus Penicillium.
There are notable unique features in fungal cell walls and membranes. Fungal cell walls contain chitin, as opposed to the cellulose
found in the cell walls of plants and many protists. Additionally, whereas animals have cholesterol in their cell membranes, fungal
cell membranes have different sterols called ergosterols. Ergosterols are often exploited as targets for antifungal drugs.
Fungal life cycles are unique and complex. Fungi reproduce sexually either through cross- or self-fertilization. Haploid fungi can
form hyphae that have gametes at the tips. Two different mating types (represented as “+ type” and “– type”) are involved. The
cytoplasms of the + and – type gametes fuse (in an event called plasmogamy), producing a cell with two distinct nuclei (a
dikaryotic cell). Later, the nuclei fuse (in an event called karyogamy) to create a diploid zygote. The zygote undergoes meiosis to
form spores that germinate to start the haploid stage, which eventually creates more haploid mycelia. Depending on the taxonomic
group, these sexually produced spores are known as zygospores (in Zygomycota), ascospores (in Ascomycota), or basidiospores (in
Basidiomycota).
Fungi may also exhibit asexual reproduction by mitosis, mitosis with budding, fragmentation of hyphae, and formation of asexual
spores by mitosis. These spores are specialized cells that, depending on the organism, may have unique characteristics for survival,
reproduction, and dispersal. Fungi exhibit several types of asexual spores and these can be important in classification.

Molds
Fungi have well-defined characteristics that set them apart from other organisms. Most multicellular fungal bodies, commonly
called molds, are made up of filaments called hyphae. Hyphae can form a tangled network called a mycelium and form the thallus
(body) of fleshy fungi. Hyphae that have walls between the cells are called septate hyphae; hyphae that lack walls and cell
membranes between the cells are called nonseptate or coenocytic hyphae). (Figure 5.2.15). Macroscopically these create a velvety
appearance as the colony grows.

Figure 5.2.15 : Multicellular fungi (molds) form hyphae, which may be septate or nonseptate. Unicellular fungi (yeasts) cells form
pseudohyphae from individual yeast cells.

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Figure 5.2.16: These images show asexually produced spores. (a) This brightfield micrograph shows the release of spores from a
sporangium at the end of a hypha called a sporangiophore. The organism is a Mucor sp. fungus, a mold often found indoors. (b)
Sporangia grow at the ends of stalks, which appear as the white fuzz seen on this bread mold, Rhizopus stolonifer. The tips of bread
mold are the dark, spore-containing sporangia. (credit a: modification of work by Centers for Disease Control and Prevention;
credit b right: modification of work by “Andrew”/Flickr)

Yeasts
In contrast to molds, yeasts are unicellular fungi. The budding yeasts reproduce asexually by budding off a smaller daughter cell;
the resulting cells may sometimes stick together as a short chain or pseudohypha (Figure 5.2.15). Candida albicans is a common
yeast that forms pseudohyphae; it is associated with various infections in humans, including vaginal yeast infections, oral thrush,
and candidiasis of the skin.
Saccharomyces yeasts, including the baker’s yeast S. cerevisiae, are unicellular ascomycetes with haploid and diploid stages
(Figure 5.2.17). This and other Saccharomyces species are used for brewing beer. Cryptococcus neoformans, a fungus commonly
found as a yeast in the environment, can cause serious lung infections when inhaled by individuals with weakened immune
systems.

Figure 5.2.17 : (a) This brightfield micrograph shows ascospores being released from asci in the fungus Talaromyces flavus var.
flavus. (b) This electron micrograph shows the conidia (spores) borne on the conidiophore of Aspergillus, a type of toxic fungus
found mostly in soil and plants. (c) This brightfield micrograph shows the yeast Candida albicans, the causative agent of
candidiasis and thrush. (credit a, b, c: modification of work by Centers for Disease Control and Prevention)

Dimorphic
Some fungi are dimorphic, having more than one appearance during their life cycle. These dimorphic fungi may be able to appear
as yeasts or molds, which can be important for infectivity but a pain when trying to determine the organism responsible for disease.
They are capable of changing their appearance in response to environmental changes such as nutrient availability or fluctuations in
temperature, growing as a mold, for example, at 25 °C (77 °F), and as yeast cells at 37 °C (98.6 °F). This ability helps dimorphic
fungi to survive in diverse environments. Histoplasma capsulatum, the pathogen that causes histoplasmosis, a lung infection, is an
example of a dimorphic fungus (Figure 5.2.18).

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 Figure 5.2.18 : Histoplasma capsulatum is a dimorphic fungus that grows in soil exposed to bird feces or bat feces (guano) (top
left). It can change forms to survive at different temperatures. In the outdoors, it typically grows as a mycelium (as shown in the
micrograph, bottom left), but when the spores are inhaled (right), it responds to the high internal temperature of the body (37 °C
[98.6 °F]) by turning into a yeast that can multiply in the lungs, causing the chronic lung disease histoplasmosis. (credit:
modification of work by Centers for Disease Control and Prevention)

Eukaryotic Pathogens in Eukaryotic Hosts

When we think about antimicrobial medications, antibiotics such as penicillin often come to mind. Penicillin and related
antibiotics interfere with the synthesis of peptidoglycan cell walls, which effectively targets bacterial cells. These antibiotics
are useful because humans (like all eukaryotes) do not have peptidoglycan cell walls.
Developing medications that are effective against eukaryotic cells but not harmful to human cells is more difficult. Despite
huge morphological differences, the cells of humans, fungi, helminths and protists are similar in terms of their ribosomes,
cytoskeletons, and cell membranes. As a result, it is more challenging to develop medications that target protozoans and fungi
in the same way that antibiotics target prokaryotes.
Fungicides have relatively limited modes of action. Because fungi have ergosterols (instead of cholesterol) in their cell
membranes, the different enzymes involved in sterol production can be a target of some medications. The azole and
morpholine fungicides interfere with the synthesis of membrane sterols. These are used widely in agriculture (fenpropimorph)
and clinically (e.g., miconazole). Some antifungal medications target the chitin cell walls of fungi. Despite the success of these
compounds in targeting fungi, antifungal medications for systemic infections still tend to have more toxic side effects than
antibiotics for bacteria.

Clinical Focus: Resolution

Sarah is relieved the ringworm is not an actual worm, but wants to know what it really is. The physician explains that
ringworm is a fungus. He tells her that she will not see mushrooms popping out of her skin, because this fungus is more like
the invisible part of a mushroom that hides in the soil. He reassures her that they are going to get the fungus out of her.

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 The doctor cleans and then carefully scrapes the lesion to place a specimen on a slide. By looking at it under a microscope, the
physician is able to confirm that a fungal infection is responsible for Sarah’s lesion. Even if the pathogen resembled a helminth
under the microscope, the presence of cell walls would rule out the possibility because animal cells lack cell walls. The doctor
prescribes an antifungal cream for Sarah’s mother to apply to the ringworm. Sarah’s mother asks, “What should we do if it
doesn’t go away?”
The doctor explains that ringworm is a general term for a condition caused by multiple species. The first step is to take a
scraping for examination under the microscope, which the doctor has already done. He explains that he has identified the
infection as a fungus, and that the antifungal cream works against the most common fungi associated with ringworm. However,
the cream may not work against some species of fungus.
If the cream is not working after a couple of weeks, Sarah should come in for another visit, at which time the doctor will take
steps to identify the species of the fungus.
Positive identification of dermatophytes requires culturing. For this purpose, Sabouraud’s agar may be used. In the case of
Sarah’s infection, which cleared up within 2 weeks of treatment, the culture would have a granular texture and would appear
pale pink on top and red underneath. These features suggest that the fungus is Trichophyton rubrum, a common cause of
ringworm.

Key Concepts and Summary

Protists are a diverse, polyphyletic group of eukaryotic organisms.
Protists may be unicellular or multicellular. They vary in how they get their nutrition, morphology, method of locomotion, and
mode of reproduction.
The protozoa group include important pathogens and parasites.
Algae are a diverse group of photosynthetic eukaryotic protists
Algae may be unicellular or multicellular. Large, multicellular algae are called seaweeds but are not plants and lack plant-like
tissues and organs
Helminth parasites are included within the study of microbiology because they are often identified by looking for microscopic
eggs and larvae.
The two major groups of helminth parasites are the roundworms (Nematoda) and the flatworms (Platyhelminthes).
The fungi include diverse saprotrophic eukaryotic organisms with chitin cell walls
Fungi can be unicellular or multicellular; some (like yeast) and fungal spores are microscopic, whereas some are large and
conspicuous.
Important differences in fungal cells, such as ergosterols in fungal membranes, can be targets for antifungal medications, but
similarities between human and fungal cells make it difficult to find targets for medications and these medications often have
toxic adverse effects
Footnotes
1. J. Flegr et al. “Toxoplasmosis—A Global Threat. Correlation of Latent Toxoplasmosis With Specific Disease Burden in a Set of
88 Countries.” PloS ONE 9 no. 3 (2014):e90203.
2. J. Flegr. “Effects of Toxoplasma on Human Behavior.” Schizophrenia Bull 33, no. 3 (2007):757–760.
3. Centers for Disease Control and Prevention. “Neglected Parasitic Infections (NPIs) in the United States.”
http://www.cdc.gov/parasites/npi/. Last updated July 10, 2014.
4. Centers for Disease Control and Prevention. “Fact Sheet: Neglected Parasitic Infections in the United States.”
www.cdc.gov/parasites/resourc..._factsheet.pdf
5. Won K, Kruszon-Moran D, Schantz P, Jones J. “National seroprevalence and risk factors for zoonotic Toxocara spp. infection.”
In: Abstracts of the 56th American Society of Tropical Medicine and Hygiene; Philadelphia, Pennsylvania; 2007 Nov 4-8.
6. Fenwick, A. “The global burden of neglected tropical diseases.” Public health 126 no.3 (Mar 2012): 233–6.
7. de Silva, N., et. al. (2003). “Soil-transmitted helminth infections: updating the global picture”. Trends in Parasitology 19
(December 2003): 547–51.
8. World Health Organization. “South Sudan Reports Zero Cases of Guinea-Worm Disease for Seventh Consecutive Month.”
2016. http://www.who.int/dracunculiasis/no...ive_months/en/. Accessed May 2, 2016.

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Contributors and Attributions
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 5.2: Classifying Eukaryotic Microbes and Examples is shared under a CC BY license and was authored, remixed, and/or curated
by OpenStax.

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Chapter 5 Exercises
Review Questions for Chapter 5

Multiple Choice

1) Whose proposal of the endosymbiotic theory of mitochondrial and chloroplast origin was ultimately accepted by the greater
scientific community?
A. Rudolf Virchow
B. Ignaz Semmelweis
C. Lynn Margulis
D. Theodor Schwann

2) Which of the following organelles is not part of the endomembrane system?

A. endoplasmic reticulum
B. Golgi apparatus
C. lysosome
D. peroxisome

3) Which type of cytoskeletal fiber is important in the formation of the nuclear lamina?
A. microfilaments
B. intermediate filaments
C. microtubules
D. fibronectin

4) Sugar groups may be added to proteins in which of the following?

A. smooth endoplasmic reticulum
B. rough endoplasmic reticulum
C. Golgi apparatus
D. lysosome

5) Which of the following structures of a eukaryotic cell is not likely derived from endosymbiotic bacterium?
A. mitochondrial DNA
B. mitochondrial ribosomes
C. inner membrane
D. outer membrane

6) Which type of nutrient uptake involves the engulfment of small dissolved molecules into vesicles?
A. active transport
B. pinocytosis
C. receptor-mediated endocytosis
D. facilitated diffusion

7) Which of the following is not composed of microtubules?

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A. desmosomes
B. centrioles
C. eukaryotic flagella
D. eukaryotic cilia

8) Which genus includes the causative agent for malaria?

A. Euglena
B. Paramecium
C. Plasmodium
D. Trypanosoma

9) Which protist is a concern because of its ability to contaminate water supplies and cause diarrheal illness?
A. Plasmodium vivax
B. Toxoplasma gondii
C. Giardia lamblia
D. Trichomonas vaginalis

10) A fluke is classified within which of the following?

A. Nematoda
B. Rotifera
C. Platyhelminthes
D. Annelida

11) A nonsegmented worm is found during a routine colonoscopy of an individual who reported having abdominal cramps, nausea,
and vomiting. This worm is likely which of the following?
A. nematode
B. fluke
C. trematode
D. annelid

12) A segmented worm has male and female reproductive organs in each segment. Some use hooks to attach to the intestinal wall.
Which type of worm is this?
A. fluke
B. nematode
C. cestode
D. annelid

13) Mushrooms are a type of which of the following?

A. conidia
B. ascus
C. polar tubule
D. basidiocarp

14) Which of the following is the most common cause of human yeast infections?
A. Candida albicans

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B. Blastomyces dermatitidis
C. Cryptococcus neoformans
D. Aspergillus fumigatus

15) Which of the following is an ascomycete fungus associated with bat droppings that can cause a respiratory infection if inhaled?
A. Candida albicans
B. Histoplasma capsulatum
C. Rhizopus stolonifera
D. Trichophyton rubrum

16) Which polysaccharide found in red algal cell walls is a useful solidifying agent?
A. chitin
B. cellulose
C. phycoerythrin
D. agar

17) Which is the term for the hard outer covering of some dinoflagellates?
A. theca
B. thallus
C. mycelium
D. shell

18) Which protists are associated with red tides?

A. red algae
B. brown algae
C. dinoflagellates
D. green algae

19) You encounter a lichen with leafy structures. Which term describes this lichen?
A. crustose
B. foliose
C. fruticose
D. agarose

20) Which of the following is the term for the outer layer of a lichen?
A. the cortex
B. the medulla
C. the thallus
D. the theca

21) The fungus in a lichen is which of the following?

A. a basidiomycete
B. an ascomycete
C. a zygomycete
D. an apicomplexan

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Fill-in-the-Blanks

22) Peroxisomes typically produce _____________, a harsh chemical that helps break down molecules.

23) Microfilaments are composed of _____________ monomers.

24) The plasma membrane of a protist is called the __________.

25) Animals belong to the same supergroup as the kingdom __________.

26) Flukes are in class _________.

27) A species of worm in which there are distinct male and female individuals is described as _________.

28) Nonseptate hyphae are also called _________.

29) Unicellular fungi are called _________.

30) Some fungi have proven medically useful because they can be used to produce _________.

31) Structures in chloroplasts used to synthesize and store starch are called ________.

32) Algae with chloroplasts with three or four membranes are a result of ________ ________.

Short Answer

33) What evidence exists that supports the endosymbiotic theory?

34) What existing evidence supports the theory that mitochondria are of prokaryotic origin?

35) Why do eukaryotic cells require an endomembrane system?

36) Name at least two ways that prokaryotic flagella are different from eukaryotic flagella.

37) What are kinetoplastids?

38) Aside from a risk of birth defects, what other effect might a toxoplasmosis infection have?

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39) What is the function of the ciliate macronucleus?

40) What is the best defense against tapeworm infection?

41) Which genera of fungi are common dermatophytes (fungi that cause skin infections)?

42) What is a dikaryotic cell?

43) What is a distinctive feature of diatoms?

44) Why are algae not considered parasitic?

45) Which groups contain the multicellular algae?

Critical Thinking

46) Label the lettered parts of this eukaryotic cell.

47) How are peroxisomes more like mitochondria than like the membrane-bound organelles of the endomembrane system? How do
they differ from mitochondria?

48) Why must the functions of both lysosomes and peroxisomes be compartmentalized?

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49) The protist shown has which of the following?
A. pseudopodia
B. flagella
C. a shell
D. cilia

(credit: modification of work by Richard Robinson)

50) Protist taxonomy has changed greatly in recent years as relationships have been re-examined using newer approaches. How do
newer approaches differ from older approaches?

51) What characteristics might make you think a protist could be pathogenic? Are certain nutritional characteristics, methods of
locomotion, or morphological differences likely to be associated with the ability to cause disease?

52) Given the life cycle of the Schistosoma parasite, suggest a method of prevention of the disease.

53) Which of the drawings shows septate hyphae?

54) Explain the benefit of research into the pathways involved in the synthesis of chitin in fungi.

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 CHAPTER OVERVIEW

6: Mechanisms of Microbial Genetics

6.1: Using Microbiology to Discover the Secrets of Life
6.2: Structure and Replication of DNA
6.3: Structure and Transcription of RNA
6.4: Protein Synthesis (Translation)
6.5: Mutations
6.6: How Asexual Prokaryotes Achieve Genetic Diversity
6.7: Gene Regulation and Operon Theory
Chapter 6 Exercises

Thumbnail: DNA Double Helix. (Public Domain; Apers0n).

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1
6.1: Using Microbiology to Discover the Secrets of Life
Learning Objectives
Describe the discovery of nucleic acid and nucleotides
Explain the historical experiments that led to the characterization of DNA
Describe how microbiology and microorganisms have been used to discover the biochemistry of genes
Explain how scientists established the link between DNA and heredity

Clinical Focus: part 1

Alex is a 22-year-old college student who vacationed in Puerta Vallarta, Mexico, for spring break. Unfortunately, two
days after flying home to Ohio, he began to experience abdominal cramping and extensive watery diarrhea.
Because of his discomfort, he sought medical attention at a large Cincinnati hospital nearby.

Exercise 6.1.1

What types of infections or other conditions may be responsible?

Through the early 20th century, DNA was not yet recognized as the genetic material responsible for heredity, the passage of traits
from one generation to the next. In fact, much of the research was dismissed until the mid-20th century. The scientific community
believed, incorrectly, that the process of inheritance involved a blending of parental traits that produced an intermediate physical
appearance in offspring; this hypothetical process appeared to be correct because of what we know now as continuous variation,
which results from the action of many genes to determine a particular characteristic, like human height. Offspring appear to be a
“blend” of their parents’ traits when we look at characteristics that exhibit continuous variation. The blending theory of inheritance
asserted that the original parental traits were lost or absorbed by the blending in the offspring, but we now know that this is not the
case.
Two separate lines of research, begun in the mid to late 1800s, ultimately led to the discovery and characterization of DNA and the
foundations of genetics, the science of heredity. These lines of research began to converge in the 1920s, and research using
microbial systems ultimately resulted in significant contributions to elucidating the molecular basis of genetics.

Discovery and Characterization of DNA

Modern understanding of DNA has evolved from the discovery of nucleic acid to the development of the double-helix model. In
the 1860s, Friedrich Miescher (1844–1895), a physician by profession, was the first person to isolate phosphorus-rich chemicals
from leukocytes (white blood cells) from the pus on used bandages from a local surgical clinic. He named these chemicals (which
would eventually be known as RNA and DNA) “nuclein” because they were isolated from the nuclei of the cells. His student
Richard Altmann (1852–1900) subsequently termed it “nucleic acid” 20 years later when he discovered the acidic nature of
nuclein. In the last two decades of the 19th century, German biochemist Albrecht Kossel (1853–1927) isolated and characterized
the five different nucleotide bases composing nucleic acid. These are adenine, guanine, cytosine, thymine (in DNA), and uracil (in
RNA). Kossell received the Nobel Prize in Physiology or Medicine in 1910 for his work on nucleic acids and for his considerable
work on proteins, including the discovery of histidine. Despite the discoveries in the late 1800s and early 1900's, scientists did not
make the association with heredity for many more decades. To make this connection, scientists, including a number of
microbiologists, performed many experiments on plants, animals, and bacteria.

The Chromosomal Theory of Inheritance

Mendel's famous experiment with pea plant genetics in the 1860's was carried out long before chromosomes were visualized under
a microscope. However, with the improvement of microscopic techniques during the late 1800s, cell biologists could stain and
visualize subcellular structures with dyes and observe their actions during meiosis. They were able to observe chromosomes
replicating, condensing from an amorphous nuclear mass into distinct X-shaped bodies and migrating to separate cellular poles.
The speculation that chromosomes might be the key to understanding heredity led several scientists to examine Mendel’s
publications and re-evaluate his model in terms of the behavior of chromosomes during mitosis and meiosis.
Other famous experiments that contributed to our understanding of chromosomes included:

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 In 1902, Theodor Boveri (1862–1915) observed that in sea urchins, nuclear components (chromosomes) determined proper
embryonic development.
That same year, Walter Sutton (1877–1916) observed the separation of chromosomes into daughter cells during meiosis.
Thomas Hunt Morgan (1866–1945) and his colleagues spent several years carrying out crosses with the fruit fly, Drosophila
melanogaster. They performed meticulous microscopic observations of fly chromosomes and correlated these observations with
resulting fly characteristics.
In the late 1920s, Barbara McClintock (1902–1992) developed chromosomal staining techniques to visualize and differentiate
between the different chromosomes of maize (corn). In the 1940s and 1950s, she identified a breakage event on chromosome 9,
which she named the dissociation locus (Ds), which could change position within the chromosome. This lead to the finding of
jumping genes, which we now call transposons. Today, we know that transposons are mobile segments of DNA that can move
within the genome of an organism. They can regulate gene expression, protein expression, and virulence (ability to cause
disease).
Together, these observations led to the development of the Chromosomal Theory of Inheritance, which identified chromosomes as
the genetic material responsible for Mendelian inheritance.

Microbes and Viruses in Genetic Research

Microbiologists have also played a crucial part in our understanding of genetics. Experimental organisms such as Mendel’s garden
peas, Morgan’s fruit flies, and McClintock’s corn had already been used successfully to pave the way for an understanding of
genetics. However, microbes and viruses were (and still are) excellent model systems for the study of genetics because, unlike peas,
fruit flies, and corn, they are propagated more easily in the laboratory, growing to high population densities in a small amount of
space and in a short time. In addition, because of their structural simplicity, microbes and viruses are more readily manipulated
genetically.
Fortunately, despite significant differences in size, structure, reproduction strategies, and other biological characteristics, there is
biochemical unity among all organisms; they have in common the same underlying molecules responsible for heredity and the use
of genetic material to give cells their varying characteristics. In the words of French scientist Jacques Monod, “What is true for E.
coli is also true for the elephant,” meaning that the biochemistry of life has been maintained throughout evolution and is shared in
all forms of life, from simple unicellular organisms to large, complex organisms. This biochemical continuity makes microbes
excellent models to use for genetic studies.
In a clever set of experiments in the 1930s and 1940s, German scientist Joachim Hämmerling (1901–1980), using the single-celled
alga Acetabularia as a microbial model, established that the genetic information in a eukaryotic cell is housed within the nucleus.
Acetabularia spp. are unusually large algal cells that grow asymmetrically, forming a “foot” containing the nucleus, which is used
for substrate attachment; a stalk; and an umbrella-like cap—structures that can all be easily seen with the naked eye. In an early set
of experiments, Hämmerling removed either the cap or the foot of the cells and observed whether new caps or feet were
regenerated (Figure 6.1.1). He found that when the foot of these cells was removed, new feet did not grow; however, when caps
were removed from the cells, new caps were regenerated. This suggested that the hereditary information was located in the
nucleus-containing foot of each cell.

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 Figure 6.1.1 : (a) The cells of the single-celled alga Acetabularia measure 2–6 cm and have a cell morphology that can be observed
with the naked eye. Each cell has a cap, a stalk, and a foot, which contains the nucleus. (b) Hämmerling found that if he removed
the cap, a new cap would regenerate; but if he removed the foot, a new foot would not regenerate. He concluded that the genetic
information needed for regeneration was found in the nucleus. (credit a: modification of work by James St. John)
Another microbial model, the red bread mold Neurospora crassa, was used by George Beadle and Edward Tatum to demonstrate
the relationship between genes and the proteins they encode. Beadle had worked with fruit flies in Morgan’s laboratory but found

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them too complex to perform certain types of experiments. N. crassa, on the other hand, is a simpler organism and has the ability to
grow on a minimal medium because it contains enzymatic pathways that allow it to use the medium to produce its own vitamins
and amino acids.
Beadle and Tatum irradiated the mold with X-rays to induce changes to a sequence of nucleic acids, called mutations. They mated
the irradiated mold spores and attempted to grow them on both a complete medium and a minimal medium. They looked for
mutants that grew on a complete medium, supplemented with vitamins and amino acids, but did not grow on the minimal medium
lacking these supplements. Such molds theoretically contained mutations in the genes that encoded biosynthetic pathways. Upon
finding such mutants, they systematically tested each to determine which vitamin or amino acid it was unable to produce (Figure
6.1.2) and published this work in 1941.

Figure 6.1.2 : Beadle and Tatum’s experiment involved the mating of irradiated and nonirradiated mold spores. These spores were
grown on both complete medium and a minimal medium to determine which amino acid or vitamin the mutant was unable to
produce on its own.
Subsequent work by Beadle, Tatum, and colleagues showed that they could isolate different classes of mutants that required a
particular supplement, like the amino acid arginine. With some knowledge of the arginine biosynthesis pathway, they identified
three classes of arginine mutants by supplementing the minimal medium with intermediates (citrulline or ornithine) in the pathway.
The three mutants differed in their abilities to grow in each of the media, which led the group of scientists to propose, in 1945, that
each type of mutant had a defect in a different gene in the arginine biosynthesis pathway. This led to the so-called one gene–one
enzyme hypothesis, which suggested that each gene encodes one enzyme.
Subsequent knowledge about the processes of transcription and translation led scientists to revise this to the “one gene–one
polypeptide” hypothesis. Although there are some genes that do not encode polypeptides (but rather encode for transfer RNAs
[tRNAs] or ribosomal RNAs [rRNAs], which we will discuss later), the one gene–one enzyme hypothesis is true in many cases,
especially in microbes. Beadle and Tatum’s discovery of the link between genes and corresponding characteristics earned them the
1958 Nobel Prize in Physiology and Medicine and has since become the basis for modern molecular genetics.

Exercise 6.1.2
1. What organism did Morgan and his colleagues use to develop the Chromosomal Theory of Inheritance? What traits did they
track?
2. What did Hämmerling prove with his experiments on Acetabularia?

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DNA as the Molecule Responsible for Heredity
By the beginning of the 20th century, a great deal of work had already been done on characterizing DNA and establishing the
foundations of genetics, including attributing heredity to chromosomes found within the nucleus. Despite all of this research, it was
not until well into the 20th century that these lines of research converged and scientists began to consider that DNA could be the
genetic material that offspring inherited from their parents. DNA, containing only four different nucleotides, was thought to be
structurally too simple to encode such complex genetic information. Instead, protein was thought to have the complexity required
to serve as cellular genetic information because it is composed of 20 different amino acids that could be combined in a huge variety
of combinations. Microbiologists played a pivotal role in the research that determined that DNA is the molecule responsible for
heredity.

Griffith’s Transformation Experiments

British bacteriologist Frederick Griffith (1879–1941) was perhaps the first person to show that hereditary information could be
transferred from one cell to another “horizontally” (between members of the same generation), rather than “vertically” (from parent
to offspring). In 1928, he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up
by a cell, thereby changing its characteristics.3 He was working with two strains of Streptococcus pneumoniae, a bacterium that
causes pneumonia: a rough (R) strain and a smooth (S) strain. The R strain is nonpathogenic and lacks a capsule on its outer
surface; as a result, colonies from the R strain appear rough when grown on plates. The S strain is pathogenic and has a capsule
outside its cell wall, allowing it to escape phagocytosis by the host immune system. The capsules cause colonies from the S strain
to appear smooth when grown on plates.
In a series of experiments, Griffith analyzed the effects of live R, live S, and heat-killed S strains of S. pneumoniae on live mice
(Figure 6.1.3). When mice were injected with the live S strain, the mice died. When he injected the mice with the live R strain or
the heat-killed S strain, the mice survived. But when he injected the mice with a mixture of live R strain and heat-killed S strain,
the mice died. Upon isolating the live bacteria from the dead mouse, he only recovered the S strain of bacteria. When he then
injected this isolated S strain into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S
strain into the live R strain and “transformed” it into the pathogenic S strain; he called this the “transforming principle.” These
experiments are now famously known as Griffith’s transformation experiments.

Figure 6.1.3 : In his famous series of experiments, Griffith used two strains of S. pneumoniae. The S strain is pathogenic and causes
death. Mice injected with the nonpathogenic R strain or the heat-killed S strain survive. However, a combination of the heat-killed
S strain and the live R strain causes the mice to die. The S strain recovered from the dead mouse showed that something had passed
from the heat-killed S strain to the R strain, transforming the R strain into an S strain in the process.
In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty were interested in exploring Griffith’s transforming principle
further. They isolated the S strain from infected dead mice, heat-killed it, and inactivated various components of the S extract,
conducting a systematic elimination study (Figure 6.1.4). They used enzymes that specifically degraded proteins, RNA, and DNA

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and mixed the S extract with each of these individual enzymes. Then, they tested each extract/enzyme combination’s resulting
ability to transform the R strain, as observed by the diffuse growth of the S strain in culture media and confirmed visually by
growth on plates. They found that when DNA was degraded, the resulting mixture was no longer able to transform the R strain
bacteria, whereas no other enzymatic treatment was able to prevent transformation. This led them to conclude that DNA was the
transforming principle. Despite their results, many scientists did not accept their conclusion, instead believing that there were
protein contaminants within their extracts.

Figure 6.1.4 : Oswald Avery, Colin MacLeod, and Maclyn McCarty followed up on Griffith’s experiment and experimentally
determined that the transforming principle was DNA.

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 Exercise P ageI ndex3

How did Avery, MacLeod, and McCarty’s experiments show that DNA was the transforming principle first described by
Griffith?

Hershey and Chase’s Proof of DNA as Genetic Material

Alfred Hershey and Martha Chase performed their own experiments in 1952 and were able to provide confirmatory evidence that
DNA, not protein, was the genetic material (Figure 6.1.5).4 Hershey and Chase were studying a bacteriophage, a virus that infects
bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic
material, either DNA or RNA (see Viruses). The particular bacteriophage they were studying was the T2 bacteriophage, which
infects E. coli cells. As we now know today, T2 attaches to the surface of the bacterial cell and then it injects its nucleic acids inside
the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a
large number of bacteriophages.
Hershey and Chase labeled the protein coat in one batch of phage using radioactive sulfur, 35S, because sulfur is found in the amino
acids methionine and cysteine but not in nucleic acids. They labeled the DNA in another batch using radioactive phosphorus, 32P,
because phosphorus is found in DNA and RNA but not typically in protein. Each batch of phage was allowed to infect the cells
separately. After infection, Hershey and Chase put each phage bacterial suspension in a blender, which detached the phage coats
from the host cell, and spun down the resulting suspension in a centrifuge. The heavier bacterial cells settled down and formed a
pellet, whereas the lighter phage particles stayed in the supernatant. In the tube with the protein labeled, the radioactivity remained
only in the supernatant. In the tube with the DNA labeled, the radioactivity was detected only in the bacterial cells. Hershey and
Chase concluded that it was the phage DNA that was injected into the cell that carried the information to produce more phage
particles, thus proving that DNA, not proteins, was the source of the genetic material. As a result of their work, the scientific
community more broadly accepted DNA as the molecule responsible for heredity.

Figure 6.1.5 : Martha Chase and Alfred Hershey conducted an experiment separately labeling the DNA and proteins of the T2
bacteriophage to determine which component was the genetic material responsible for the production of new phage particles.
By the time Hershey and Chase published their experiment in the early 1950s, microbiologists and other scientists had been
researching heredity for over 80 years. Building on one another’s research during that time culminated in the general agreement
that DNA was the genetic material responsible for heredity (Figure 6.1.9). This knowledge set the stage for the age of molecular
biology to come and the significant advancements in biotechnology and systems biology that we are experiencing today.

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To learn more about the experiments involved in the history of genetics and the discovery of DNA as the genetic material of cells,
visit this website from the DNA Learning Center.

Exercise 6.1.4

How did Hershey and Chase use microbes to prove that DNA is genetic material?

Figure 6.1.6 : A timeline of key events leading up to the identification of DNA as the molecule responsible for heredity.

Key Concepts and Summary

DNA was discovered and characterized long before its role in heredity was understood. Microbiologists played significant roles
in demonstrating that DNA is the hereditary information found within cells.
In the 1850s and 1860s, Gregor Mendel experimented with true-breeding garden peas to demonstrate the heritability of
specific observable traits.
In 1869, Friedrich Miescher isolated and purified a compound rich in phosphorus from the nuclei of white blood cells; he
named the compound nuclein. Miescher’s student Richard Altmann discovered its acidic nature, renaming it nucleic acid.
Albrecht Kossell characterized the nucleotide bases found within nucleic acids.
Although Walter Sutton and Theodor Boveri proposed the Chromosomal Theory of Inheritance in 1902, it was not
scientifically demonstrated until the 1915 publication of the work of Thomas Hunt Morgan and his colleagues.
Using Acetabularia, a large algal cell, as his model system, Joachim Hämmerling demonstrated in the 1930s and 1940s that the
nucleus was the location of hereditary information in these cells.
In the 1940s, George Beadle and Edward Tatum used the mold Neurospora crassa to show that each protein’s production was
under the control of a single gene, demonstrating the “one gene–one enzyme” hypothesis.
In 1928, Frederick Griffith showed that dead encapsulated bacteria could pass genetic information to live nonencapsulated
bacteria and transform them into harmful strains.
In 1944, Oswald Avery, Colin McLeod, and Maclyn McCarty identified the compound as DNA.
The nature of DNA as the molecule that stores genetic information was unequivocally demonstrated in the experiment of Alfred
Hershey and Martha Chase published in 1952. Labeled DNA from bacterial viruses entered and infected bacterial cells, giving
rise to more viral particles. The labeled protein coats did not participate in the transmission of genetic information.

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Footnotes
1. J.G. Mendel. “Versuche über Pflanzenhybriden.” Verhandlungen des naturforschenden Vereines in Brünn, Bd. Abhandlungen 4
(1865):3–7. (For English translation, see http://www.mendelweb.org/Mendel.plain.html)
2. G.W. Beadle, E.L. Tatum. “Genetic Control of Biochemical Reactions in Neurospora.” Proceedings of the National Academy of
Sciences 27 no. 11 (1941):499–506.
3. F. Griffith. “The Significance of Pneumococcal Types.” Journal of Hygiene 27 no. 2 (1928):8–159.
4. A.D. Hershey, M. Chase. “Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage.” Journal of
General Physiology 36 no. 1 (1952):39–56.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 6.1: Using Microbiology to Discover the Secrets of Life is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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6.2: Structure and Replication of DNA
Learning Objectives
Describe the biochemical structure of deoxyribonucleotides
Identify the base pairs used in the synthesis of deoxyribonucleotides
Explain why the double helix of DNA is described as antiparallel
Explain the meaning of semiconservative DNA replication
Explain why DNA replication is bidirectional and includes both a leading and lagging strand
Explain why Okazaki fragments are formed
Describe the process of DNA replication and the functions of the enzymes involved
Identify the differences between DNA replication in bacteria and eukaryotes
Explain the process of rolling circle replication

Like other macromolecules, nucleic acids are composed of monomers, called nucleotides, which are polymerized to form large
strands. Each nucleic acid strand contains certain nucleotides that appear in a certain order within the strand, called its base
sequence. The base sequence of deoxyribonucleic acid (DNA) is responsible for carrying and retaining the hereditary information
in a cell. Now, we will discuss in detail the ways in which DNA uses its own base sequence to direct its own synthesis, as well as
the synthesis of RNA and proteins, which, in turn, gives rise to products with diverse structure and function. In this section, we will
discuss the basic structure and function of DNA.

DNA Nucleotides
The building blocks of nucleic acids are nucleotides. Nucleotides that compose DNA are called deoxyribonucleotides. The three
components of a deoxyribonucleotide are a five-carbon sugar called deoxyribose, a phosphate group, and a nitrogenous base, a
nitrogen-containing ring structure that is responsible for complementary base pairing between nucleic acid strands (Figure 6.2.1).
The carbon atoms of the five-carbon deoxyribose are numbered 1ʹ, 2ʹ, 3ʹ, 4ʹ, and 5ʹ (1ʹ is read as “one prime”).

Figure 6.2.1 : (a) Each deoxyribonucleotide is made up of a sugar called deoxyribose, a phosphate group, and a nitrogenous base—
in this case, adenine. (b) The five carbons within deoxyribose are designated as 1ʹ, 2ʹ, 3ʹ, 4ʹ, and 5ʹ.
The deoxyribonucleotide is named according to the nitrogenous bases (Figure 6.2.2). The nitrogenous bases adenine (A) and
guanine (G) are the purines; they have a double-ring structure with a six-carbon ring fused to a five-carbon ring. The pyrimidines,
cytosine (C) and thymine (T), are smaller nitrogenous bases that have only a six-carbon ring structure.

Figure 6.2.2 : Nitrogenous bases within DNA are categorized into the two-ringed purines adenine and guanine and the single-ringed
pyrimidines cytosine and thymine. Thymine is unique to DNA.

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Individual nucleotides with three phosphates combine with each other by covalent bonds known as 5ʹ-3ʹ phosphodiester bonds, or
linkages whereby the phosphate group attached to the 5ʹ carbon of the sugar of one nucleotide bonds to the hydroxyl group of the
3ʹ carbon of the sugar of the next nucleotide. Phosphodiester bonding between nucleotides forms the sugar-phosphate backbone,
the alternating sugar-phosphate structure composing the framework of a nucleic acid strand (Figure 6.2.3). During the
polymerization process, deoxynucleotide triphosphates (dNTP) are used. To construct the sugar-phosphate backbone, the two
terminal phosphates are released from the dNTP as a pyrophosphate. The resulting strand of nucleic acid has a free phosphate
group at the 5ʹ carbon end and a free hydroxyl group at the 3ʹ carbon end. The two unused phosphate groups from the nucleotide
triphosphate are released as pyrophosphate during phosphodiester bond formation. Pyrophosphate is subsequently hydrolyzed,
releasing the energy used to drive nucleotide polymerization.

Figure 6.2.3 : Phosphodiester bonds form between the phosphate group attached to the 5ʹ carbon of one nucleotide and the hydroxyl
group of the 3ʹ carbon in the next nucleotide, bringing about polymerization of nucleotides in to nucleic acid strands. Note the 5ʹ
and 3ʹ ends of this nucleic acid strand.

Exercise 6.2.1

What is meant by the 5ʹ and 3ʹ ends of a nucleic acid strand?

Discovering the Double Helix

By the early 1950s, considerable evidence had accumulated indicating that DNA was the genetic material of cells, and now the race
was on to discover its three-dimensional structure. Around this time, Austrian biochemist Erwin Chargaff1(1905–2002) examined
the content of DNA in different species and discovered that adenine, thymine, guanine, and cytosine were not found in equal
quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of
adenine was very close to equaling the amount of thymine, and the amount of cytosine was very close to equaling the amount of
guanine, or A = T and G = C. These relationships are also known as Chargaff’s rules.
Other scientists were also actively exploring this field during the mid-20th century. In 1952, American scientist Linus Pauling
(1901–1994) was the world’s leading structural chemist and odds-on favorite to solve the structure of DNA. Pauling had earlier
discovered the structure of protein α helices, using X-ray diffraction, and, based upon X-ray diffraction images of DNA made in his
laboratory, he proposed a triple-stranded model of DNA.2 At the same time, British researchers Rosalind Franklin (1920–1958) and
her graduate student R.G. Gosling were also using X-ray diffraction to understand the structure of DNA (Figure 6.2.4). It was
Franklin’s scientific expertise that resulted in the production of more well-defined X-ray diffraction images of DNA that would
clearly show the overall double-helix structure of DNA.

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 Figure 6.2.4 : The X-ray diffraction pattern of DNA shows its helical nature. (credit: National Institutes of Health)
James Watson (1928–), an American scientist, and Francis Crick (1916–2004), a British scientist, were working together in the
1950s to discover DNA’s structure. They used Chargaff’s rules and Franklin and Wilkins’ X-ray diffraction images of DNA fibers
to piece together the purine-pyrimidine pairing of the double helical DNA molecule (Figure 6.2.5). In April 1953, Watson and
Crick published their model of the DNA double helix in Nature.3 The same issue additionally included papers by Wilkins and
colleagues,4 as well as by Franklin and Gosling,5 each describing different aspects of the molecular structure of DNA. In 1962,
James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Physiology and Medicine. Unfortunately, by
then Franklin had died, and Nobel prizes at the time were not awarded posthumously. Work continued, however, on learning about
the structure of DNA. In 1973, Alexander Rich (1924–2015) and colleagues were able to analyze DNA crystals to confirm and
further elucidate DNA structure.6

Figure 6.2.5 : In 1953, James Watson and Francis Crick built this model of the structure of DNA, shown here on display at the
Science Museum in London.

Exercise 6.2.2

Which scientists are given most of the credit for describing the molecular structure of DNA?

DNA Structure
Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix.
The two DNA strands are antiparallel, such that the 3ʹ end of one strand faces the 5ʹ end of the other (Figure 6.2.6). The 3ʹ end of
each strand has a free hydroxyl group, while the 5ʹ end of each strand has a free phosphate group. The sugar and phosphate of the
polymerized nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside. These nitrogenous
bases on the interior of the molecule interact with each other, base pairing.

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Analysis of the diffraction patterns of DNA has determined that there are approximately 10 bases per turn in DNA. The
asymmetrical spacing of the sugar-phosphate backbones generates major grooves (where the backbone is far apart) and minor
grooves (where the backbone is close together) (Figure 6.2.6). These grooves are locations where proteins can bind to DNA. The
binding of these proteins can alter the structure of DNA, regulate replication, or regulate transcription of DNA into RNA.

Figure 6.2.6 : Watson and Crick proposed the double helix model for DNA. (a) The sugar-phosphate backbones are on the outside
of the double helix and purines and pyrimidines form the “rungs” of the DNA helix ladder. (b) The two DNA strands are
antiparallel to each other. (c) The direction of each strand is identified by numbering the carbons (1 through 5) in each sugar
molecule. The 5ʹ end is the one where carbon #5 is not bound to another nucleotide; the 3ʹ end is the one where carbon #3 is not
bound to another nucleotide.
Base pairing takes place between a purine and pyrimidine. In DNA, adenine (A) and thymine (T) are complementary base pairs,
and cytosine (C) and guanine (G) are also complementary base pairs, explaining Chargaff’s rules (Figure 6.2.7). The base pairs are
stabilized by hydrogen bonds; adenine and thymine form two hydrogen bonds between them, whereas cytosine and guanine form
three hydrogen bonds between them.

Figure 6.2.7 : Hydrogen bonds form between complementary nitrogenous bases on the interior of DNA.
In the laboratory, exposing the two DNA strands of the double helix to high temperatures or to certain chemicals can break the
hydrogen bonds between complementary bases, thus separating the strands into two separate single strands of DNA (single-
stranded DNA [ssDNA]). This process is called DNA denaturation and is analogous to protein denaturation. The ssDNA strands
can also be put back together as double-stranded DNA (dsDNA), through reannealing or renaturing by cooling or removing the
chemical denaturants, allowing these hydrogen bonds to reform. The ability to artificially manipulate DNA in this way is the basis
for several important techniques in biotechnology (Figure 6.2.8). Because of the additional hydrogen bonding between the C = G
base pair, DNA with a high GC content is more difficult to denature than DNA with a lower GC content.

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 Figure 6.2.8 : In the laboratory, the double helix can be denatured to single-stranded DNA through exposure to heat or chemicals,
and then renatured through cooling or removal of chemical denaturants to allow the DNA strands to reanneal. (credit: modification
of work by Hernández-Lemus E, Nicasio-Collazo LA, Castañeda-Priego R)

View an animation on DNA structure from the DNA Learning Center to learn more.

Exercise 6.2.3

What are the two complementary base pairs of DNA and how are they bonded together?

PAVING THE WAY FOR WOMEN IN SCIENCE AND HEALTH PROFESSIONS

Historically, women have been underrepresented in the sciences and in medicine, and often their pioneering contributions have
gone relatively unnoticed. For example, although Rosalind Franklin performed the X-ray diffraction studies demonstrating the
double helical structure of DNA, it is Watson and Crick who became famous for this discovery, building on her data. There still
remains great controversy over whether their acquisition of her data was appropriate and whether personality conflicts and
gender bias contributed to the delayed recognition of her significant contributions. Similarly, Barbara McClintock did
pioneering work in maize (corn) genetics from the 1930s through 1950s, discovering transposons (jumping genes), but she was
not recognized until much later, receiving a Nobel Prize in Physiology or Medicine in 1983 (Figure 6.2.9).
Today, women still remain underrepresented in many fields of science and medicine. While more than half of the
undergraduate degrees in science are awarded to women, only 46% of doctoral degrees in science are awarded to women. In
academia, the number of women at each level of career advancement continues to decrease, with women holding less than one-
third of the positions of Ph.D.-level scientists in tenure-track positions, and less than one-quarter of the full professorships at 4-
year colleges and universities.7 Even in the health professions, like nearly all other fields, women are often underrepresented in
many medical careers and earn significantly less than their male counterparts, as shown in a 2013 study published by the
Journal of the American Medical Association.8
Why do such disparities continue to exist and how do we break these cycles? The situation is complex and likely results from
the combination of various factors, including how society conditions the behaviors of girls from a young age and supports their
interests, both professionally and personally. Some have suggested that women do not belong in the laboratory, including
Nobel Prize winner Tim Hunt, whose 2015 public comments suggesting that women are too emotional for science9 were met
with widespread condemnation.
Perhaps girls should be supported more from a young age in the areas of science and math (Figure 6.2.9). Science, technology,
engineering, and mathematics (STEM) programs sponsored by the American Association of University Women (AAUW)10
and National Aeronautics and Space Administration (NASA)11 are excellent examples of programs that offer such support.
Contributions by women in science should be made known more widely to the public, and marketing targeted to young girls
should include more images of historically and professionally successful female scientists and medical professionals,
encouraging all bright young minds, including girls and women, to pursue careers in science and medicine.

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 Figure 6.2.9 : (a) Barbara McClintock’s work on maize genetics in the 1930s through 1950s resulted in the discovery of
transposons, but its significance was not recognized at the time. (b) Efforts to appropriately mentor and to provide continued
societal support for women in science and medicine may someday help alleviate some of the issues preventing gender equality
at all levels in science and medicine. (credit a: modification of work by Smithsonian Institution; credit b: modification of work
by Haynie SL, Hinkle AS, Jones NL, Martin CA, Olsiewski PJ, Roberts MF)

Clinical Focus: part 2

Based upon his symptoms, Alex’s physician suspects that he is suffering from a foodborne illness that he acquired during his
travels. Possibilities include bacterial infection (e.g., enterotoxigenic E. coli, Vibrio cholerae, Campylobacter jejuni,
Salmonella), viral infection (rotavirus or norovirus), or protozoan infection (Giardia lamblia, Cryptosporidium parvum, or
Entamoeba histolytica).
His physician orders a stool sample to identify possible causative agents (e.g., bacteria, cysts) and to look for the presence of
blood because certain types of infectious agents (like C. jejuni, Salmonella, and E. histolytica) are associated with the
production of bloody stools.
Alex’s stool sample showed neither blood nor cysts. Following analysis of his stool sample and based upon his recent travel
history, the hospital physician suspected that Alex was suffering from traveler’s diarrhea caused by enterotoxigenic E. coli
(ETEC), the causative agent of most traveler’s diarrhea. To verify the diagnosis and rule out other possibilities, Alex’s
physician ordered a diagnostic lab test of his stool sample to look for DNA sequences encoding specific virulence factors of
ETEC. The physician instructed Alex to drink lots of fluids to replace what he was losing and discharged him from the
hospital.
ETEC produces several plasmid-encoded virulence factors that make it pathogenic compared with typical E. coli. These
include the secreted toxins heat-labile enterotoxin (LT) and heat-stabile enterotoxin (ST), as well as colonization factor (CF).
Both LT and ST cause the excretion of chloride ions from intestinal cells to the intestinal lumen, causing a consequent loss of
water from intestinal cells, resulting in diarrhea. CF encodes a bacterial protein that aids in allowing the bacterium to adhere to
the lining of the small intestine.

Exercise 6.2.4

Why did Alex’s physician use genetic analysis instead of either isolation of bacteria from the stool sample or direct Gram
stain of the stool sample alone?

DNA Function
DNA stores the information needed to build and control the cell. The transmission of this information from mother to daughter
cells is called vertical gene transfer and it occurs through the process of DNA replication. DNA is replicated when a cell makes a
duplicate copy of its DNA, then the cell divides, resulting in the correct distribution of one DNA copy to each resulting cell. DNA
can also be enzymatically degraded and used as a source of nucleotides for the cell. Unlike other macromolecules, DNA does not
serve a structural role in cells.
The elucidation of the structure of the double helix by James Watson and Francis Crick in 1953 provided a hint as to how DNA is
copied during the process of replication. Separating the strands of the double helix would provide two templates for the synthesis of

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new complementary strands, but exactly how new DNA molecules were constructed was still unclear. In one model,
semiconservative replication, the two strands of the double helix separate during DNA replication, and each strand serves as a
template from which the new complementary strand is copied; after replication, each double-stranded DNA includes one parental
or “old” strand and one “new” strand. There were two competing models also suggested: conservative and dispersive, which are
shown in Figure 6.2.10.

Figure 6.2.10 : There were three models suggested for DNA replication. In the conservative model, parental DNA strands (blue)
remained associated in one DNA molecule while new daughter strands (red) remained associated in newly formed DNA molecules.
In the semiconservative model, parental strands separated and directed the synthesis of a daughter strand, with each resulting DNA
molecule being a hybrid of a parental strand and a daughter strand. In the dispersive model, all resulting DNA strands have regions
of double-stranded parental DNA and regions of double-stranded daughter DNA.
Matthew Meselson (1930–) and Franklin Stahl (1929–) devised an experiment in 1958 to test which of these models correctly
represents DNA replication. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N)
that was incorporated into nitrogenous bases and, eventually, into the DNA. This labeled the parental DNA. The E. coli culture was
then shifted into a medium containing 14N and allowed to grow for one generation. The cells were harvested and the DNA was
isolated. The DNA was separated by ultracentrifugation, during which the DNA formed bands according to its density. DNA grown
in 15N would be expected to form a band at a higher density position than that grown in 14N. Meselson and Stahl noted that after
one generation of growth in 14N, the single band observed was intermediate in position in between DNA of cells grown exclusively
in 15N or 14N. This suggested either a semiconservative or dispersive mode of replication. Some cells were allowed to grow for one
more generation in 14N and spun again. The DNA harvested from cells grown for two generations in 14N formed two bands: one
DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These
results could only be explained if DNA replicates in a semiconservative manner. Therefore, the other two models were ruled out.
As a result of this experiment, we now know that during DNA replication, each of the two strands that make up the double helix
serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. The
resulting DNA molecules have the same sequence and are divided equally into the two daughter cells. This means the middle
explanation from Figure 6.2.10 is correct.
DNA Replication in Bacteria
DNA replication has been well studied in bacteria primarily because of the small size of the genome and the mutants that are
available. E. coli has 4.6 million base pairs (Mbp) in a single circular chromosome and all of it is replicated in approximately 42
minutes, starting from a single origin of replication and proceeding around the circle bidirectionally (i.e., in both directions). This
means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs with few errors.
DNA replication uses a large number of proteins and enzymes (Table 6.2.1). One of the key players is the enzyme DNA
polymerase, also known as DNA pol. In bacteria, three main types of DNA polymerases are known: DNA pol I, DNA pol II, and
DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily
required for repair. DNA pol III adds deoxyribonucleotides each complementary to a nucleotide on the template strand, one by one

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to the 3’-OH group of the growing DNA chain. The addition of these nucleotides requires energy. This energy is present in the
bonds of three phosphate groups attached to each nucleotide (a triphosphate nucleotide), similar to how energy is stored in the
phosphate bonds of adenosine triphosphate (ATP) (Figure 6.2.11). When the bond between the phosphates is broken and
diphosphate is released, the energy released allows for the formation of a covalent phosphodiester bond by dehydration synthesis
between the incoming nucleotide and the free 3’-OH group on the growing DNA strand.

Figure 6.2.11 : This structure shows the guanosine triphosphate deoxyribonucleotide that is incorporated into a growing DNA
strand by cleaving the two end phosphate groups from the molecule and transferring the energy to the sugar phosphate bond. The
other three nucleotides form analogous structures.
Initiation
The initiation of replication occurs at specific nucleotide sequence called the origin of replication, where various proteins bind to
begin the replication process. E. coli has a single origin of replication (as do most prokaryotes), called oriC, on its one
chromosome. The origin of replication is approximately 245 base pairs long and is rich in adenine-thymine (AT) sequences.
Some of the proteins that bind to the origin of replication are important in making single-stranded regions of DNA accessible for
replication. Chromosomal DNA is typically wrapped around histones (in eukaryotes and archaea) or histone-like proteins (in
bacteria), and is supercoiled, or extensively wrapped and twisted on itself. This packaging makes the information in the DNA
molecule inaccessible. However, enzymes can change the shape and supercoiling of the chromosome. For bacterial DNA
replication to begin, the supercoiled chromosome is relaxed. An enzyme called helicase then separates the DNA strands by
breaking the hydrogen bonds between the nitrogenous base pairs. Recall that AT sequences have fewer hydrogen bonds and, hence,
have weaker interactions than guanine-cytosine (GC) sequences. These enzymes require ATP hydrolysis. As the DNA opens up, Y-
shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication, allowing for
bidirectional replication and formation of a structure that looks like a bubble when viewed with a transmission electron microscope;
as a result, this structure is called a replication bubble. The DNA near each replication fork is coated with single-stranded binding
proteins to prevent the single-stranded DNA from rewinding into a double helix.
Once single-stranded DNA is accessible at the origin of replication, DNA replication can begin. However, DNA pol III is able to
add nucleotides only in the 5’ to 3’ direction (a new DNA strand can be only extended in this direction). This is because DNA
polymerase requires a free 3’-OH group to which it can add nucleotides by forming a covalent phosphodiester bond between the 3’-
OH end and the 5’ phosphate of the next nucleotide. This also means that it cannot add nucleotides if a free 3’-OH group is not
available, which is the case for a single strand of DNA. The problem is solved with the help of an RNA sequence that provides the
free 3’-OH end. Because this sequence allows the start of DNA synthesis, it is appropriately called the primer. The primer is five to
10 nucleotides long and complementary to the parental or template DNA. It is synthesized by RNA primase, which is an RNA
polymerase. Unlike DNA polymerases, RNA polymerases do not need a free 3’-OH group to synthesize an RNA molecule. Now
that the primer provides the free 3’-OH group, DNA polymerase III can now extend this RNA primer, adding DNA nucleotides one
by one that are complementary to the template strand.
Elongation
During elongation in DNA replication, the addition of nucleotides occurs at its maximal rate of about 1000 nucleotides per second.
DNA polymerase III can only extend in the 5’ to 3’ direction, which poses a problem at the replication fork. The DNA double helix
is antiparallel; that is, one strand is oriented in the 5’ to 3’ direction and the other is oriented in the 3’ to 5’ direction. During
replication, one strand, which is complementary to the 3’ to 5’ parental DNA strand, is synthesized continuously toward the
replication fork because polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the
leading strand. The other strand, complementary to the 5’ to 3’ parental DNA, grows away from the replication fork, so the
polymerase must move back toward the replication fork to begin adding bases to a new primer, again in the direction away from the
replication fork. It does so until it bumps into the previously synthesized strand and then it moves back again (Figure 6.2.12).
These steps produce small DNA sequence fragments known as Okazaki fragments, each separated by RNA primer. Okazaki

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fragments are named after the Japanese research team and married couple Reiji and Tsuneko Okazaki, who first discovered them in
1966. The strand with the Okazaki fragments is known as the lagging strand, and its synthesis is said to be discontinuous.

Figure 6.2.12 : At the origin of replication, topoisomerase II relaxes the supercoiled chromosome. Two replication forks are formed
by the opening of the double-stranded DNA at the origin, and helicase separates the DNA strands, which are coated by single-
stranded binding proteins to keep the strands separated. DNA replication occurs in both directions. An RNA primer complementary
to the parental strand is synthesized by RNA primase and is elongated by DNA polymerase III through the addition of nucleotides
to the 3’-OH end. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in
short stretches called Okazaki fragments. RNA primers within the lagging strand are removed by the exonuclease activity of DNA
polymerase I, and the Okazaki fragments are joined by DNA ligase.
The leading strand can be extended from one primer alone, whereas the lagging strand needs a new primer for each of the short
Okazaki fragments. The overall direction of the lagging strand will be 3’ to 5’, and that of the leading strand 5’ to 3’. A protein
called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped
protein that binds to the DNA and holds the polymerase in place. As synthesis proceeds, the RNA primers are replaced by DNA by
the exonuclease activity of DNA polymerase I, and the gaps are filled in. Any nicks that remain between the newly synthesized
DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase, stabilizing the
sugar-phosphate backbone of the DNA molecule.
Termination

Once the complete chromosome has been replicated, termination of DNA replication must occur. Although much is known about
initiation of replication, less is known about the termination process. Following replication, the resulting complete circular
genomes of prokaryotes are concatenated, meaning that the circular DNA chromosomes are interlocked and must be separated from

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each other. This is accomplished through the activity of bacterial topoisomerase IV, which introduces double-stranded breaks into
DNA molecules, allowing them to separate from each other; the enzyme then reseals the circular chromosomes. The resolution of
concatemers is an issue unique to prokaryotic DNA replication because of their circular chromosomes. Because both bacterial
DNA gyrase and topoisomerase IV are distinct from their eukaryotic counterparts, these enzymes serve as targets for a class of
antimicrobial drugs called quinolones.

Exercise 6.2.5

1. Which enzyme breaks the hydrogen bonds holding the two strands of DNA together so that replication can occur?
2. Is it the lagging strand or the leading strand that is synthesized in the direction toward the opening of the replication fork?
3. Which enzyme is responsible for removing the RNA primers in newly replicated bacterial DNA?

DNA Replication in Eukaryotes

Eukaryotic genomes are much more complex and larger than prokaryotic genomes and are typically composed of multiple linear
chromosomes (Table 6.2.2). The human genome, for example, has 3 billion base pairs per haploid set of chromosomes, and 6
billion base pairs are inserted during replication. There are multiple origins of replication on each eukaryotic chromosome (Figure
6.2.13); the human genome has 30,000 to 50,000 origins of replication. The rate of replication is approximately 100 nucleotides

per second—10 times slower than prokaryotic replication.

Figure 6.2.13 : Eukaryotic chromosomes are typically linear, and each contains multiple origins of replication.
The essential steps of replication in eukaryotes are the same as in prokaryotes. Before replication can start, the DNA has to be made
available as a template. Eukaryotic DNA is highly supercoiled and packaged, which is facilitated by many proteins, including
histones. At the origin of replication, a prereplication complex composed of several proteins, including helicase, forms and recruits
other enzymes involved in the initiation of replication, including topoisomerase to relax supercoiling, single-stranded binding
protein, RNA primase, and DNA polymerase. Following initiation of replication, in a process similar to that found in prokaryotes,
elongation is facilitated by eukaryotic DNA polymerases. The leading strand is continuously synthesized by the eukaryotic
polymerase enzyme pol δ, while the lagging strand is synthesized by pol ε. A sliding clamp protein holds the DNA polymerase in
place so that it does not fall off the DNA. The enzyme ribonuclease H (RNase H), instead of a DNA polymerase as in bacteria,
removes the RNA primer, which is then replaced with DNA nucleotides. The gaps that remain are sealed by DNA ligase.

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Because eukaryotic chromosomes are linear, one might expect that their replication would be more straightforward. As in
prokaryotes, the eukaryotic DNA polymerase can add nucleotides only in the 5’ to 3’ direction. In the leading strand, synthesis
continues until it reaches either the end of the chromosome or another replication fork progressing in the opposite direction. On the
lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork
reaches the end of the linear chromosome, there is no place to make a primer for the DNA fragment to be copied at the end of the
chromosome. These ends thus remain unpaired and, over time, they may get progressively shorter as cells continue to divide.
The ends of the linear chromosomes are known as telomeres and consist of noncoding repetitive sequences. The telomeres protect
coding sequences from being lost as cells continue to divide. In humans, a six base-pair sequence, TTAGGG, is repeated 100 to
1000 times to form the telomere. The discovery of the enzyme telomerase clarified our understanding of how chromosome ends are
maintained. Telomerase contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and
complementary bases to the RNA template are added on the 3’ end of the DNA strand. Once the 3’ end of the lagging strand
template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. In
this way, the ends of the chromosomes are replicated. In humans, telomerase is typically active in germ cells and adult stem cells; it
is not active in adult somatic cells and may be associated with the aging of these cells. Eukaryotic microbes including fungi and
protozoans also produce telomerase to maintain chromosomal integrity. For her discovery of telomerase and its action, Elizabeth
Blackburn (1948–) received the Nobel Prize for Medicine or Physiology in 2009.

Exercise 6.2.3

1. How does the origin of replication differ between eukaryotes and prokaryotes?
2. What polymerase enzymes are responsible for DNA synthesis during eukaryotic replication?
3. What is found at the ends of the chromosomes in eukaryotes and why?

Key Concepts and Summary

Nucleic acids are composed of nucleotides, each of which contains a pentose sugar, a phosphate group, and a nitrogenous
base. Deoxyribonucleotides within DNA contain deoxyribose as the pentose sugar.
DNA contains the pyrimidines cytosine and thymine, and the purines adenine and guanine.
Nucleotides are linked together by phosphodiester bonds between the 5ʹ phosphate group of one nucleotide and the 3ʹ hydroxyl
group of another. A nucleic acid strand has a free phosphate group at the 5ʹ end and a free hydroxyl group at the 3ʹ end.
Chargaff discovered that the amount of adenine is approximately equal to the amount of thymine in DNA, and that the amount
of the guanine is approximately equal to cytosine. These relationships were later determined to be due to complementary base
pairing.
Watson and Crick, building on the work of Chargaff, Franklin and Gosling, and Wilkins, proposed the double helix model and
base pairing for DNA structure.
DNA is composed of two complementary strands oriented antiparallel to each other with the phosphodiester backbones on
the exterior of the molecule. The nitrogenous bases of each strand face each other and complementary bases hydrogen bond to
each other, stabilizing the double helix.
Heat or chemicals can break the hydrogen bonds between complementary bases, denaturing DNA. Cooling or removing
chemicals can lead to renaturation or reannealing of DNA by allowing hydrogen bonds to reform between complementary
bases.
DNA stores the instructions needed to build and control the cell. This information is transmitted from parent to offspring
through vertical gene transfer.
The DNA replication process is semiconservative, which results in two DNA molecules, each having one parental strand of
DNA and one newly synthesized strand.
In bacteria, the initiation of replication occurs at the origin of replication, where supercoiled DNA is unwound by DNA
gyrase, made single-stranded by helicase, and bound by single-stranded binding protein to maintain its single-stranded state.
Primase synthesizes a short RNA primer, providing a free 3’-OH group to which DNA polymerase III can add DNA
nucleotides.
During elongation, the leading strand of DNA is synthesized continuously from a single primer. The lagging strand is
synthesized discontinuously in short Okazaki fragments, each requiring its own primer. The RNA primers are removed and
replaced with DNA nucleotides by bacterial DNA polymerase I, and DNA ligase seals the gaps between these fragments.

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 Termination of replication in bacteria involves the resolution of circular DNA concatemers by topoisomerase IV to release the
two copies of the circular chromosome.
Eukaryotes typically have multiple linear chromosomes, each with multiple origins of replication. Overall, replication in
eukaryotes is similar to that in prokaryotes.
The linear nature of eukaryotic chromosomes necessitates telomeres to protect genes near the end of the chromosomes.
Telomerase extends telomeres, preventing their degradation, in some cell types.

Footnotes
1. N. Kresge et al. “Chargaff's Rules: The Work of Erwin Chargaff.” Journal of Biological Chemistry 280 (2005):e21.
2. L. Pauling, “A Proposed Structure for the Nucleic Acids.” Proceedings of the National Academy of Science of the United States
of America 39 no. 2 (1953):84–97.
3. J.D. Watson, F.H.C. Crick. “A Structure for Deoxyribose Nucleic Acid.” Nature 171 no. 4356 (1953):737–738.
4. M.H.F. Wilkins et al. “Molecular Structure of Deoxypentose Nucleic Acids.” Nature 171 no. 4356 (1953):738–740.
5. R. Franklin, R.G. Gosling. “Molecular Configuration in Sodium Thymonucleate.” Nature 171 no. 4356 (1953):740–741.
6. R.O. Day et al. “A Crystalline Fragment of the Double Helix: The Structure of the Dinucleoside Phosphate Guanylyl-3',5'-
Cytidine.” Proceedings of the National Academy of Sciences of the United States of America 70 no. 3 (1973):849–853.
7. N.H. Wolfinger “For Female Scientists, There's No Good Time to Have Children.” The Atlantic July 29, 2013.
www.theatlantic.com/sexes/arc...ildren/278165/.
8. S.A. Seabury et al. “Trends in the Earnings of Male and Female Health Care Professionals in the United States, 1987 to 2010.”
Journal of the American Medical Association Internal Medicine 173 no. 18 (2013):1748–1750.
9. E. Chung. “Tim Hunt, Sexism and Science: The Real 'Trouble With Girls' in Labs.” CBC News Technology and Science, June
12, 2015. http://www.cbc.ca/news/technology/ti...labs-1.3110133. Accessed 8/4/2016.
10. American Association of University Women. “Building a STEM Pipeline for Girls and Women.” www.aauw.org/what-we-
do/stem-education/. Accessed June 10, 2016.
11. National Aeronautics and Space Administration. “Outreach Programs: Women and Girls Initiative.”
http://women.nasa.gov/outreach-programs/. Accessed June 10, 2016.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 6.2: Structure and Replication of DNA is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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6.3: Structure and Transcription of RNA
Learning Objectives
Describe the biochemical structure of ribonucleotides
Describe the similarities and differences between RNA and DNA
Explain how RNA is synthesized using DNA as a template
Distinguish between transcription in prokaryotes and eukaryote
Describe the functions of the three main types of RNA used in protein synthesis
Explain how RNA can serve as hereditary information

Structurally speaking, ribonucleic acid (RNA), is quite similar to DNA. However, whereas DNA molecules are typically long and
double stranded, RNA molecules are much shorter and are typically single stranded. RNA molecules perform a variety of roles in
the cell but are mainly involved in the process of protein synthesis (translation) and its regulation.

RNA Structure
RNA is typically single stranded and is made of ribonucleotides that are linked by phosphodiester bonds. A ribonucleotide in the
RNA chain contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and a phosphate group. The
subtle structural difference between the sugars gives DNA added stability, making DNA more suitable for storage of genetic
information, whereas the relative instability of RNA makes it more suitable for its more short-term functions.

Figure 6.3.1 : (a) Ribonucleotides contain the pentose sugar ribose instead of the deoxyribose found in deoxyribonucleotides. (b)
RNA contains the pyrimidine uracil in place of thymine found in DNA.
The RNA-specific pyrimidine uracil forms a complementary base pair with adenine and is used instead of the thymine used in
DNA. Even though RNA is single stranded, most types of RNA molecules show extensive intramolecular base pairing between
complementary sequences within the RNA strand, creating a predictable three-dimensional structure essential for their function
(Figure 6.3.1 and Figure 6.3.2).

Figure 6.3.2 : (a) DNA is typically double stranded, whereas RNA is typically single stranded. (b) Although it is single stranded,
RNA can fold upon itself, with the folds stabilized by short areas of complementary base pairing within the molecule, forming a
three-dimensional structure.

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 Exercise 6.3.1

How does the structure of RNA differ from the structure of DNA?

Functions of RNA
DNA serves two essential functions that deal with cellular information. First, DNA is the genetic material responsible for
inheritance and is passed from parent to offspring for all life on earth. To preserve the integrity of this genetic information, DNA
must be replicated with great accuracy, with minimal errors that introduce changes to the DNA sequence. A genome contains the
full complement of DNA within a cell and is organized into smaller, discrete units called genes that are arranged on chromosomes
and plasmids. The second function of DNA is to direct and regulate the construction of the proteins necessary to a cell for growth
and reproduction in a particular cellular environment.
In 1961, French scientists François Jacob and Jacques Monod hypothesized the existence of an intermediary between DNA and its
protein products, which they called messenger RNA.1 Evidence supporting their hypothesis was gathered soon afterwards showing
that information from DNA is transmitted to the ribosome for protein synthesis using RNA. There are three main types of RNA
directly involved in protein synthesis are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). If DNA
serves as the complete library of cellular information, mRNA serves as a photocopy of specific information needed at a particular
point in time that serves as the instructions to make a protein. The other types, rRNA and tRNA serve to aid the decoding of mRNA
into the correct amino acid sequence which then folds into a protein. Proteins within a cell have many functions, including building
cellular structures and serving as enzyme catalysts for cellular chemical reactions that give cells their specific characteristics.
A gene is composed of DNA that is “read” or transcribed to produce an RNA (any type of RNA) molecule during the process of
transcription. The processes of transcription and translation are collectively referred to as gene expression. Gene expression is the
synthesis of a specific protein with a sequence of amino acids that is encoded in the gene. The flow of genetic information from
DNA to RNA to protein is described by the central dogma (Figure 6.3.3). This central dogma of molecular biology further
elucidates the mechanism behind Beadle and Tatum’s “one gene-one enzyme” hypothesis.

Figure 6.3.3 : The central dogma states that DNA encodes messenger RNA, which, in turn, encodes protein.

Genotype vs Phenotype
A cell’s genotype is the full collection of genes it contains, whereas its phenotype is the set of observable characteristics that result
from those genes. The phenotype is the product of the array of proteins being produced by the cell at a given time, which is
influenced by the cell’s genotype as well as interactions with the cell’s environment. Genes code for proteins that have functions in
the cell. Production of a specific protein encoded by an individual gene often results in a distinct phenotype for the cell compared
with the phenotype without that protein. For this reason, it is also common to refer to the genotype of an individual gene and its
phenotype. Although a cell’s genotype remains constant, not all genes are used to direct the production of their proteins
simultaneously. Genes that are always expressed are known as constitutive genes; some constitutive genes are known as
housekeeping genes because they are necessary for the basic functions of the cell.
Cells carefully regulate expression of most of their genes, only using genes to make specific proteins when those proteins are
needed (Figure 6.3.4). If a cell requires a certain protein to be synthesized, the gene for this product is “turned on” and the mRNA
is synthesized through the process of transcription. The mRNA then interacts with ribosomes and other cellular machinery to direct
the synthesis of the protein it encodes during the process of translation. The mRNA is relatively unstable and short-lived in the cell,
especially in prokaryotic cells, ensuring that proteins are only made when needed.

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 Figure 6.3.4 : Phenotype is determined by the specific genes within a genotype that are expressed under specific conditions.
Although multiple cells may have the same genotype, they may exhibit a wide range of phenotypes resulting from differences in
patterns of gene expression in response to different environmental conditions.

Exercise 6.3.2

1. What are the two functions of DNA?

2. Distinguish between the genotype and phenotype of a cell.
3. How can cells have the same genotype but differ in their phenotype?

Transcription
During the process of transcription, the information encoded within the DNA sequence of one or more genes is transcribed into a
strand of RNA, also called an RNA transcript. The resulting single-stranded RNA molecule, composed of ribonucleotides
containing the bases adenine (A), cytosine (C), guanine (G), and uracil (U), acts as a mobile molecular copy of the original DNA
sequence. Transcription in prokaryotes and in eukaryotes requires the DNA double helix to partially unwind in the region of RNA
synthesis. The unwound region is called a transcription bubble. Transcription of a particular gene always proceeds from one of the
two DNA strands that acts as a template, the so-called antisense strand. The RNA product is complementary to the template strand
of DNA and is almost identical to the nontemplate DNA strand, or the sense strand. The only difference is that in RNA, all of the T
nucleotides are replaced with U nucleotides; during RNA synthesis, U is incorporated when there is an A in the complementary
antisense strand.
Transcription in Bacteria
Bacteria use the same RNA polymerase to transcribe all of their genes. Like DNA polymerase, RNA polymerase adds nucleotides
one by one to the 3’-OH group of the growing nucleotide chain. One critical difference in activity between DNA polymerase and
RNA polymerase is the requirement for a 3’-OH onto which to add nucleotides: DNA polymerase requires such a 3’-OH group,
thus necessitating a primer, whereas RNA polymerase does not. During transcription, a ribonucleotide complementary to the DNA
template strand is added to the growing RNA strand and a covalent phosphodiester bond is formed by dehydration synthesis
between the new nucleotide and the last one added.
Initiation
The initiation of transcription begins at a promoter, a DNA sequence onto which the transcription machinery binds and initiates
transcription. The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5’ RNA nucleotide is
transcribed is the initiation site. Nucleotides preceding the initiation site are designated “upstream,” whereas nucleotides following
the initiation site are called “downstream” nucleotides. In most cases, promoters are located just upstream of the genes they
regulate. Although promoter sequences vary among bacterial genomes, a few elements are conserved. At the –10 and –35
(upstream) positions within the DNA prior to the initiation site (designated +1), there are two promoter consensus sequences, or
regions that are similar across all promoters and across various bacterial species. The –10 consensus sequence, called the TATA
box, is TATAAT.
Elongation
The elongation in transcription phase begins when the a RNA polymerase subunit dissociates from the polymerase, allowing the
core enzyme to synthesize RNA complementary to the DNA template in a 5’ to 3’ direction at a rate of approximately 40
nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind
it (Figure 6.3.5).

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 Figure 6.3.5 : During elongation, the bacterial RNA polymerase tracks along the DNA template, synthesizes mRNA in the 5’ to 3’
direction, and unwinds and rewinds the DNA as it is read.
Termination
Once a gene is transcribed, the bacterial polymerase must dissociate from the DNA template and liberate the newly made RNA.
This is referred to as termination of transcription. The DNA template includes repeated nucleotide sequences that act as termination
signals, causing RNA polymerase to stall and release from the DNA template, freeing the RNA transcript.

Exercise 6.3.3

1. Where does σ factor of RNA polymerase bind DNA to start transcription?

2. What occurs to initiate the polymerization activity of RNA polymerase?
3. Where does the signal to end transcription come from?

Transcription in Eukaryotes
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few significant differences.
Eukaryotes use three different polymerases, RNA polymerases I, II, and III, all structurally distinct from the bacterial RNA
polymerase. Each transcribes a different subset of genes. Interestingly, archaea contain a single RNA polymerase that is more
closely related to eukaryotic RNA polymerase II than to its bacterial counterpart. Eukaryotic mRNAs are also usually
monocistronic, meaning that they each encode only a single polypeptide, whereas prokaryotic mRNAs of bacteria and archaea are
commonly polycistronic, meaning that they encode multiple polypeptides.
The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus, which influences the
ease of use of RNA molecules for protein synthesis. With the genes bound in a nucleus, the eukaryotic cell must transport protein-
encoding RNA molecules to the cytoplasm to be translated. Protein-encoding primary transcripts, the RNA molecules directly
synthesized by RNA polymerase, must undergo several processing steps to protect these RNA molecules from degradation during
the time they are transferred from the nucleus to the cytoplasm and translated into a protein. For example, eukaryotic mRNAs may
last for several hours, whereas the typical prokaryotic mRNA lasts no more than 5 seconds.
The primary transcript (also called pre-mRNA) is first coated with RNA-stabilizing proteins to protect it from degradation while it
is processed and exported out of the nucleus. The first type of processing begins while the primary transcript is still being
synthesized; a special nucleotide, called the 5’ cap, is added to the 5’ end of the growing transcript. In addition to preventing
degradation, factors involved in subsequent protein synthesis recognize the cap, which helps initiate translation by ribosomes. Once
elongation is complete, another processing enzyme then adds a string of approximately 200 adenine nucleotides to the 3’ end,
called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals to cellular factors that the
transcript needs to be exported to the cytoplasm.
Eukaryotic genes that encode polypeptides are composed of coding sequences called exons (ex-on signifies that they are expressed)
and intervening sequences called introns (int-ron denotes their intervening role). Transcribed RNA sequences corresponding to
introns do not encode regions of the functional polypeptide and are removed from the pre-mRNA during processing. It is essential

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that all of the intron-encoded RNA sequences are completely and precisely removed from a pre-mRNA before protein synthesis so
that the exon-encoded RNA sequences are properly joined together to code for a functional polypeptide. If the process errs by even
a single nucleotide, the sequences of the rejoined exons would be shifted, and the resulting polypeptide would be nonfunctional,
discussed later in the mutations section. The process of removing intron-encoded RNA sequences and reconnecting those encoded
by exons is called RNA splicing and is facilitated by the action of a spliceosome containing small nuclear ribonucleo proteins
(snRNPs). Intron-encoded RNA sequences are removed from the pre-mRNA while it is still in the nucleus. Although they are not
translated, introns appear to have various functions, including gene regulation and mRNA transport. On completion of these
modifications, the mature transcript, the mRNA that encodes a polypeptide, is transported out of the nucleus, destined for the
cytoplasm for translation. Introns can be spliced out differently, resulting in various exons being included or excluded from the
final mRNA product. This process is known as alternative splicing. The advantage of alternative splicing is that different types of
mRNA transcripts can be generated, all derived from the same DNA sequence. In recent years, it has been shown that some archaea
also have the ability to splice their pre-mRNA.
An illustration shows that before R N A processing, there is a primary R N A transcript including five boxes labeled, left to right, as exon 1, intron, exon 2, intron, and exon 3. After R N A processing, there is a
spliced R N A with these parts, left to right are a 5 prime cap, a 5 prime untranslated region, exon 1, exon 2, exon 3, a 3 prime untranslated region, and a poly a tail.

Figure 6.3.6: Eukaryotic mRNA contains introns that must be spliced out. A 5' cap and 3' poly-A tail are also added.
Visualize how mRNA splicing happens by watching the process in action in this video. See how introns are removed during RNA
splicing here.

Exercise 6.3.4

1. In eukaryotic cells, how is the RNA transcript from a gene for a protein modified after it is transcribed?
2. Do exons or introns contain information for protein sequences?

Role of the other RNAs

The two other type of RNA, rRNA and tRNA, are stable types of RNA. In prokaryotes and eukaryotes, tRNA and rRNA are
encoded in the DNA, then copied into long RNA molecules that are cut to release smaller fragments containing the individual
mature RNA species. In eukaryotes, synthesis, cutting, and assembly of rRNA into ribosomes takes place in the nucleolus region of
the nucleus, but these activities occur in the cytoplasm of prokaryotes. Neither of these types of RNA carries instructions to direct
the synthesis of a polypeptide, but they play other important roles in protein synthesis.
Ribosomes are composed of rRNA and protein. As its name suggests, rRNA is a major constituent of ribosomes, composing up to
about 60% of the ribosome by mass and providing the location where the mRNA binds. The rRNA ensures the proper alignment of
the mRNA, tRNA, and the ribosomes; the rRNA of the ribosome also has an enzymatic activity (peptidyl transferase) and catalyzes
the formation of the peptide bonds between two aligned amino acids during protein synthesis. Although rRNA had long been
thought to serve primarily a structural role, its catalytic role within the ribosome was proven in 2000.2 Scientists in the laboratories
of Thomas Steitz (1940–) and Peter Moore(1939–) at Yale University were able to crystallize the ribosome structure from
Haloarcula marismortui, a halophilic archaeon isolated from the Dead Sea. Because of the importance of this work, Steitz shared
the 2009 Nobel Prize in Chemistry with other scientists who made significant contributions to the understanding of ribosome
structure.
Transfer RNA is the third main type of RNA and one of the smallest, usually only 70–90 nucleotides long. It carries the correct
amino acid to the site of protein synthesis in the ribosome. It is the base pairing between the tRNA and mRNA that allows for the
correct amino acid to be inserted in the polypeptide chain being synthesized (Figure 6.3.7). Any mutations in the tRNA or rRNA
can result in global problems for the cell because both are necessary for proper protein synthesis (Table 6.3.1).

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 Figure 6.3.7 : A tRNA molecule is a single-stranded molecule that exhibits significant intracellular base pairing, giving it its
characteristic three-dimensional shape.
Table 6.3.1 : Structure and Function of RNA
mRNA rRNA tRNA

Short (70-90 nucleotides), stable

Short, unstable, single-stranded Longer, stable RNA molecules RNA with extensive
Structure RNAcorresponding to a gene composing 60% of ribosome’s intramolecular base pairing;
encoded within DNA mass contains an amino acid binding
site and an mRNA binding site

Ensures the proper alignment of

Serves as intermediary between
mRNA, tRNA, and ribosome Carries the correct amino acid to
DNA and protein; used by
Function during protein synthesis; catalyzes the site of protein synthesis in the
ribosome to direct synthesis of
peptide bond formation between ribosome
protein it encodes
amino acids

Exercise 6.3.5

What are the functions of the three major types of RNA molecules involved in protein synthesis?

RNA as Hereditary Information

Although RNA does not serve as the hereditary information in cells, RNA does hold this function for many viruses that do not
contain DNA. Thus, RNA clearly does have the additional capacity to serve as genetic information. Although RNA is typically
single stranded within cells, there is significant diversity in viruses. Rhinoviruses, which cause the common cold; influenza viruses;
and the Ebola virus are single-stranded RNA viruses. Rotaviruses, which cause severe gastroenteritis in children and other
immunocompromised individuals, are examples of double-stranded RNA viruses. Because double-stranded RNA is uncommon in
eukaryotic cells, its presence serves as an indicator of viral infection.

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Key Concepts and Summary
Ribonucleic acid (RNA) is typically single stranded and contains ribose as its pentose sugar and the pyrimidine uracil instead
of thymine. An RNA strand can undergo significant intramolecular base pairing to take on a three-dimensional structure.
During transcription, the information encoded in DNA is used to make RNA.
RNA polymerase synthesizes RNA, using the antisense strand of the DNA as template by adding complementary RNA
nucleotides to the 3’ end of the growing strand.
RNA polymerase binds to DNA at a sequence called a promoter during the initiation of transcription.
Genes encoding proteins of related functions are frequently transcribed under the control of a single promoter in prokaryotes,
resulting in the formation of a polycistronic mRNA molecule that encodes multiple polypeptides.
Unlike DNA polymerase, RNA polymerase does not require a 3’-OH group to add nucleotides, so a primer is not needed
during initiation.
Termination of transcription in bacteria occurs when the RNA polymerase encounters specific DNA sequences that lead to
stalling of the polymerase. This results in release of RNA polymerase from the DNA template strand, freeing the RNA
transcript.
Eukaryotes have three different RNA polymerases. Eukaryotes also have monocistronic mRNA, each encoding only a single
polypeptide.
Eukaryotic primary transcripts are processed in several ways, including the addition of a 5’ cap and a 3′-poly-A tail, as well as
splicing, to generate a mature mRNA molecule that can be transported out of the nucleus and that is protected from
degradation.
There are three main types of RNA, all involved in protein synthesis.
Messenger RNA (mRNA) serves as the intermediary between DNA and the synthesis of protein products during translation.
Ribosomal RNA (rRNA) is a type of stable RNA that is a major constituent of ribosomes. It ensures the proper alignment of the
mRNA and the ribosomes during protein synthesis and catalyzes the formation of the peptide bonds between two aligned amino
acids during protein synthesis.
Transfer RNA (tRNA) is a small type of stable RNA that carries an amino acid to the corresponding site of protein synthesis in
the ribosome. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the
polypeptide chain being synthesized.
Although RNA is not used for long-term genetic information in cells, many viruses do use RNA as their genetic material.

Footnotes
1. 1 A. Rich. “The Era of RNA Awakening: Structural Biology of RNA in the Early Years.” Quarterly Reviews of Biophysics 42
no. 2 (2009):117–137.
2. 2 P. Nissen et al. “The Structural Basis of Ribosome Activity in Peptide Bond Synthesis.” Science 289 no. 5481 (2000):920–
930.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 6.3: Structure and Transcription of RNA is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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6.4: Protein Synthesis (Translation)
Learning Objectives
Describe the genetic code and explain why it is considered almost universal
Explain the process of translation and the functions of the molecular machinery of translation
Compare translation in eukaryotes and prokaryotes

The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more
mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional
(e.g., enzymes) and structural elements. The process of translation, or protein synthesis, the second part of gene expression,
involves the decoding by a ribosome of an mRNA message into a polypeptide product.

The Genetic Code

Translation of the mRNA template converts nucleotide-based genetic information into the “language” of amino acids to create a
protein product. A protein sequence consists of 20 commonly occurring amino acids. Each amino acid is defined within the mRNA
by a triplet of nucleotides called a codon. The relationship between an mRNA codon and its corresponding amino acid is called the
genetic code.
The three-nucleotide code means that there is a total of 64 possible combinations (43, with four different nucleotides possible at
each of the three different positions within the codon). This number is greater than the number of amino acids and a given amino
acid is encoded by more than one codon (Figure 6.4.1). This redundancy in the genetic code is called degeneracy. Typically,
whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing
polypeptide, the third position, called the wobble position, is less critical. In some cases, if the nucleotide in the third position is
changed, the same amino acid is still incorporated.

Figure 6.4.1 : This figure shows the genetic code for translating each nucleotide triplet in mRNA into an amino acid or a
termination signal in a nascent protein. The first letter of a codon is shown vertically on the left, the second letter of a codon is
shown horizontally across the top, and the third letter of a codon is shown vertically on the right. (credit: modification of work by
National Institutes of Health)

Whereas 61 of the 64 possible triplets code for amino acids, three of the 64 codons do not code for an amino acid; they terminate
protein synthesis, releasing the polypeptide from the translation machinery. These are called stop codons or nonsense codons.

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Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also typically serves as
the start codon to initiate translation. The reading frame, the way nucleotides in mRNA are grouped into codons, for translation is
set by the AUG start codon near the 5’ end of the mRNA. Each set of three nucleotides following this start codon is a codon in the
mRNA message.The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic code for
protein synthesis, which is powerful evidence that all extant life on earth shares a common origin. However, unusual amino acids
such as selenocysteine and pyrrolysine have been observed in archaea and bacteria. In the case of selenocysteine, the codon used is
UGA (normally a stop codon). However, UGA can encode for selenocysteine using a stem-loop structure (known as the
selenocysteine insertion sequence, or SECIS element), which is found at the 3’ untranslated region of the mRNA. Pyrrolysine uses
a different stop codon, UAG. The incorporation of pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a
CUA anticodon

Exercise 6.4.1

1. How many bases are in each codon?

2. What amino acid is coded for by the codon AAU?
3. What happens when a stop codon is reached?

The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition
of each component varies across taxa; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNAs) and
polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are
comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various
enzymatic factors.

Ribosomes
A ribosome is a complex macromolecule composed of catalytic rRNAs (called ribozymes) and structural rRNAs, as well as many
distinct polypeptides. Mature rRNAs make up approximately 50% of each ribosome. Prokaryotes have 70S ribosomes, whereas
eukaryotes have 80S ribosomes in the cytoplasm and rough endoplasmic reticulum, and 70S ribosomes in mitochondria and
chloroplasts. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the
initiation of translation. In E. coli, the small subunit is described as 30S (which contains the 16S rRNA subunit), and the large
subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of 70S (Svedberg units are not additive). Eukaryote
ribosomes have a small 40S subunit (which contains the 18S rRNA subunit) and a large 60S subunit (which contains the 5S, 5.8S
and 28S rRNA subunits), for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large
subunit binds tRNAs (discussed in the next subsection).
Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the
mRNA from 5’ to 3’ and synthesizing the polypeptide from the N terminus to the C terminus. The complete structure containing an
mRNA with multiple associated ribosomes is called a polyribosome (or polysome). In both bacteria and archaea, before
transcriptional termination occurs, each protein-encoding transcript is already being used to begin synthesis of numerous copies of
the encoded polypeptide(s) because the processes of transcription and translation can occur concurrently, forming polyribosomes
(Figure 6.4.2). The reason why transcription and translation can occur simultaneously is because both of these processes occur in
the same 5’ to 3’ direction, they both occur in the cytoplasm of the cell, and because the RNA transcript is not processed once it is
transcribed. This allows a prokaryotic cell to respond to an environmental signal requiring new proteins very quickly. In contrast, in
eukaryotic cells, simultaneous transcription and translation is not possible. Although polyribosomes also form in eukaryotes, they
cannot do so until RNA synthesis is complete and the RNA molecule has been modified and transported out of the nucleus.

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 Figure 6.4.2 : In prokaryotes, multiple RNA polymerases can transcribe a single bacterial gene while numerous ribosomes
concurrently translate the mRNA transcripts into polypeptides. In this way, a specific protein can rapidly reach a high concentration
in the bacterial cell.

Transfer RNAs
Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the
cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific
codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules
that actually “translate” the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising
that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA
synthetases, ribosomes, and mRNA.
Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule
hydrogen bond with each other (Figure 6.4.3). This shape positions the amino-acid binding site, called the CCA amino acid
binding end, which is a cytosine-cytosine-adenine sequence at the 3’ end of the tRNA, and the anticodon at the other end. The
anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.
An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA
molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one
type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by
the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is
released.

Figure 6.4.3 : (a) After folding caused by intramolecular base pairing, a tRNA molecule has one end that contains the anticodon,
which interacts with the mRNA codon, and the CCA amino acid binding end. (b) A space-filling model is helpful for visualizing
the three-dimensional shape of tRNA. (c) Simplified models are useful when drawing complex processes such as protein synthesis.

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 Exercise 6.4.2
1. Describe the structure and composition of the prokaryotic ribosome.
2. In what direction is the mRNA template read?
3. Describe the structure and function of a tRNA.

The Mechanism of Protein Synthesis

Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative
prokaryote, and specify any differences between bacterial and eukaryotic translation.

Initiation
The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small
30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate
(GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine(fMet-tRNAfMet) (Figure 6.4.4).
The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its
involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E.
coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal
binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This
interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal
subunit then binds to the initiation complex, forming an intact ribosome.
In eukaryotes, initiation complex formation is similar, with the following differences:
The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5’ cap of the
eukaryotic mRNA, then tracks along the mRNA in the 5’ to 3’ direction until the AUG start codon is recognized. At this point,
the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

Figure 6.4.4 : Translation in bacteria begins with the formation of the initiation complex, which includes the small ribosomal
subunit, the mRNA, the initiator tRNA carrying N-formyl-methionine, and initiation factors. Then the 50S subunit binds, forming
an intact ribosome.

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Elongation
In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli, the binding of the 50S ribosomal
subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming
charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with
the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated
tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During
initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the
A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.
Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation
event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal
movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. Peptide
bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid
attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that
is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide
chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is
expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation,
require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation
apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10
seconds.

Termination
The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no
complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and
eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large
ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation
initiation complex.
In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are
illustrated in Figure 6.4.5 and listed in Figure 6.4.6.

Figure 6.4.5 : (a) In prokaryotes, the processes of transcription and translation occur simultaneously in the cytoplasm, allowing for
a rapid cellular response to an environmental cue. (b) In eukaryotes, transcription is localized to the nucleus and translation is
localized to the cytoplasm, separating these processes and necessitating RNA processing for stability.

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 Figure 6.4.6 : Comparison of translation in bacteria versus eukaryotes

Protein Targeting, Folding, and Modification

During and after translation, polypeptides may need to be modified before they are biologically active. Post-translational
modifications include:
1. removal of translated signal sequences—short tails of amino acids that aid in directing a protein to a specific cellular
compartment
2. proper “folding” of the polypeptide and association of multiple polypeptide subunits, often facilitated by chaperone proteins,
into a distinct three-dimensional structure
3. proteolytic processing of an inactive polypeptide to release an active protein component, and
4. various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of individual amino acids.

Exercise 6.4.3
1. What are the components of the initiation complex for translation in prokaryotes?
2. What are two differences between initiation of prokaryotic and eukaryotic translation?
3. What occurs at each of the three active sites of the ribosome?
4. What causes termination of translation?

Key Concepts and Summary

In translation, polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match
mRNA codons to specific amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.
The genetic code is degenerate in that several mRNA codons code for the same amino acids. The genetic code is almost
universal among living organisms.
Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large subunit and a small subunit of
differing sizes between the two groups. Each subunit is composed of rRNA and protein. Organelle ribosomes in eukaryotic cells
resemble prokaryotic ribosomes.
Some 60 to 90 species of tRNA exist in bacteria. Each tRNA has a three-nucleotide anticodon as well as a binding site for a
cognate amino acid. All tRNAs with a specific anticodon will carry the same amino acid.
Initiation of translation occurs when the small ribosomal subunit binds with initiation factors and an initiator tRNA at the
start codon of an mRNA, followed by the binding to the initiation complex of the large ribosomal subunit.
In prokaryotic cells, the start codon codes for N-formyl-methionine carried by a special initiator tRNA. In eukaryotic cells, the
start codon codes for methionine carried by a special initiator tRNA. In addition, whereas ribosomal binding of the mRNA in
prokaryotes is facilitated by the Shine-Dalgarno sequence within the mRNA, eukaryotic ribosomes bind to the 5’ cap of the
mRNA.

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 During the elongation stage of translation, a charged tRNA binds to mRNA in the A site of the ribosome; a peptide bond is
catalyzed between the two adjacent amino acids, breaking the bond between the first amino acid and its tRNA; the ribosome
moves one codon along the mRNA; and the first tRNA is moved from the P site of the ribosome to the E site and leaves the
ribosomal complex.
Termination of translation occurs when the ribosome encounters a stop codon, which does not code for a tRNA. Release
factors cause the polypeptide to be released, and the ribosomal complex dissociates.
In prokaryotes, transcription and translation may be coupled, with translation of an mRNA molecule beginning as soon as
transcription allows enough mRNA exposure for the binding of a ribosome, prior to transcription termination. Transcription and
translation are not coupled in eukaryotes because transcription occurs in the nucleus, whereas translation occurs in the
cytoplasm or in association with the rough endoplasmic reticulum.
Polypeptides often require one or more post-translational modifications to become biologically active.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 6.4: Protein Synthesis (Translation) is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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6.5: Mutations
Learning Objectives
Compare point mutations and frameshift mutations
Describe the differences between missense, nonsense, and silent mutations
Explain how different mutagens act
Compare different types o f repair mechanisms
Explain why the Ames test can be used to detect carcinogens
Analyze sequences of DNA and identify examples of types of mutations

A mutation is a heritable change in the DNA sequence of an organism. The resulting organism, called a mutant, may have a
recognizable change in phenotype compared to the wild type, which is the phenotype most commonly observed in nature. A change
in the DNA sequence is conferred to mRNA through transcription, and may lead to an altered amino acid sequence in a protein on
translation. Because proteins carry out the vast majority of cellular functions, a change in amino acid sequence in a protein may
lead to an altered phenotype for the cell and organism.

Effects of Mutations on DNA Sequence

There are several types of mutations that are classified according to how the DNA molecule is altered. One type, called a point
mutation, affects a single base and most commonly occurs when one base is substituted or replaced by another. Mutations also
result from the addition of one or more bases, known as an insertion, or the removal of one or more bases, known as a deletion.

Exercise 6.5.1

What type of a mutation occurs when a gene has two fewer nucleotides in its sequence?

Effects of Mutations on Protein Structure and Function

Point mutations may have a wide range of effects on protein function (Figure 6.5.1). As a consequence of the degeneracy of the
genetic code, a point mutation will commonly result in the same amino acid being incorporated into the resulting polypeptide
despite the sequence change. This change would have no effect on the protein’s structure, and is thus called a silent mutation. A
missense mutation results in a different amino acid being incorporated into the resulting polypeptide. The effect of a missense
mutation depends on how chemically different the new amino acid is from the wild-type amino acid. The location of the changed
amino acid within the protein also is important. For example, if the changed amino acid is part of the enzyme’s active site, then the
effect of the missense mutation may be significant. Many missense mutations result in proteins that are still functional, at least to
some degree. Sometimes the effects of missense mutations may be only apparent under certain environmental conditions; such
missense mutations are called conditional mutations. Rarely, a missense mutation may be beneficial. Under the right environmental
conditions, this type of mutation may give the organism that harbors it a selective advantage. Yet another type of point mutation,
called a nonsense mutation, converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon).
Nonsense mutations result in the synthesis of proteins that are shorter than the wild type and typically not functional.
Deletions and insertions also cause various effects. Because codons are triplets of nucleotides, insertions or deletions in groups of
three nucleotides may lead to the insertion or deletion of one or more amino acids and may not cause significant effects on the
resulting protein’s functionality. However, frameshift mutations, caused by insertions or deletions of a number of nucleotides that
are not a multiple of three are extremely problematic because a shift in the reading frame results (Figure 6.5.1). Because ribosomes
read the mRNA in triplet codons, frameshift mutations can change every amino acid after the point of the mutation. The new
reading frame may also include a stop codon before the end of the coding sequence. Consequently, proteins made from genes
containing frameshift mutations are nearly always nonfunctional.

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 Figure 6.5.1 : Mutations can lead to changes in the protein sequence encoded by the DNA.

Exercise 6.5.2

1. What are the reasons a nucleotide change in a gene for a protein might not have any effect on the phenotype of that gene?
2. Is it possible for an insertion of three nucleotides together after the fifth nucleotide in a protein-coding gene to produce a
protein that is shorter than normal? How or how not?

A Beneficial Mutation

Since the first case of infection with human immunodeficiency virus (HIV) was reported in 1981, nearly 40 million people
have died from HIV infection,1 the virus that causes acquired immune deficiency syndrome (AIDS). The virus targets helper T
cells that play a key role in bridging the innate and adaptive immune response, infecting and killing cells normally involved in
the body’s response to infection. There is no cure for HIV infection, but many drugs have been developed to slow or block the

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progression of the virus. Although individuals around the world may be infected, the highest prevalence among people 15–49
years old is in sub-Saharan Africa, where nearly one person in 20 is infected, accounting for greater than 70% of the infections
worldwide2 (Figure 6.5.2). Unfortunately, this is also a part of the world where prevention strategies and drugs to treat the
infection are the most lacking.

Figure 6.5.2 : HIV is highly prevalent in sub-Saharan Africa, but its prevalence is quite low in some other parts of the world.
In recent years, scientific interest has been piqued by the discovery of a few individuals from northern Europe who are resistant
to HIV infection. In 1998, American geneticist Stephen J. O’Brien at the National Institutes of Health (NIH) and colleagues
published the results of their genetic analysis of more than 4,000 individuals. These indicated that many individuals of
Eurasian descent (up to 14% in some ethnic groups) have a deletion mutation, called CCR5-delta 32, in the gene encoding
CCR5. CCR5 is a coreceptor found on the surface of T cells that is necessary for many strains of the virus to enter the host cell.
The mutation leads to the production of a receptor to which HIV cannot effectively bind and thus blocks viral entry. People
homozygous for this mutation have greatly reduced susceptibility to HIV infection, and those who are heterozygous have some
protection from infection as well.
It is not clear why people of northern European descent, specifically, carry this mutation, but its prevalence seems to be highest
in northern Europe and steadily decreases in populations as one moves south. Research indicates that the mutation has been
present since before HIV appeared and may have been selected for in European populations as a result of exposure to the
plague or smallpox. This mutation may protect individuals from plague (caused by the bacterium Yersinia pestis) and smallpox
(caused by the variola virus) because this receptor may also be involved in these diseases. The age of this mutation is a matter
of debate, but estimates suggest it appeared between 1875 years to 225 years ago, and may have been spread from Northern
Europe through Viking invasions.
This exciting finding has led to new avenues in HIV research, including looking for drugs to block CCR5 binding to HIV in
individuals who lack the mutation. Although DNA testing to determine which individuals carry the CCR5-delta 32 mutation is
possible, there are documented cases of individuals homozygous for the mutation contracting HIV. For this reason, DNA
testing for the mutation is not widely recommended by public health officials so as not to encourage risky behavior in those

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 who carry the mutation. Nevertheless, inhibiting the binding of HIV to CCR5 continues to be a valid strategy for the
development of drug therapies for those infected with HIV.

Causes of Mutations
Mistakes in the process of DNA replication can cause spontaneous mutations to occur. The error rate of DNA polymerase is one
incorrect base per billion base pairs replicated. Exposure to mutagens can cause induced mutations, which are various types of
chemical agents or radiation (Table 6.5.1). Exposure to a mutagen can increase the rate of mutation more than 1000-fold. Mutagens
are often also carcinogens, agents that cause cancer. However, whereas nearly all carcinogens are mutagenic, not all mutagens are
necessarily carcinogens.
Chemical Mutagens
Various types of chemical mutagens interact directly with DNA either by acting as nucleoside analogs or by modifying nucleotide
bases. Chemicals called nucleoside analogs are structurally similar to normal nucleotide bases and can be incorporated into DNA
during replication (Figure 6.5.3). These base analogs induce mutations because they often have different base-pairing rules than the
bases they replace. Other chemical mutagens can modify normal DNA bases, resulting in different base-pairing rules. For example,
nitrous acid deaminates cytosine, converting it to uracil. Uracil then pairs with adenine in a subsequent round of replication,
resulting in the conversion of a GC base pair to an AT base pair. Nitrous acid also deaminates adenine to hypoxanthine, which base
pairs with cytosine instead of thymine, resulting in the conversion of a TA base pair to a CG base pair.

Figure 6.5.3 : (a) 2-aminopurine nucleoside (2AP) structurally is a nucleoside analog to adenine nucleoside, whereas 5-bromouracil
(5BU) is a nucleoside analog to thymine nucleoside. 2AP base pairs with C, converting an AT base pair to a GC base pair after
several rounds of replication. 5BU pairs with G, converting an AT base pair to a GC base pair after several rounds of replication.
(b) Nitrous acid is a different type of chemical mutagen that modifies already existing nucleoside bases like C to produce U, which
base pairs with A. This chemical modification, as shown here, results in converting a CG base pair to a TA base pair.
Chemical mutagens known as intercalating agents work differently. These molecules slide between the stacked nitrogenous bases
of the DNA double helix, distorting the molecule and creating atypical spacing between nucleotide base pairs (Figure 6.5.4). As a
result, during DNA replication, DNA polymerase may either skip replicating several nucleotides (creating a deletion) or insert extra
nucleotides (creating an insertion). Either outcome may lead to a frameshift mutation. Combustion products like polycyclic

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aromatic hydrocarbons are particularly dangerous intercalating agents that can lead to mutation-caused cancers. The intercalating
agents ethidium bromide and acridine orange are commonly used in the laboratory to stain DNA for visualization and are potential
mutagens.

Figure 6.5.4 : Intercalating agents, such as acridine, introduce atypical spacing between base pairs, resulting in DNA polymerase
introducing either a deletion or an insertion, leading to a potential frameshift mutation.
Radiation
Exposure to either ionizing or nonionizing radiation can each induce mutations in DNA, although by different mechanisms. Strong
ionizing radiation like X-rays and gamma rays can cause single- and double-stranded breaks in the DNA backbone through the
formation of hydroxyl radicals on radiation exposure (Figure 6.5.5). Ionizing radiation can also modify bases; for example, the
deamination of cytosine to uracil, analogous to the action of nitrous acid.3 Ionizing radiation exposure is used to kill microbes to
sterilize medical devices and foods, because of its dramatic nonspecific effect in damaging DNA, proteins, and other cellular
components.
Nonionizing radiation, like ultraviolet light, is not energetic enough to initiate these types of chemical changes. However,
nonionizing radiation can induce dimer formation between two adjacent pyrimidine bases, commonly two thymines, within a
nucleotide strand. During thymine dimer formation, the two adjacent thymines become covalently linked and, if left unrepaired,
both DNA replication and transcription are stalled at this point. DNA polymerase may proceed and replicate the dimer incorrectly,
potentially leading to frameshift or point mutations.

Figure 6.5.5 : (a) Ionizing radiation may lead to the formation of single-stranded and double-stranded breaks in the sugar-phosphate
backbone of DNA, as well as to the modification of bases (not shown). (b) Nonionizing radiation like ultraviolet light can lead to
the formation of thymine dimers, which can stall replication and transcription and introduce frameshift or point mutations.
Table 6.5.1 : A Summary of Mutagenic Agents

Mutagenic Agents Mode of Action Effect on DNA Resulting Type of Mutation

Nucleoside analogs

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 Mutagenic Agents Mode of Action Effect on DNA Resulting Type of Mutation

Is inserted in place of A but base

2-aminopurine Converts AT to GC base pair Point
pairs with C

Is inserted in place of T but base

5-bromouracil Converts AT to GC base pair Point
pairs with G

Nucleotide-modifying agent

Nitrous oxide Deaminates C to U Converts GC to AT base pair Point

Intercalating agents

Acridine orange, ethidium Distorts double helix, creates

Introduces small deletions and
bromide, polycyclic aromatic unusual spacing between Frameshift
insertions
hydrocarbons nucleotides

Ionizing radiation

Causes single- and double-strand Repair mechanisms may introduce

X-rays, γ-rays Forms hydroxyl radicals
DNA breaks mutations

Modifies bases (e.g., deaminating

X-rays, γ-rays Converts GC to AT base pair Point
C to U)

Nonionizing radiation

Forms pyrimidine (usually

Ultraviolet Causes DNA replication errors Frameshift or point
thymine) dimers

Exercise 6.5.3

1. How does a base analog introduce a mutation?

2. How does an intercalating agent introduce a mutation?
3. What type of mutagen causes thymine dimers?

DNA Repair
The process of DNA replication is highly accurate, but mistakes can occur spontaneously or be induced by mutagens. Uncorrected
mistakes can lead to serious consequences for the phenotype. Cells have developed several repair mechanisms to minimize the
number of mutations that persist.
Proofreading

Most of the mistakes introduced during DNA replication are promptly corrected by most DNA polymerases through a function
called proofreading. In proofreading, the DNA polymerase reads the newly added base, ensuring that it is complementary to the
corresponding base in the template strand before adding the next one. If an incorrect base has been added, the enzyme makes a cut
to release the wrong nucleotide and a new base is added.
Mismatch Repair
Some errors introduced during replication are corrected shortly after the replication machinery has moved. This mechanism is
called mismatch repair. The enzymes involved in this mechanism recognize the incorrectly added nucleotide, excise it, and replace
it with the correct base. One example is the methyl-directed mismatch repair in E. coli. The DNA is hemimethylated. This means
that the parental strand is methylated while the newly synthesized daughter strand is not. It takes several minutes before the new
strand is methylated. Proteins MutS, MutL, and MutH bind to the hemimethylated site where the incorrect nucleotide is found.
MutH cuts the nonmethylated strand (the new strand). An exonuclease removes a portion of the strand (including the incorrect
nucleotide). The gap formed is then filled in by DNA pol III and ligase.

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Repair of Thymine Dimers
Because the production of thymine dimers is common (many organisms cannot avoid ultraviolet light), mechanisms have evolved
to repair these lesions. In nucleotide excision repair (also called dark repair), enzymes remove the pyrimidine dimer and replace it
with the correct nucleotides (Figure 6.5.6). In E. coli, the DNA is scanned by an enzyme complex. If a distortion in the double
helix is found that was introduced by the pyrimidine dimer, the enzyme complex cuts the sugar-phosphate backbone several bases
upstream and downstream of the dimer, and the segment of DNA between these two cuts is then enzymatically removed. DNA pol
I replaces the missing nucleotides with the correct ones and DNA ligase seals the gap in the sugar-phosphate backbone.

Figure 6.5.6 : Bacteria have two mechanisms for repairing thymine dimers. (a) In nucleotide excision repair, an enzyme complex
recognizes the distortion in the DNA complex around the thymine dimer and cuts and removes the damaged DNA strand. The
correct nucleotides are replaced by DNA pol I and the nucleotide strand is sealed by DNA ligase. (b) In photoreactivation, the
enzyme photolyase binds to the thymine dimer and, in the presence of visible light, breaks apart the dimer, restoring the base
pairing of the thymines with complementary adenines on the opposite DNA strand.
The direct repair (also called light repair) of thymine dimers occurs through the process of photoreactivation in the presence of
visible light. An enzyme called photolyase recognizes the distortion in the DNA helix caused by the thymine dimer and binds to the

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dimer. Then, in the presence of visible light, the photolyase enzyme changes conformation and breaks apart the thymine dimer,
allowing the thymines to again correctly base pair with the adenines on the complementary strand. Photoreactivation appears to be
present in all organisms, with the exception of placental mammals, including humans. Photoreactivation is particularly important
for organisms chronically exposed to ultraviolet radiation, like plants, photosynthetic bacteria, algae, and corals, to prevent the
accumulation of mutations caused by thymine dimer formation

Exercise 6.5.4

1. During mismatch repair, how does the enzyme recognize which is the new and which is the old strand?
2. How does an intercalating agent introduce a mutation?
3. What type of mutation does photolyase repair?

Identifying Bacterial Mutants

One common technique used to identify bacterial mutants is called replica plating. This technique is used to detect nutritional
mutants, called auxotrophs, which have a mutation in a gene encoding an enzyme in the biosynthesis pathway of a specific nutrient,
such as an amino acid. As a result, whereas wild-type cells retain the ability to grow normally on a medium lacking the specific
nutrient, auxotrophs are unable to grow on such a medium. During replica plating (Figure 6.5.7), a population of bacterial cells is
mutagenized and then plated as individual cells on a complex nutritionally complete plate and allowed to grow into colonies. Cells
from these colonies are removed from this master plate, often using sterile velvet. This velvet, containing cells, is then pressed in
the same orientation onto plates of various media. At least one plate should also be nutritionally complete to ensure that cells are
being properly transferred between the plates. The other plates lack specific nutrients, allowing the researcher to discover various
auxotrophic mutants unable to produce specific nutrients. Cells from the corresponding colony on the nutritionally complete plate
can be used to recover the mutant for further study.

Figure 6.5.7 : Identification of auxotrophic mutants, like histidine auxotrophs, is done using replica plating. After mutagenesis,
colonies that grow on nutritionally complete medium but not on medium lacking histidine are identified as histidine auxotrophs.

Exercise 6.5.5

Why are cells plated on a nutritionally complete plate in addition to nutrient-deficient plates when looking for a mutant?

The Ames Test

The Ames test, developed by Bruce Ames (1928–) in the 1970s, is a method that uses bacteria for rapid, inexpensive screening of
the carcinogenic potential of new chemical compounds. The test measures the mutation rate associated with exposure to the

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compound, which, if elevated, may indicate that exposure to this compound is associated with greater cancer risk. The Ames test
uses as the test organism a strain of Salmonella typhimurium that is a histidine auxotroph, unable to synthesize its own histidine
because of a mutation in an essential gene required for its synthesis. After exposure to a potential mutagen, these bacteria are plated
onto a medium lacking histidine, and the number of mutants regaining the ability to synthesize histidine is recorded and compared
with the number of such mutants that arise in the absence of the potential mutagen (Figure 6.5.8). Chemicals that are more
mutagenic will bring about more mutants with restored histidine synthesis in the Ames test. Because many chemicals are not
directly mutagenic but are metabolized to mutagenic forms by liver enzymes, rat liver extract is commonly included at the start of
this experiment to mimic liver metabolism. After the Ames test is conducted, compounds identified as mutagenic are further tested
for their potential carcinogenic properties by using other models, including animal models like mice and rats.

Figure 6.5.8 : The Ames test is used to identify mutagenic, potentially carcinogenic chemicals. A Salmonella histidine auxotroph is
used as the test strain, exposed to a potential mutagen/carcinogen. The number of reversion mutants capable of growing in the
absence of supplied histidine is counted and compared with the number of natural reversion mutants that arise in the absence of the
potential mutagen.

Exercise 6.5.6

1. What mutation is used as an indicator of mutation rate in the Ames test?

2. Why can the Ames test work as a test for carcinogenicity?

Key Concepts and Summary

A mutation is a heritable change in DNA. A mutation may lead to a change in the amino-acid sequence of a protein, possibly
affecting its function.
A point mutation affects a single base pair. A point mutation may cause a silent mutation if the mRNA codon codes for the
same amino acid, a missense mutation if the mRNA codon codes for a different amino acid, or a nonsense mutation if the
mRNA codon becomes a stop codon.
Missense mutations may retain function, depending on the chemistry of the new amino acid and its location in the protein.
Nonsense mutations produce truncated and frequently nonfunctional proteins.
A frameshift mutation results from an insertion or deletion of a number of nucleotides that is not a multiple of three. The
change in reading frame alters every amino acid after the point of the mutation and results in a nonfunctional protein.
Spontaneous mutations occur through DNA replication errors, whereas induced mutations occur through exposure to a
mutagen.
Mutagenic agents are frequently carcinogenic but not always. However, nearly all carcinogens are mutagenic.

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 Chemical mutagens include base analogs and chemicals that modify existing bases. In both cases, mutations are introduced after
several rounds of DNA replication.
Ionizing radiation, such as X-rays and γ-rays, leads to breakage of the phosphodiester backbone of DNA and can also
chemically modify bases to alter their base-pairing rules.
Nonionizing radiation like ultraviolet light may introduce pyrimidine (thymine) dimers, which, during DNA replication and
transcription, may introduce frameshift or point mutations.
Cells have mechanisms to repair naturally occurring mutations. DNA polymerase has proofreading activity. Mismatch repair is
a process to repair incorrectly incorporated bases after DNA replication has been completed.
Pyrimidine dimers can also be repaired. In nucleotide excision repair (dark repair), enzymes recognize the distortion
introduced by the pyrimidine dimer and replace the damaged strand with the correct bases, using the undamaged DNA strand as
a template. Bacteria and other organisms may also use direct repair, in which the photolyase enzyme, in the presence of visible
light, breaks apart the pyrimidines.
Through comparison of growth on the complete plate and lack of growth on media lacking specific nutrients, specific loss-of-
function mutants called auxotrophs can be identified.
The Ames test is an inexpensive method that uses auxotrophic bacteria to measure mutagenicity of a chemical compound.
Mutagenicity is an indicator of carcinogenic potential.

Footnotes
1. 1 World Health Organization. “ Global Health Observatory (GHO) Data, HIV/AIDS.” http://www.who.int/gho/hiv/en/.
Accessed August 5, 2016.
2. 2 World Health Organization. “ Global Health Observatory (GHO) Data, HIV/AIDS.” http://www.who.int/gho/hiv/en/.
Accessed August 5, 2016.
3. 3 K.R. Tindall et al. “Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage.”
Genetics 118 no. 4 (1988):551–560.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 6.5: Mutations is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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6.6: How Asexual Prokaryotes Achieve Genetic Diversity
Learning Objectives
Compare the processes of transformation, transduction, and conjugation
Explain how asexual gene transfer results in prokaryotic genetic diversity
Explain the structure and consequences for bacterial genetic diversity of transposons

Typically, when we consider genetic transfer, we think of vertical gene transfer, the transmission of genetic information from
generation to generation. Vertical gene transfer is by far the main mode of transmission of genetic information in all cells. In
sexually reproducing organisms, crossing-over events and independent assortment of individual chromosomes during meiosis
contribute to genetic diversity in the population. Genetic diversity is also introduced during sexual reproduction, when the genetic
information from two parents, each with different complements of genetic information, are combined, producing new combinations
of parental genotypes in the diploid offspring. The occurrence of mutations also contributes to genetic diversity in a population.
Genetic diversity of offspring is useful in changing or inconsistent environments and may be one reason for the evolutionary
success of sexual reproduction.
When prokaryotes and eukaryotes reproduce asexually, they transfer a nearly identical copy of their genetic material to their
offspring through vertical gene transfer. Although asexual reproduction produces more offspring more quickly, any benefits of
diversity among those offspring are lost. How then do organisms whose dominant reproductive mode is asexual create genetic
diversity? In prokaryotes, horizontal gene transfer (HGT), the introduction of genetic material from one organism to another
organism within the same generation, is an important way to introduce genetic diversity. HGT allows even distantly related species
to share genes, influencing their phenotypes. It is thought that HGT is more prevalent in prokaryotes but that only a small fraction
of the prokaryotic genome may be transferred by this type of transfer at any one time. As the phenomenon is investigated more
thoroughly, it may be revealed to be even more common. Many scientists believe that HGT and mutation are significant sources of
genetic variation, the raw material for the process of natural selection, in prokaryotes. Although HGT is more common among
evolutionarily related organisms, it may occur between any two species that live together in a natural community.
HGT in prokaryotes is known to occur by the three primary mechanisms that are illustrated in Figure 6.6.1:
1. Transformation: naked DNA is taken up from the environment
2. Transduction: genes are transferred between cells in a virus
3. Conjugation: use of a hollow tube called a conjugation pilus to transfer genes between cells

Figure 6.6.1 : There are three prokaryote-specific mechanisms leading to horizontal gene transfer in prokaryotes. a) In
transformation, the cell takes up DNA directly from the environment. The DNA may remain separate as a plasmid or be
incorporated into the host genome. b) In transduction, a bacteriophage injects DNA that is a hybrid of viral DNA and DNA from a
previously infected bacterial cell. c) In conjugation, DNA is transferred between cells through a cytoplasmic bridge after a
conjugation pilus draws the two cells close enough to form the bridge.

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 Exercise 6.6.1

1. What are three ways sexual reproduction introduces genetic variation into offspring?
2. What is a benefit of asexual reproduction?
3. What are the three mechanisms of horizontal gene transfer in prokaryotes?

Transformation
Frederick Griffith was the first to demonstrate the process of transformation. In 1928, he showed that live, nonpathogenic
Streptococcus pneumoniae bacteria could be transformed into pathogenic bacteria through exposure to a heat-killed pathogenic
strain. He concluded that some sort of agent, which he called the “transforming principle,” had been passed from the dead
pathogenic bacteria to the live, nonpathogenic bacteria. In 1944, Oswald Avery (1877–1955), Colin MacLeod (1909–1972), and
Maclyn McCarty (1911–2005) demonstrated that the transforming principle was DNA.
In transformation, the prokaryote takes up naked DNA found in its environment and that is derived from other cells that have lysed
on death and released their contents, including their genome, into the environment. Many bacteria are naturally competent,
meaning that they actively bind to environmental DNA, transport it across their cell envelopes into their cytoplasm, and make it
single stranded. Typically, double-stranded foreign DNA within cells is destroyed by nucleases as a defense against viral infection.
However, these nucleases are usually ineffective against single-stranded DNA, so this single-stranded DNA within the cell has the
opportunity to recombine into the bacterial genome. A molecule of DNA that contains fragments of DNA from different organisms
is called recombinant DNA. If the bacterium incorporates the new DNA into its own genome through recombination, the bacterial
cell may gain new phenotypic properties. For example, if a nonpathogenic bacterium takes up DNA for a toxin gene from a
pathogen and then incorporates it into its chromosome, it, too, may become pathogenic. Plasmid DNA may also be taken up by
competent bacteria and confer new properties to the cell. Overall, transformation in nature is a relatively inefficient process because
environmental DNA levels are low because of the activity of nucleases that are also released during cellular lysis. Additionally,
genetic recombination is inefficient at incorporating new DNA sequences into the genome.
In nature, bacterial transformation is an important mechanism for the acquisition of genetic elements encoding virulence factors
and antibiotic resistance. Genes encoding resistance to antimicrobial compounds have been shown to be widespread in nature, even
in environments not influenced by humans. These genes, which allow microbes living in mixed communities to compete for limited
resources, can be transferred within a population by transformation, as well as by the other processes of HGT. In the laboratory, we
can exploit the natural process of bacterial transformation for genetic engineering to make a wide variety of medicinal products, as
discussed in chapter 10.

Exercise 6.6.2

Why does a bacterial cell make environmental DNA brought into the cell into a single-stranded form?

Transduction
Viruses that infect bacteria (bacteriophages) may also move short pieces of chromosomal DNA from one bacterium to another in a
process called transduction (see chapter 9 for more details). In generalized transduction, any piece of chromosomal DNA may be
transferred to a new host cell by accidental packaging of chromosomal DNA into a phage head during phage assembly. By contrast,
specialized transduction results from the imprecise excision of an embedded (lysogenic) prophage from the bacterial chromosome
such that it carries with it a piece of the bacterial chromosome from either side of the phage’s integration site to a new host cell. As
a result, the host may acquire new properties. This process is called lysogenic conversion. Of medical significance, a lysogenic
phage may carry with it a virulence gene to its new host. Once inserted into the new host’s chromosome, the new host may gain
pathogenicity. Several pathogenic bacteria, including Corynebacterium diphtheriae (the causative agent of diphtheria) and
Clostridium botulinum (the causative agent of botulism), are virulent because of the introduction of toxin-encoding genes by
lysogenic bacteriophages, affirming the clinical relevance of transduction in the exchange of genes involved in infectious disease.
Archaea have their own viruses that translocate genetic material from one individual to another.

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 Exercise 6.6.3

1. What is the agent of transduction of prokaryotic cells?

2. In specialized transduction, where does the transducing piece of DNA come from?

The Clinical Consequences of Transduction

Paul, a 23-year-old relief worker from Atlanta, traveled to Haiti in 2011 to provide aid following the 2010 earthquake. After
working there for several weeks, he suddenly began experiencing abdominal distress, including severe cramping, nausea,
vomiting, and watery diarrhea. He also began to experience intense muscle cramping. At a local clinic, the physician suspected
that Paul’s symptoms were caused by cholera because there had been a cholera outbreak after the earthquake. Because cholera
is transmitted by the fecal-oral route, breaches in sanitation infrastructure, such as often occur following natural disasters, may
precipitate outbreaks. The physician confirmed the presumptive diagnosis using a cholera dipstick test. He then prescribed Paul
a single dose of doxycycline, as well as oral rehydration salts, instructing him to drink significant amounts of clean water.
Cholera is caused by the gram-negative curved rod Vibrio cholerae (Figure 6.6.2). Its symptoms largely result from the
production of the cholera toxin (CT), which ultimately activates a chloride transporter to pump chloride ions out of the
epithelial cells into the gut lumen. Water then follows the chloride ions, causing the prolific watery diarrhea characteristic of
cholera. The gene encoding the cholera toxin is incorporated into the bacterial chromosome of V. cholerae through infection of
the bacterium with the lysogenic filamentous CTX phage, which carries the CT gene and introduces it into the chromosome on
integration of the prophage. Thus, pathogenic strains of V. cholerae result from horizontal gene transfer by specialized
transduction.

Figure 6.6.2 : A scanning electron micrograph of Vibrio cholerae shows its characteristic curved rod shape.

Exercise 6.6.4

1. Why are outbreaks of cholera more common as a result of a natural disaster?

2. Why is muscle cramping a common symptom of cholera? Why is treatment with oral rehydration salts so important for
the treatment of cholera?
3. In areas stricken by cholera, what are some strategies that people could use to prevent disease transmission?

Conjugation
In conjugation, DNA is directly transferred from one prokaryote to another by means of a conjugation pilus, which brings the
organisms into contact with one another. In E. coli, the genes encoding the ability to conjugate are located on a bacterial plasmid
called the F plasmid, also known as the fertility factor, and the conjugation pilus is called the F pilus. The F-plasmid genes encode
both the proteins composing the F pilus and those involved in rolling circle replication of the plasmid. Cells containing the F
plasmid, capable of forming an F pilus, are called F+ cells or donor cells, and those lacking an F plasmid are called F− cells or
recipient cells.

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Conjugation of the F Plasmid
During typical conjugation in E. coli, the F pilus of an F+ cell comes into contact with an F– cell and retracts, bringing the two cell
envelopes into contact (Figure 6.6.3). Then a cytoplasmic bridge forms between the two cells at the site of the conjugation pilus.
As rolling circle replication of the F plasmid occurs in the F+ cell, a single-stranded copy of the F plasmid is transferred through the
cytoplasmic bridge to the F− cell, which then synthesizes the complementary strand, making it double stranded. The F− cell now
becomes an F+ cell capable of making its own conjugation pilus. Eventually, in a mixed bacterial population containing both F+ and
F− cells, all cells will become F+ cells. Genes on the E. coli F plasmid also encode proteins preventing conjugation between F+
cells.

Figure 6.6.3 : Typical conjugation of the F plasmid from an F+ cell to an F− cell is brought about by the conjugation pilus bringing
the two cells into contact. A single strand of the F plasmid is transferred to the F− cell, which is then made double stranded.

Conjugation of F’ and Hfr Cells

Although typical conjugation in E. coli results in the transfer of the F plasmid DNA only, conjugation may also transfer
chromosomal DNA. This is because the F plasmid occasionally integrates into the bacterial chromosome through recombination
between the plasmid and the chromosome, forming an Hfr cell (Figure 6.6.4). “Hfr” refers to the high frequency of recombination
seen when recipient F− cells receive genetic information from Hfr cells through conjugation. Similar to the imprecise excision of a
prophage during specialized transduction, the integrated F plasmid may also be imprecisely excised from the chromosome,
producing an F’ plasmid that carries with it some chromosomal DNA adjacent to the integration site. On conjugation, this DNA is
introduced to the recipient cell and may be either maintained as part of the F’ plasmid or be recombined into the recipient cell’s
bacterial chromosome.
Hfr cells may also treat the bacterial chromosome like an enormous F plasmid and attempt to transfer a copy of it to a recipient F−
cell. Because the bacterial chromosome is so large, transfer of the entire chromosome takes a long time (Figure 6.6.5). However,
contact between bacterial cells during conjugation is transient, so it is unusual for the entire chromosome to be transferred. Host
chromosomal DNA near the integration site of the F plasmid, is more likely to be transferred and recombined into a recipient cell’s
chromosome than host genes farther away.

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 Figure 6.6.4 : (a) The F plasmid can occasionally integrate into the bacterial chromosome, producing an Hfr cell. (b) Imprecise
excision of the F plasmid from the chromosome of an Hfr cell may lead to the production of an F’ plasmid that carries
chromosomal DNA adjacent to the integration site. This F’ plasmid can be transferred to an F− cell by conjugation.

Consequences and Applications of Conjugation

Plasmids are an important type of extrachromosomal (non-required) DNA element in bacteria and, in those cells that harbor them,
are considered to be part of the bacterial genome. From a clinical perspective, plasmids often code for genes involved in virulence.
For example, genes encoding proteins that make a bacterial cell resistant to a particular antibiotic are encoded on R plasmids. R
plasmids, in addition to their genes for antimicrobial resistance, contain genes that control conjugation and transfer of the plasmid.
R plasmids are able to transfer between cells of the same species and between cells of different species. Single R plasmids
commonly contain multiple genes conferring resistance to multiple antibiotics.
Genes required for the production of various toxins and molecules important for colonization during infection may also be found
encoded on plasmids. For example, verotoxin-producing strains of E. coli (VTEC) appear to have acquired the genes encoding the
Shiga toxin from its gram-negative relative Shigella dysenteriae through the acquisition of a large plasmid encoding this toxin.
VTEC causes severe diarrheal disease that may result in hemolytic uremic syndrome(HUS), which may be lead to kidney failure
and death.
In nonclinical settings, bacterial genes that encode metabolic enzymes needed to degrade specialized atypical compounds like
polycyclic aromatic hydrocarbons (PAHs) are also frequently encoded on plasmids. Additionally, certain plasmids have the ability
to move from bacterial cells to other cell types, like those of plants and animals, through mechanisms distinct from conjugation.
Such mechanisms and their use in genetic engineering are covered in chapter 10.

Exercise 6.6.5

1. What type of replication occurs during conjugation?

2. What occurs to produce an Hfr E. coli cell?
3. What types of traits are encoded on plasmids?

Transposition
Genetic elements called transposons (transposable elements), or “jumping genes,” are molecules of DNA that include special
inverted repeat sequences at their ends and a gene encoding the enzyme transposase (Figure 6.6.5). Transposons allow the entire
sequence to independently excise from one location in a DNA molecule and integrate into the DNA elsewhere through a process
called transposition. Transposons were originally discovered in maize (corn) by American geneticist Barbara McClintock (1902–
1992) in the 1940s. Transposons have since been found in all types of organisms, both prokaryotes and eukaryotes. Thus, unlike the
three previous mechanisms discussed, transposition is not prokaryote-specific. Most transposons are nonreplicative, meaning they
move in a “cut-and-paste” fashion. Some may be replicative, however, retaining their location in the DNA while making a copy to
be inserted elsewhere (“copy and paste”). Because transposons can move within a DNA molecule, from one DNA molecule to
another, or even from one cell to another, they have the ability to introduce genetic diversity. Movement within the same DNA
molecule can alter phenotype by inactivating or activating a gene.
Transposons may also carry with them additional genes, moving these genes from one location to another with them. For example,
bacterial transposons can relocate antibiotic resistance genes, moving them from chromosomes to plasmids. This mechanism has
been shown to be responsible for the co-localization of multiple antibiotic resistance genes on a single R plasmid in Shigella strains
causing bacterial dysentery. Such an R plasmid can then be easily transferred among a bacterial population through the process of
conjugation.

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 Figure 6.6.5 : Transposons are segments of DNA that have the ability to move from one location to another because they code for
the enzyme transposase. In this example, a nonreplicative transposon has disrupted gene B. The consequence of that the
transcription of gene B may now have been interrupted.

Exercise 6.6.6

What are two ways a transposon can affect the phenotype of a cell it moves to?

Table 6.6.1 summarizes the processes discussed in this section.

Table 6.6.1 : Summary of Mechanisms of Genetic Diversity in Prokaryotes
Term Definition

Conjugation Transfer of DNA through direct contact using a conjugation pilus

Mechanism of horizontal gene transfer in bacteria in which genes are

Transduction
transferred through viral infection
Mechanism of horizontal gene transfer in which naked environmental
Transformation
DNA is taken up by a bacterial cell
Process whereby DNA independently excises from one location in a
Transposition
DNA molecule and integrates elsewhere

Clinical Focus: Resolution

Within 24 hours, the results of the diagnostic test analysis of Alex’s stool sample revealed that it was positive for heat-labile
enterotoxin (LT), heat-stabile enterotoxin (ST), and colonization factor (CF), confirming the hospital physician’s suspicion of
ETEC. During a follow-up with Alex’s family physician, this physician noted that Alex’s symptoms were not resolving quickly
and he was experiencing discomfort that was preventing him from returning to classes. The family physician prescribed Alex a
course of ciprofloxacin to resolve his symptoms. Fortunately, the ciprofloxacin resolved Alex’s symptoms within a few days.
Alex likely got his infection from ingesting contaminated food or water. Emerging industrialized countries like Mexico are still
developing sanitation practices that prevent the contamination of water with fecal material. Travelers in such countries should
avoid the ingestion of undercooked foods, especially meats, seafood, vegetables, and unpasteurized dairy products. They
should also avoid use of water that has not been treated; this includes drinking water, ice cubes, and even water used for
brushing teeth. Using bottled water for these purposes is a good alternative. Good hygiene (handwashing) can also aid the
prevention of an ETEC infection. Alex had not been careful about his food or water consumption, which led to his illness.
Alex’s symptoms were very similar to those of cholera, caused by the gram-negative bacterium Vibrio cholerae, which also
produces a toxin similar to ST and LT. At some point in the evolutionary history of ETEC, a nonpathogenic strain of E. coli
similar to those typically found in the gut may have acquired the genes encoding the ST and LT toxins from V. cholerae. The
fact that the genes encoding those toxins are encoded on extrachromosomal plasmids in ETEC supports the idea that these
genes were acquired by E. coli and are likely maintained in bacterial populations through horizontal gene transfer.

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Key Concepts and Summary
Horizontal gene transfer is an important way for asexually reproducing organisms like prokaryotes to acquire new traits.
There are three mechanisms of horizontal gene transfer typically used by bacteria: transformation, transduction, and
conjugation.
Transformation allows for competent cells to take up naked DNA, released from other cells on their death, into their cytoplasm,
where it may recombine with the host genome.
In generalized transduction, any piece of chromosomal DNA may be transferred by accidental packaging of the degraded host
chromosome into a phage head. In specialized transduction, only chromosomal DNA adjacent to the integration site of a
lysogenic phage may be transferred as a result of imprecise excision of the prophage.
Conjugation is mediated by the F plasmid, which encodes a conjugation pilus that brings an F plasmid-containing F+ cell into
contact with an F- cell.
The rare integration of the F plasmid into the bacterial chromosome, generating an Hfr cell, allows for transfer of chromosomal
DNA from the donor to the recipient. Additionally, imprecise excision of the F plasmid from the chromosome may generate an
F’ plasmid that may be transferred to a recipient by conjugation.
Conjugation transfer of R plasmids is an important mechanism for the spread of antibiotic resistance in bacterial communities.
Transposons are molecules of DNA with inverted repeats at their ends that also encode the enzyme transposase, allowing for
their movement from one location in DNA to another. Although found in both prokaryotes and eukaryotes, transposons are
clinically relevant in bacterial pathogens for the movement of virulence factors, including antibiotic resistance genes.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 6.6: How Asexual Prokaryotes Achieve Genetic Diversity is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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6.7: Gene Regulation and Operon Theory
Learning Objectives
Compare inducible operons and repressible operons
Describe why regulation of operons is important

Genomic DNA contains both structural genes, which encode products that serve as cellular structures or enzymes, and regulatory
genes, which encode products that regulate gene expression. The expression of a gene is a highly regulated process. Whereas
regulating gene expression in multicellular organisms allows for cellular differentiation, in single-celled organisms like
prokaryotes, it primarily ensures that a cell’s resources are not wasted making proteins (especially enzymes- which use up energy
as they are synthesized and as they are speeding up chemical reactions) that the cell does not need at that time.
Each nucleated cell in a multicellular organism contains copies of the same DNA. Similarly, all cells in two pure bacterial cultures
inoculated from the same starting colony contain the same DNA, with the exception of changes that arise from spontaneous
mutations. If each cell in a multicellular organism has the same DNA, then how is it that cells in different parts of the organism’s
body exhibit different characteristics? Similarly, how is it that the same bacterial cells within two pure cultures exposed to different
environmental conditions can exhibit different phenotypes? In both cases, each genetically identical cell does not turn on, or
express, the same set of genes. Only a subset of proteins in a cell at a given time is expressed.
Elucidating the mechanisms controlling gene expression is important to the understanding of human health. Malfunctions in this
process in humans lead to the development of cancer and other diseases. Understanding the interaction between the gene
expression of a pathogen and that of its human host is important for the understanding of a particular infectious disease. Gene
regulation involves a complex web of interactions within a given cell among signals from the cell’s environment, signaling
molecules within the cell, and the cell’s DNA. These interactions lead to the expression of some genes and the suppression of
others, depending on circumstances.
Prokaryotes and eukaryotes share some similarities in their mechanisms to regulate gene expression; however, gene expression in
eukaryotes is more complicated because of the temporal and spatial separation between the processes of transcription and
translation. Thus, although most regulation of gene expression occurs through transcriptional control in prokaryotes, regulation of
gene expression in eukaryotes occurs at the transcriptional level and post-transcriptionally (after the primary transcript has been
made).

Prokaryotic Gene Regulation

In bacteria and archaea, structural proteins with related functions are usually encoded together within the genome in a block called
an operon and are transcribed together under the control of a single promoter, resulting in the formation of a polycistronic transcript
(Figure 6.7.1). In this way, regulation of the transcription of all of the structural genes encoding the enzymes that catalyze the many
steps in a single biochemical pathway can be controlled simultaneously, because they will either all be needed at the same time, or
none will be needed. For example, in E. coli, all of the structural genes that encode enzymes needed to use lactose as an energy
source lie next to each other in the lactose (or lac) operon under the control of a single promoter, the lac promoter. French scientists
François Jacob (1920–2013) and Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes
into operons, through their studies on the lac operon of E. coli. For this work, they won the Nobel Prize in Physiology or Medicine
in 1965. Although eukaryotic genes are not organized into operons, prokaryotic operons are excellent models for learning about
gene regulation generally. There are some gene clusters in eukaryotes that function similar to operons. Many of the principles can
be applied to eukaryotic systems and contribute to our understanding of changes in gene expression in eukaryotes that can result
pathological changes such as cancer.
Each operon includes DNA sequences that influence its own transcription; these are located in a region called the regulatory region.
The regulatory region includes the promoter and the region surrounding the promoter, to which transcription factors, proteins
encoded by regulatory genes, can bind. Transcription factors influence the binding of RNA polymerase to the promoter and allow
its progression to transcribe structural genes. A repressor is a transcription factor that suppresses transcription of a gene in response
to an external stimulus by binding to a DNA sequence within the regulatory region called the operator, which is located between
the RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene. Repressor binding
physically blocks RNA polymerase from transcribing structural genes. Conversely, an activator is a transcription factor that
increases the transcription of a gene in response to an external stimulus by facilitating RNA polymerase binding to the promoter.

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An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses transcription by interacting
with a repressor or an activator.
In prokaryotes, there are examples of operons whose gene products are required rather consistently and whose expression,
therefore, is unregulated. Such operons are constitutively expressed, meaning they are transcribed and translated continuously to
provide the cell with constant intermediate levels of the protein products. Such genes encode enzymes involved in housekeeping
functions required for cellular maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core
metabolism. In contrast, there are other prokaryotic operons that are expressed only when needed and are regulated by repressors,
activators, and inducers.

Figure 6.7.1 : In prokaryotes, structural genes of related function are often organized together on the genome and transcribed
together under the control of a single promoter. The operon’s regulatory region includes both the promoter and the operator. If a
repressor binds to the operator, then the structural genes will not be transcribed. Alternatively, activators may bind to the regulatory
region, enhancing transcription.

Exercise 6.7.1
1. What are the parts in the DNA sequence of an operon?
2. What types of regulatory molecules are there?

Regulation by Repression and Induction

Prokaryotic operons are commonly controlled by the binding of repressors to operator regions, thereby preventing the transcription
of the structural genes. Such operons are classified as either repressible operons or inducible operons. Repressible operons, like
the tryptophan (trp) operon, typically contain genes encoding enzymes required for a biosynthetic pathway. As long as the product
of the pathway, like tryptophan, continues to be required by the cell, a repressible operon will continue to be expressed. However,
when the product of the biosynthetic pathway begins to accumulate in the cell, removing the need for the cell to continue to make
more, the expression of the operon is repressed. Conversely, inducible operons, like the lac operon of E. coli, often contain genes
encoding enzymes in a pathway involved in the metabolism of a specific substrate like lactose. These enzymes are only required
when that substrate is available, thus expression of the operons is typically induced only in the presence of the substrate.
The trp Operon: A Repressible Operon
E. coli can synthesize tryptophan using enzymes that are encoded by five structural genes located next to each other in the trp
operon (Figure 6.7.2). When environmental tryptophan is low, the operon is turned on. This means that transcription is initiated,
the genes are expressed, and tryptophan is synthesized. However, if tryptophan is present in the environment, the trp operon is
turned off. Transcription does not occur and tryptophan is not synthesized.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and
tryptophan is synthesized. However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor
molecule, which changes its shape, allowing it to bind to the trp operator. This binding of the active form of the trp repressor to the
operator blocks RNA polymerase from transcribing the structural genes, stopping expression of the operon. Thus, the actual
product of the biosynthetic pathway controlled by the operon regulates the expression of the operon.

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 Figure 6.7.2 : The five structural genes needed to synthesize tryptophan in E. coli are located next to each other in the trp operon.
When tryptophan is absent, the repressor protein does not bind to the operator, and the genes are transcribed. When tryptophan is
plentiful, tryptophan binds the repressor protein at the operator sequence. This physically blocks the RNA polymerase from
transcribing the tryptophan biosynthesis genes.

Watch this video to learn more about the trp operon.

The lac Operon: An Inducible Operon
The lac operon is an example of an inducible operon that is also subject to activation in the absence of glucose (Figure 6.7.3). The
lac operon encodes three structural genes necessary to acquire and process the disaccharide lactose from the environment, breaking
it down into the simple sugars glucose and galactose. For the lac operon to be expressed, lactose must be present. This makes sense
for the cell because it would be energetically wasteful to create the enzymes to process lactose if lactose was not available.
In the absence of lactose, the lac repressor is bound to the operator region of the lac operon, physically preventing RNA
polymerase from transcribing the structural genes. However, when lactose is present, the lactose inside the cell is converted to
allolactose. Allolactose serves as an inducer molecule, binding to the repressor and changing its shape so that it is no longer able to
bind to the operator DNA. Removal of the repressor in the presence of lactose allows RNA polymerase to move through the
operator region and begin transcription of the lac structural genes.

Figure 6.7.3 : The three structural genes that are needed to degrade lactose in E. coli are located next to each other in the lac operon.
When lactose is absent, the repressor protein binds to the operator, physically blocking the RNA polymerase from transcribing the
lac structural genes. When lactose is available, a lactose molecule binds the repressor protein, preventing the repressor from
binding to the operator sequence, and the genes are transcribed.
The lac Operon: Activation by Catabolite Activator Protein
Bacteria typically have the ability to use a variety of substrates as carbon sources. However, because glucose is usually preferable
to other substrates, bacteria have mechanisms to ensure that alternative substrates are only used when glucose has been depleted.
Additionally, bacteria have mechanisms to ensure that the genes encoding enzymes for using alternative substrates are expressed

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only when the alternative substrate is available. In the 1940s, Jacques Monod was the first to demonstrate the preference for certain
substrates over others through his studies of E. coli’s growth when cultured in the presence of two different substrates
simultaneously. Such studies generated diauxic growth curves, like the one shown in Figure 6.7.4. Although the preferred substrate
glucose is used first, E. coli grows quickly and the enzymes for lactose metabolism are absent. However, once glucose levels are
depleted, growth rates slow, inducing the expression of the enzymes needed for the metabolism of the second substrate, lactose.
Notice how the growth rate in lactose is slower, as indicated by the lower steepness of the growth curve.

Figure 6.7.4: When grown in the presence of two substrates, E. coli uses the preferred substrate (in this case glucose) until it is
depleted. Then, enzymes needed for the metabolism of the second substrate are expressed and growth resumes, although at a slower
rate.

The ability to switch from glucose use to another substrate like lactose is a consequence of the activity of an enzyme called Enzyme
IIA (EIIA). When glucose levels drop, cells produce less ATP from catabolism, and EIIA becomes phosphorylated. Phosphorylated
EIIA activates adenylyl cyclase, an enzyme that converts some of the remaining ATP to cyclic AMP (cAMP), a cyclic derivative of
AMP and important signaling molecule involved in glucose and energy metabolism in E. coli. As a result, cAMP levels begin to
rise in the cell (Figure 6.7.5).The lac operon also plays a role in this switch from using glucose to using lactose. When glucose is
scarce, the accumulating cAMP caused by increased adenylyl cyclase activity binds to catabolite activator protein (CAP), also
known as cAMP receptor protein (CRP). The complex binds to the promoter region of the lac operon (Figure 6.7.6). In the
regulatory regions of these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter.
Binding of the CAP-cAMP complex to this site increases the binding ability of RNA polymerase to the promoter region to initiate
the transcription of the structural genes. Thus, in the case of the lac operon, for transcription to occur, lactose must be present
(removing the lac repressor protein) and glucose levels must be depleted (allowing binding of an activating protein). When glucose
levels are high, there is catabolite repression of operons encoding enzymes for the metabolism of alternative substrates. Because of
low cAMP levels under these conditions, there is an insufficient amount of the CAP-cAMP complex to activate transcription of
these operons. See Table 6.7.1 for a summary of the regulation of the lac operon.

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 Figure 6.7.5 : When ATP levels decrease due to depletion of glucose, some remaining ATP is converted to cAMP by adenylyl
cyclase. Thus, increased cAMP levels signal glucose depletion.

Figure 6.7.6 : (a) In the presence of cAMP, CAP binds to the promoters of operons, like the lac operon, that encode genes for
enzymes for the use of alternate substrates. (b) For the lac operon to be expressed, there must be activation by cAMP-CAP as well
as removal of the lac repressor from the operator.
Table 6.7.1 : Conditions Affecting Transcription of the lac Operon
Glucose CAP binds Lactose Repressor binds Transcription

+ – – + No

+ – + – Some

– + – + No

– + + – Yes

Watch an animated tutorial about the workings of lac operon here.

Exercise 6.7.2

1. What affects the binding of the trp operon repressor to the operator?
2. How and when is the behavior of the lac repressor protein altered?
3. In addition to being repressible, how else is the lac operon regulated?

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Global Responses of Prokaryotes
In prokaryotes, there are also several higher levels of gene regulation that have the ability to control the transcription of many
related operons simultaneously in response to an environmental signal. A group of operons all controlled simultaneously is called a
regulon.
Alarmones
When sensing impending stress, prokaryotes alter the expression of a wide variety of operons to respond in coordination. They do
this through the production of alarmones, which are small intracellular nucleotide derivatives. Alarmones change which genes are
expressed and stimulate the expression of specific stress-response genes. The use of alarmones to alter gene expression in response
to stress appears to be important in pathogenic bacteria. On encountering host defense mechanisms and other harsh conditions
during infection, many operons encoding virulence genes are upregulated in response to alarmone signaling. Knowledge of these
responses is key to being able to fully understand the infection process of many pathogens and to the development of therapies to
counter this process.
Alternate σ Factors
Since the σ subunit of bacterial RNA polymerase confers specificity as to which promoters should be transcribed, altering the σ
factor used is another way for bacteria to quickly and globally change what regulons are transcribed at a given time. The σ factor
recognizes sequences within a bacterial promoter, so different σ factors will each recognize slightly different promoter sequences.
In this way, when the cell senses specific environmental conditions, it may respond by changing which σ factor it expresses,
degrading the old one and producing a new one to transcribe the operons encoding genes whose products will be useful under the
new environmental condition. For example, in sporulating bacteria of the genera Bacillus and Clostridium (which include many
pathogens), a group of σ factors controls the expression of the many genes needed for sporulation in response to sporulation-
stimulating signals.

Exercise 6.7.3

1. What is the name given to a collection of operons that can be regulated as a group?
2. What type of stimulus would trigger the transcription of a different σ factor?

Other Factors Affecting Gene Expression in Prokaryotes and Eukaryotes

Although most gene expression is regulated at the level of transcription initiation in prokaryotes, there are also mechanisms to
control both the completion of transcription as well as translation concurrently. Since their discovery, these mechanisms have been
shown to control the completion of transcription and translation of many prokaryotic operons. Because these mechanisms link the
regulation of transcription and translation directly, they are specific to prokaryotes, because these processes are physically
separated in eukaryotes.
Although the focus on our discussion of transcriptional control used prokaryotic operons as examples, eukaryotic transcriptional
control is similar in many ways. As in prokaryotes, eukaryotic transcription can be controlled through the binding of transcription
factors including repressors and activators. Interestingly, eukaryotic transcription can be influenced by the binding of proteins to
regions of DNA, called enhancers, rather far away from the gene, through DNA looping facilitated between the enhancer and the
promoter (Figure 6.7.7). Overall, regulating transcription is a highly effective way to control gene expression in both prokaryotes
and eukaryotes. However, the control of gene expression in eukaryotes in response to environmental and cellular stresses can be
accomplished in additional ways without the binding of transcription factors to regulatory regions.

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 Figure 6.7.7 : In eukaryotes, an enhancer is a DNA sequence that promotes transcription. Each enhancer is made up of short DNA
sequences called distal control elements. Activators bound to the distal control elements interact with mediator proteins and
transcription factors. Two different genes may have the same promoter but different distal control elements, enabling differential
gene expression.

DNA-Level Control
In eukaryotes, the DNA molecules or associated histones can be chemically modified in such a way as to influence transcription;
this is called epigenetic regulation. Methylation of certain cytosine nucleotides in DNA in response to environmental factors has
been shown to influence use of such DNA for transcription, with DNA methylation commonly correlating to lowered levels of gene
expression. Additionally, in response to environmental factors, histone proteins for packaging DNA can also be chemically
modified in multiple ways, including acetylation and deacetylation, influencing the packaging state of DNA and thus affecting the
availability of loosely wound DNA for transcription. These chemical modifications can sometimes be maintained through multiple
rounds of cell division, making at least some of these epigenetic changes heritable.
Because different regions of DNA are packaged differently, some regions of chromosomal DNA are more accessible to enzymes
and thus may be used more readily as templates for gene expression. Interestingly, several bacteria, including Helicobacter pylori
and Shigella flexneri, have been shown to induce epigenetic changes in their hosts upon infection, leading to chromatin remodeling
that may cause long-term effects on host immunity.1

Noncoding DNA
In addition to genes, a genome also contains many regions of noncoding DNA that do not encode proteins or stable RNA products.
Noncoding DNA is commonly found in areas prior to the start of coding sequences of genes as well as in intergenic regions (i.e.,
DNA sequences located between genes) Figure 6.7.8.

Figure 6.7.8 : Chromosomes typically have a significant amount of noncoding DNA, often found in intergenic regions.
Prokaryotes appear to use their genomes very efficiently, with only an average of 12% of the genome being taken up by noncoding
sequences. In contrast, noncoding DNA can represent about 98% of the genome in eukaryotes, as seen in humans, but the
percentage of noncoding DNA varies between species.2 These noncoding DNA regions were once referred to as “junk DNA”;
however, this terminology is no longer widely accepted because scientists have since found roles for some of these regions, many
of which contribute to the regulation of transcription or translation through the production of small noncoding RNA molecules,

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DNA packaging, and chromosomal stability. Although scientists may not fully understand the roles of all noncoding regions of
DNA, it is generally believed that they do have purposes within the cell.
This video describes how epigenetic regulation controls gene expression.

Exercise 6.7.4
1. Describe the function of an enhancer.
2. Describe two mechanisms of epigenetic regulation in eukaryotes.
3. What is the role of noncoding DNA?

Key Concepts and Summary

Gene expression is a tightly regulated process.
Gene expression in prokaryotes is largely regulated at the point of transcription. Gene expression in eukaryotes is additionally
regulated post-transcriptionally.
Prokaryotic structural genes of related function are often organized into operons, all controlled by transcription from a single
promoter. The regulatory region of an operon includes the promoter itself and the region surrounding the promoter to which
transcription factors can bind to influence transcription.
Although some operons are constitutively expressed, most are subject to regulation through the use of transcription factors
(repressors and activators). A repressor binds to an operator, a DNA sequence within the regulatory region between the RNA
polymerase binding site in the promoter and first structural gene, thereby physically blocking transcription of these operons. An
activator binds within the regulatory region of an operon, helping RNA polymerase bind to the promoter, thereby enhancing
the transcription of this operon. An inducerinfluences transcription through interacting with a repressor or activator.
The trp operon is a classic example of a repressible operon. When tryptophan accumulates, tryptophan binds to a repressor,
which then binds to the operator, preventing further transcription.
The lac operon is a classic example an inducible operon. When lactose is present in the cell, it is converted to allolactose.
Allolactose acts as an inducer, binding to the repressor and preventing the repressor from binding to the operator. This allows
transcription of the structural genes.
The lac operon is also subject to activation. When glucose levels are depleted, some cellular ATP is converted into cAMP,
which binds to the catabolite activator protein (CAP). The cAMP-CAP complex activates transcription of the lac operon.
When glucose levels are high, its presence prevents transcription of the lac operon and other operons by catabolite repression.
Small intracellular molecules called alarmones are made in response to various environmental stresses, allowing bacteria to
control the transcription of a group of operons, called a regulon.
Bacteria have the ability to change which σ factor of RNA polymerase they use in response to environmental conditions to
quickly and globally change which regulons are transcribed.
There are additional points of regulation of gene expression in prokaryotes and eukaryotes. In eukaryotes, epigenetic
regulation by chemical modification of DNA or histones, and regulation of RNA processing are two methods.

Footnotes
1. H. Bierne et al. “Epigenetics and Bacterial Infections.” Cold Spring Harbor Perspectives in Medicine 2 no. 12 (2012):a010272.
2. R.J. Taft et al. “The Relationship between Non-Protein-Coding DNA and Eukaryotic Complexity.” Bioessays 29 no. 3
(2007):288–299.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 6.7: Gene Regulation and Operon Theory is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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Chapter 6 Exercises
Review questions for Chapter 6

Multiple Choice

1) Frederick Griffith infected mice with a combination of dead R and live S bacterial strains. What was the outcome, and why did it
occur?
1. The mice will live. Transformation was not required.
2. The mice will die. Transformation of genetic material from R to S was required.
3. The mice will live. Transformation of genetic material from S to R was required.
4. The mice will die. Transformation was not required.

2) Why was the alga Acetabularia a good model organism for Joachim Hämmerling to use to identify the location of genetic
material?
1. It lacks a nuclear membrane.
2. It self-fertilizes.
3. It is a large, asymmetrical, single cell easy to see with the naked eye.
4. It makes a protein capsid.

3) Which of the following best describes the results from Hershey and Chase’s experiment using bacterial viruses with 35S-labeled
proteins or 32P-labeled DNA that are consistent with protein being the molecule responsible for hereditary?
1. After infection with the 35S-labeled viruses and centrifugation, only the pellet would be radioactive.
2. After infection with the 35S-labeled viruses and centrifugation, both the pellet and the supernatant would be radioactive.
3. After infection with the 32P-labeled viruses and centrifugation, only the pellet would be radioactive.
4. After infection with the 32P-labeled viruses and centrifugation, both the pellet and the supernatant would be radioactive.

4) Which method did Morgan and colleagues use to show that hereditary information was carried on chromosomes?
1. statistical predictions of the outcomes of crosses using true-breeding parents
2. correlations between microscopic observations of chromosomal movement and the characteristics of offspring
3. transformation of nonpathogenic bacteria to pathogenic bacteria
4. mutations resulting in distinct defects in metabolic enzymatic pathways

5) According to Beadle and Tatum’s “one gene–one enzyme” hypothesis, which of the following enzymes will eliminate the
transformation of hereditary material from pathogenic bacteria to nonpathogenic bacteria?
1. carbohydrate-degrading enzymes
2. proteinases
3. ribonucleases
4. deoxyribonucleases

6) Which of the following is not found within DNA?

1. thymine
2. phosphodiester bonds
3. complementary base pairing
4. amino acids

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7) If 30% of the bases within a DNA molecule are adenine, what is the percentage of thymine?
1. 20%
2. 25%
3. 30%
4. 35%

8) Which of the following statements about base pairing in DNA is incorrect?

1. Purines always base pairs with pyrimidines.
2. Adenine binds to guanine.
3. Base pairs are stabilized by hydrogen bonds.
4. Base pairing occurs at the interior of the double helix.

9) During denaturation of DNA, which of the following happens?

1. Hydrogen bonds between complementary bases break.
2. Phosphodiester bonds break within the sugar-phosphate backbone.
3. Hydrogen bonds within the sugar-phosphate backbone break.
4. Phosphodiester bonds between complementary bases break.

10) Which of the following genes would not likely be encoded on a plasmid?
1. genes encoding toxins that damage host tissue
2. genes encoding antibacterial resistance
3. gene encoding enzymes for glycolysis
4. genes encoding enzymes for the degradation of an unusual substrate

11) Histones are DNA binding proteins that are important for DNA packaging in which of the following?
1. double-stranded and single-stranded DNA viruses
2. archaea and bacteria
3. bacteria and eukaryotes
4. eukaryotes and archaea

12) DNA does all but which of the following?

1. serves as the genetic material passed from parent to offspring
2. remains constant despite changes in environmental conditions
3. provides the instructions for the synthesis of messenger RNA
4. is read by ribosomes during the process of translation

13) According to the central dogma, which of the following represents the flow of genetic information in cells?
1. protein to DNA to RNA
2. DNA to RNA to protein
3. RNA to DNA to protein
4. DNA to protein to RNA

14) Which of the following is the enzyme that replaces the RNA nucleotides in a primer with DNA nucleotides?

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 1. DNA polymerase III
2. DNA polymerase I
3. primase
4. helicase

15) Which of the following is not involved in the initiation of replication?

1. ligase
2. DNA gyrase
3. single-stranded binding protein
4. primase

16) Which of the following enzymes involved in DNA replication is unique to eukaryotes?
1. helicase
2. DNA polymerase
3. ligase
4. telomerase

17) Which of the following would be synthesized using 5′-CAGTTCGGA-3′ as a template?

1. 3′-AGGCTTGAC-4′
2. 3′-TCCGAACTG-5′
3. 3′-GTCAAGCCT-5′
4. 3′-CAGTTCGGA-5′

18) Which of the following types of RNA codes for a protein?

1. dsRNA
2. mRNA
3. rRNA
4. tRNA

19) A nucleic acid is purified from a mixture. The molecules are relatively small, contain uracil, and most are covalently bound to
an amino acid. Which of the following was purified?
1. DNA
2. mRNA
3. rRNA
4. tRNA

20) Which of the following types of RNA is known for its catalytic abilities?
1. dsRNA
2. mRNA
3. rRNA
4. tRNA

21) Ribosomes are composed of rRNA and what other component?

1. protein
2. carbohydrates

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 3. DNA
4. mRNA

22) Which of the following may use RNA as its genome?

1. a bacterium
2. an archaeon
3. a virus
4. a eukaryote

23) During which stage of bacterial transcription is the σ subunit of the RNA polymerase involved?
1. initiation
2. elongation
3. termination
4. splicing

24) Which of the following components is involved in the initiation of transcription?

1. primer
2. origin
3. promoter
4. start codon

25) Which of the following is not a function of the 5’ cap and 3’ poly-A tail of a mature eukaryotic mRNA molecule?
1. to facilitate splicing
2. to prevent mRNA degradation
3. to aid export of the mature transcript to the cytoplasm
4. to aid ribosome binding to the transcript

26) Mature mRNA from a eukaryote would contain each of these features except which of the following?
1. exon-encoded RNA
2. intron-encoded RNA
3. 5’ cap
4. 3’ poly-A tail

27) Which of the following is the name of the three-base sequence in the mRNA that binds to a tRNA molecule?
1. P site
2. codon
3. anticodon
4. CCA binding site

28) Which component is the last to join the initiation complex during the initiation of translation?
1. the mRNA molecule
2. the small ribosomal subunit
3. the large ribosomal subunit
4. the initiator tRNA

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29) During elongation in translation, to which ribosomal site does an incoming charged tRNA molecule bind?
1. A site
2. P site
3. E site
4. B site

30) Which of the following is the amino acid that appears at the N-terminus of all newly translated prokaryotic and eukaryotic
polypeptides?
1. tryptophan
2. methionine
3. selenocysteine
4. glycine

31) When the ribosome reaches a nonsense codon, which of the following occurs?
1. a methionine is incorporated
2. the polypeptide is released
3. a peptide bond forms
4. the A site binds to a charged tRNA

32) Which of the following correctly describes the structure of the typical eukaryotic genome?
1. diploid
2. linear
3. singular
4. double stranded

33) Which of the following is typically found as part of the prokaryotic genome?
1. chloroplast DNA
2. linear chromosomes
3. plasmids
4. mitochondrial DNA

34) Serratia marcescens cells produce a red pigment at room temperature. The red color of the colonies is an example of which of
the following?
1. genotype
2. phenotype
3. change in DNA base composition
4. adaptation to the environment

35) Which of the following is a change in the sequence that leads to formation of a stop codon?
1. missense mutation
2. nonsense mutation
3. silent mutation
4. deletion mutation

36) The formation of pyrimidine dimers results from which of the following?

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 1. spontaneous errors by DNA polymerase
2. exposure to gamma radiation
3. exposure to ultraviolet radiation
4. exposure to intercalating agents

37) Which of the following is an example of a frameshift mutation?

1. a deletion of a codon
2. missense mutation
3. silent mutation
4. deletion of one nucleotide

38) Which of the following is the type of DNA repair in which thymine dimers are directly broken down by the enzyme
photolyase?
1. direct repair
2. nucleotide excision repair
3. mismatch repair
4. proofreading

39) Which of the following regarding the Ames test is true?

1. It is used to identify newly formed auxotrophic mutants.
2. It is used to identify mutants with restored biosynthetic activity.
3. It is used to identify spontaneous mutants.
4. It is used to identify mutants lacking photoreactivation activity.

40) Which is the mechanism by which improper excision of a prophage from a bacterial chromosome results in packaging of
bacterial genes near the integration site into a phage head?
1. conjugation
2. generalized transduction
3. specialized transduction
4. transformation

41) Which of the following refers to the uptake of naked DNA from the surrounding environment?
1. conjugation
2. generalized transduction
3. specialized transduction
4. transformation

42) The F plasmid is involved in which of the following processes?

1. conjugation
2. transduction
3. transposition
4. transformation

43) Which of the following refers to the mechanism of horizontal gene transfer naturally responsible for the spread of antibiotic
resistance genes within a bacterial population?

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 1. conjugation
2. generalized transduction
3. specialized transduction
4. transformation

44) An operon of genes encoding enzymes in a biosynthetic pathway is likely to be which of the following?
1. inducible
2. repressible
3. constitutive
4. monocistronic

45) An operon encoding genes that are transcribed and translated continuously to provide the cell with constant intermediate levels
of the protein products is said to be which of the following?
1. repressible
2. inducible
3. constitutive
4. activated

46) Which of the following conditions leads to maximal expression of the lac operon?
1. lactose present, glucose absent
2. lactose present, glucose present
3. lactose absent, glucose absent
4. lactose absent, glucose present

47) Which of the following is a type of regulation of gene expression unique to eukaryotes?
1. attenuation
2. use of alternate σ factor
3. chemical modification of histones
4. alarmones

Fill-in-the-Blanks
48) The process of making an RNA copy of a gene is called ________.

49) A cell’s ________ remains constant whereas its phenotype changes in response to environmental influences.

50) The enzyme responsible for relaxing supercoiled DNA to allow for the initiation of replication is called ________.

51) Unidirectional replication of a circular DNA molecule like a plasmid that involves nicking one DNA strand and displacing it
while synthesizing a new strand is called ________.

52) A ________ mRNA is one that codes for multiple polypeptides.

53) The protein complex responsible for removing intron-encoded RNA sequences from primary transcripts in eukaryotes is called
the ________.

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54) The third position within a codon, in which changes often result in the incorporation of the same amino acid into the growing
polypeptide, is called the ________.

55) The enzyme that adds an amino acid to a tRNA molecule is called ________.

56) A chemical mutagen that is structurally similar to a nucleotide but has different base-pairing rules is called a ________.

57) The enzyme used in light repair to split thymine dimers is called ________.

58) The phenotype of an organism that is most commonly observed in nature is called the ________.

59) A small DNA molecule that has the ability to independently excise from one location in a larger DNA molecule and integrate
into the DNA elsewhere is called a ________.

60) ________ is a group of mechanisms that allow for the introduction of genetic material from one organism to another organism
within the same generation.

61) The DNA sequence, to which repressors may bind, that lies between the promoter and the first structural gene is called the
________.

62) The prevention of expression of operons encoding substrate use pathways for substrates other than glucose when glucose is
present is called _______.

63) The element ____________ is unique to nucleic acids compared with other macromolecules.

64) In the late 1800s and early 1900s, the macromolecule thought to be responsible for heredity was ______________.

65) The end of a nucleic acid strand with a free phosphate group is called the ________.

66) Plasmids are typically transferred among members of a bacterial community by ________ gene transfer.

Short Answer

67) Why do bacteria and viruses make good model systems for various genetic studies?

68) Why was nucleic acid disregarded for so long as the molecule responsible for the transmission of hereditary information?

69) Bacteriophages inject their genetic material into host cells, whereas animal viruses enter host cells completely. Why was it
important to use a bacteriophage in the Hershey–Chase experiment rather than an animal virus?

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70) What is the role of phosphodiester bonds within the sugar-phosphate backbone of DNA?

71) What is meant by the term “antiparallel?”

72) Why is DNA with a high GC content more difficult to denature than that with a low GC content?

73) What are the differences between DNA nucleotides and RNA nucleotides?

74) How is the information stored within the base sequence of DNA used to determine a cell’s properties?

75) How do complementary base pairs contribute to intramolecular base pairing within an RNA molecule?

76) If an antisense RNA has the sequence 5ʹAUUCGAAUGC3ʹ, what is the sequence of the mRNA to which it will bind? Be sure
to label the 5ʹ and 3ʹ ends of the molecule you draw.

77) Why does double-stranded RNA (dsRNA) stimulate RNA interference?

78) What are some differences in chromosomal structures between prokaryotes and eukaryotes?

79) How do prokaryotes and eukaryotes manage to fit their lengthy DNA inside of cells? Why is this necessary?

80) What are some functions of noncoding DNA?

81) In the chromatin of eukaryotic cells, which regions of the chromosome would you expect to be more compact: the regions that
contain genes being actively copied into RNA or those that contain inactive genes?

82) Can two observably different cells have the same genotype? Explain.

83) Why is primase required for DNA replication?

84) What is the role of single-stranded binding protein in DNA replication?

85) Below is a DNA sequence. Envision that this is a section of a DNA molecule that has separated in preparation for replication,
so you are only seeing one DNA strand. Construct the complementary DNA sequence (indicating 5’ and 3’ ends).
DNA sequence: 3’-T A C T G A C T G A C G A T C-5’

86) What is the purpose of RNA processing in eukaryotes? Why don’t prokaryotes require similar processing?

87) Below is a DNA sequence. Envision that this is a section of a DNA molecule that has separated in preparation for transcription,
so you are only seeing the antisense strand. Construct the mRNA sequence transcribed from this template.
Antisense DNA strand: 3’-T A C T G A C T G A C G A T C-5’

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89) Why does translation terminate when the ribosome reaches a stop codon? What happens?

90) How does the process of translation differ between prokaryotes and eukaryotes?

91) What is meant by the genetic code being nearly universal?

93) Below is an antisense DNA sequence. Translate the mRNA molecule synthesized using the genetic code, recording the
resulting amino acid sequence, indicating the N and C termini.
Antisense DNA strand: 3’-T A C T G A C T G A C G A T C-5’

94) Why is it more likely that insertions or deletions will be more detrimental to a cell than point mutations?

95) Briefly describe two ways in which chromosomal DNA from a donor cell may be transferred to a recipient cell during the
process of conjugation.

96) Describe what happens when a nonsense mutation is introduced into the gene encoding transposase within a transposon.

97) What are two ways that bacteria can influence the transcription of multiple different operons simultaneously in response to a
particular environmental condition?

Critical Thinking

98) In the first figure from this chapter, if the nuclei were contained within the stalks of Acetabularia, what types of caps would
you expect from the pictured grafts?

99) Why are Hershey and Chase credited with identifying DNA as the carrier of heredity even though DNA had been discovered
many years before?

100) A certain DNA sample is found to have a makeup consisting of 22% thymine. Use Chargaff’s rules to fill in the percentages
for the other three nitrogenous bases.

101) In considering the structure of the DNA double helix, how would you expect the structure to differ if there was base pairing
between two purines? Between two pyrimidines?

102) Identify the location of mRNA, rRNA, and tRNA in the figure.

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103) Why does it make sense that tRNA and rRNA molecules are more stable than mRNA molecules?

104) A new type of bacteriophage has been isolated and you are in charge of characterizing its genome. The base composition of
the bacteriophage is A (15%), C (20%), T (35%), and G (30%). What can you conclude about the genome of the virus?

105) A pure culture of an unknown bacterium was streaked onto plates of a variety of media. You notice that the colony
morphology is strikingly different on plates of minimal media with glucose compared to that seen on trypticase soy agar plates.
How can you explain these differences in colony morphology?

106) Why was it important that Meselson and Stahl continue their experiment to at least two rounds of replication after isotopic
labeling of the starting DNA with 15N, instead of stopping the experiment after only one round of replication?

107) If deoxyribonucleotides that lack the 3’-OH groups are added during the replication process, what do you expect will occur?

108) Predict the effect of an alteration in the sequence of nucleotides in the –35 region of a bacterial promoter.

109) Label the following in the figure: ribosomal E, P, and A sites; mRNA; codons; anticodons; growing polypeptide; incoming
amino acid; direction of translocation; small ribosomal unit; large ribosomal unit.

110) Prior to the elucidation of the genetic code, prominent scientists, including Francis Crick, had predicted that each mRNA
codon, coding for one of the 20 amino acids, needed to be at least three nucleotides long. Why is it not possible for codons to be
any shorter?

111) Below are several DNA sequences that are mutated compared with the wild-type sequence: 3’-T A C T G A C T G A C G A T
C-5’. Envision that each is a section of a DNA molecule that has separated in preparation for transcription, so you are only seeing
the template strand. Construct the complementary DNA sequences (indicating 5’ and 3’ ends) for each mutated DNA sequence,
then transcribe (indicating 5’ and 3’ ends) the template strands, and translate the mRNA molecules using the genetic code,
recording the resulting amino acid sequence (indicating the N and C termini). What type of mutation is each?
each?

Mutated DNA Template Strand #1: 3’-T A C T G T C T G A C G A T C-5’

Complementary DNA sequence:
mRNA sequence transcribed from template:

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 Amino acid sequence of peptide:
Type of mutation:

Mutated DNA Template Strand #2: 3’-T A C G G A C T G A C G A T C-5’

Complementary DNA sequence:
mRNA sequence transcribed from template:
Amino acid sequence of peptide:
Type of mutation:

Mutated DNA Template Strand #3: 3’-T A C T G A C T G A C T A T C-5’

Complementary DNA sequence:
mRNA sequence transcribed from template:
Amino acid sequence of peptide:
Type of mutation:

Mutated DNA Template Strand #4: 3’-T A C G A C T G A C T A T C-5’

Complementary DNA sequence:
mRNA sequence transcribed from template:
Amino acid sequence of peptide:
Type of mutation:

112) Why do you think the Ames test is preferable to the use of animal models to screen chemical compounds for mutagenicity?

113) The following figure is from Monod’s original work on diauxic growth showing the growth of E. coli in the simultaneous
presence of xylose and glucose as the only carbon sources. Explain what is happening at points A–D with respect to the carbon
source being used for growth, and explain whether the xylose-use operon is being expressed (and why). Note that expression of the
enzymes required for xylose use is regulated in a manner similar to the expression of the enzymes required for lactose use.

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 CHAPTER OVERVIEW

7: Microbial Metabolism
7.1: Energy, Matter, and Enzymes
7.2: Catabolism of Carbohydrates
7.3: Alternate Forms of Catabolism: Fermentation, Lipids and Proteins
7.4: Photosynthesis and the Importance of Light
Chapter 7 Exercises

Thumbnail: The Krebs cycle, also known as the citric acid cycle, is summarized here. Note incoming two-carbon acetyl results in
the main outputs per turn of two CO2, three NADH, one FADH2, and one ATP (or GTP) molecules made by substrate-level
phosphorylation. Two turns of the Krebs cycle are required to process all of the carbon from one glucose molecule.

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1
7.1: Energy, Matter, and Enzymes
Learning Objectives
Define and describe metabolism
Compare and contrast autotrophs and heterotrophs
Describe the importance of oxidation-reduction reactions in metabolism
Describe why ATP, FAD, NAD+, and NADP+ are important in a cell
Identify the structure and structural components of an enzyme
Describe the differences between competitive and noncompetitive enzyme inhibitors

Clinical Focus: part 1

Hannah is a 15-month-old girl from Washington state. She is spending the summer in Gambia, where her parents are working
for a nongovernmental organization. About 3 weeks after her arrival in Gambia, Hannah’s appetite began to diminish and her
parents noticed that she seemed unusually sluggish, fatigued, and confused. She also seemed very irritable when she was
outdoors, especially during the day. When she began vomiting, her parents figured she had caught a 24-hour virus, but when
her symptoms persisted, they took her to a clinic. The local physician noticed that Hannah’s reflexes seemed abnormally slow,
and when he examined her eyes with a light, she seemed unusually light sensitive. She also seemed to be experiencing a stiff
neck.

Exercise 7.1.1

What are some possible causes of Hannah’s symptoms?

Scientists use the term bioenergetics to discuss the concept of energy flow through living systems, such as cells. We define energy
as the ability to do work. Energy exists in different forms: for example, electrical energy, light energy, and heat energy are all
different energy types. While these are all familiar energy types that one can see or feel, there is another energy type that is much
less tangible. Cells need to be able to use as many of the types of energy at its disposable as possible. In cells some of these
chemical reactions are spontaneous and release energy; whereas, others require energy to proceed. Just as living things must
continually consume food to replenish what they have used, cells must continually produce more energy to replenish that which the
many energy-requiring chemical reactions that constantly take place use. Thus energy is constantly shifted and exchanged via these
chemical reactions.
The term used to describe all of the chemical reactions inside a cell is metabolism (Figure 7.1.1). Cellular processes such as the
building or breaking down of complex molecules occur through series of stepwise, interconnected chemical reactions called
metabolic pathways. Reactions that are spontaneous and release energy are exergonic reactions, whereas endergonic reactions
require (take in) energy to proceed. The amount of energy being released or taken in by these reactions is sometimes so great that
organisms would not be able to handle the increase or decrease in energy all at once, thus metabolic pathways that split reactions
into smaller steps (and thus smaller amounts of energy) provide a way for cells to control the amount and use of that energy.
The terms endergonic and exergonic are often related to descriptions of what type of reaction is taking place. The term anabolism
usually refers to those endergonic metabolic pathways involved in biosynthesis, converting simple molecular building blocks into
more complex molecules, and fueled by the use of cellular energy. Conversely, the term catabolism usually refers to exergonic
pathways that break down complex molecules into simpler ones. Molecular energy stored in the bonds of complex molecules is
released in catabolic pathways and harvested in such a way that it can be used to produce high-energy molecules, which are used to
drive anabolic pathways. Thus, in terms of both energy and molecules, cells are continually balancing catabolism with anabolism.

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 Figure 7.1.1 : Metabolism includes catabolism and anabolism. Anabolic pathways require energy to synthesize larger molecules.
Catabolic pathways generate energy by breaking down larger molecules. Both types of pathways are required for maintaining the
cell’s energy balance.

Figure 7.1.2: Reactions classified as exergonic release energy because the products have less energy than the reactants. Endergonic
reaction are opposite, with products containing more energy than the reactants. Notice for both of these types of reactions the
energy hump (activation energy) that is need to be overcome before the reaction completes.

Classification by Carbon and Energy Source

Organisms can be identified according to the source of carbon they use for metabolism as well as their energy source. The prefixes
auto- (“self”) and hetero- (“other”) refer to the origins of the carbon sources various organisms can use. Organisms that convert
inorganic carbon dioxide (CO2) into organic carbon compounds are autotrophs. Plants and cyanobacteria are well-known examples
of autotrophs. Conversely, heterotrophs rely on more complex organic carbon compounds as nutrients; these are provided to them
initially by autotrophs. Many organisms, ranging from humans to many prokaryotes, including the well-studied Escherichia coli,
are heterotrophic.
Organisms can also be identified by the energy source they use. All energy is derived from the transfer of electrons, but the source
of electrons differs between various types of organisms. The prefixes photo- (“light”) and chemo- (“chemical”) refer to the energy
sources that various organisms use. Those that get their energy for electron transfer from light are phototrophs, whereas
chemotrophs obtain energy for electron transfer by breaking chemical bonds. There are two types of chemotrophs: organotrophs
and lithotrophs. Organotrophs, including humans, fungi, and many prokaryotes, are chemotrophs that obtain energy from organic
compounds. Lithotrophs (“litho” means “rock”) are chemotrophs that get energy from inorganic compounds, including hydrogen
sulfide (H2S) and reduced iron. Lithotrophy is unique to the microbial world.
The strategies used to obtain both carbon and energy can be combined for the classification of organisms according to nutritional
type. Most organisms are chemoheterotrophs because they use organic molecules as both their electron and carbon sources. Table
7.1.1 summarizes this and the other classifications.

Table 7.1.1 : Classifications of Organisms by Energy and Carbon Source

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 Classifications Energy Source Carbon Source Examples

Hydrogen-, sulfur-, iron-,

nitrogen-, and carbon
Chemoautotrophs Chemical Inorganic
monoxide-oxidizing
Chemotrophs bacteria
All animals, most fungi,
Chemoheterotrophs Chemical Organic compounds
protozoa, and bacteria
All plants, algae,
Photoautotrophs Light Inorganic cyanobacteria, and green
Phototrophs and purple sulfur bacteria
Green and purple nonsulfur
Photoheterotrophs Light Organic compounds
bacteria, heliobacteria

Exercise 7.1.2

1. Explain the difference between catabolism and anabolism.

2. Explain the difference between autotrophs and heterotrophs.

Oxidation and Reduction in Metabolism

The transfer of electrons between molecules is important because most of the energy stored in atoms and used to fuel cell functions
is in the form of high-energy electrons. The transfer of energy in the form of electrons allows the cell to transfer and use energy
incrementally; that is, in small packages rather than a single, destructive burst. Reactions that remove electrons from donor
molecules, leaving them oxidized, are oxidation reactions; those that add electrons to acceptor molecules, leaving them reduced,
are reduction reactions. Because electrons can move from one molecule to another, oxidation and reduction occur in tandem. These
pairs of reactions are called oxidation-reduction reactions, or redox reactions.

Energy Carriers: NAD+, NADP+, FAD, and ATP

The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of
electron carriers or in the bonds of adenosine triphosphate (ATP). In living systems, a small class of compounds functions as
mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal
electron carriers we will consider originate from the B vitamin group and are derivatives of nucleotides; they are nicotinamide
adenine dinucleotide, nicotine adenine dinucleotide phosphate, and flavin adenine dinucleotide. These compounds can be easily
reduced or oxidized. Nicotinamide adenine dinucleotide (NAD+/NADH) is the most common mobile electron carrier used in
catabolism. NAD+ is the oxidized form of the molecule; NADH is the reduced form of the molecule. Nicotine adenine dinucleotide
phosphate (NADP+), the oxidized form of an NAD+ variant that contains an extra phosphate group, is another important electron
carrier; it forms NADPH when reduced. The oxidized form of flavin adenine dinucleotide is FAD, and its reduced form is FADH2.
Both NAD+/NADH and FAD/FADH2 are extensively used in energy extraction from sugars during catabolism in
chemoheterotrophs, whereas NADP+/NADPH plays an important role in anabolic reactions and photosynthesis. Collectively,
FADH2, NADH, and NADPH are often referred to as having reducing power due to their ability to donate electrons to various
chemical reactions.
A living cell must be able to handle the energy released during catabolism in a way that enables the cell to store energy safely and
release it for use only as needed. Living cells accomplish this by using the compound adenosine triphosphate (ATP). ATP is often
called the “energy currency” of the cell, and, like currency, this versatile compound can be used to fill any energy need of the cell.
At the heart of ATP is a molecule of adenosine monophosphate (AMP), which is composed of an adenine molecule bonded to a
ribose molecule and a single phosphate group. Ribose is a five-carbon sugar found in RNA, and AMP is one of the nucleotides in
RNA. The addition of a second phosphate group to this core molecule results in the formation of adenosine diphosphate (ADP); the
addition of a third phosphate group forms ATP (Figure 7.1.3). Adding a phosphate group to a molecule, a process called
phosphorylation, requires energy. Phosphate groups are negatively charged and thus repel one another when they are arranged in
series, as they are in ADP and ATP. This repulsion makes the ADP and ATP molecules inherently unstable. Thus, the bonds
between phosphate groups (one in ADP and two in ATP) are called high-energy phosphate bonds. When these high-energy bonds

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are broken to release one phosphate (called inorganic phosphate [Pi]) or two connected phosphate groups (called pyrophosphate
[PPi]) from ATP through a process called dephosphorylation, energy is released to drive endergonic reactions (Figure 7.1.4).

Figure 7.1.3 : The energy released from dephosphorylation of ATP is used to drive cellular work, including anabolic pathways. ATP
is regenerated through phosphorylation, harnessing the energy found in chemicals or from sunlight. (credit: modification of work
by Robert Bear, David Rintoul)

Figure 7.1.4 : Exergonic reactions are coupled to endergonic ones, making the combination favorable. Here, the endergonic reaction
of ATP phosphorylation is coupled to the exergonic reactions of catabolism. Similarly, the exergonic reaction of ATP
dephosphorylation is coupled to the endergonic reaction of polypeptide formation, an example of anabolism.

Exercise 7.1.3

What is the function of an electron carrier?

Enzyme Structure and Function

A substance that helps speed up a chemical reaction is a catalyst. Catalysts are not used or changed during chemical reactions and,
therefore, are reusable. While inorganic molecules may serve as catalysts for a wide range of chemical reactions, proteins called
enzymes serve as catalysts for biochemical reactions inside cells. Enzymes thus play an important role in controlling cellular
metabolism. Enzymes are also occasionally made from RNA, or a combination of RNA and proteins (like the ribosome). This
means they are made from the information in DNA via transcription and translation.
Like all catalysts, an enzyme functions by reducing (lowering) the amount of activation energy of a chemical reaction inside the
cell. Activation energy is the energy needed to form or break chemical bonds and convert reactants to products, thus the energy
needed to get the reaction started. (Figure 7.1.5). Enzymes reduce the amount the activation energy by binding to the reactant
molecules and holding them in such a way as to speed up the reaction.
The chemical reactants to which an enzyme binds are called substrates, and the location within the enzyme where the substrate
binds is called the enzyme’s active site. The characteristics of the amino acids near the active site create a very specific chemical
environment within the active site that induces suitability to binding, albeit briefly, to a specific substrate (or substrates). Due to

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this jigsaw puzzle-like match between an enzyme and its substrates, enzymes are known for their specificity. In fact, as an enzyme
binds to its substrate(s), the enzyme structure changes slightly to find the best fit between the transition state (a structural
intermediate between the substrate and product) and the active site, just as a rubber glove molds to a hand inserted into it. This
active-site modification in the presence of substrate, along with the simultaneous formation of the transition state, is called induced
fit (Figure 7.1.6). Overall, there is a specifically matched enzyme for each substrate and, thus, for each chemical reaction; however,
there is some flexibility as well. Some enzymes have the ability to act on several different structurally related substrates (called an
easy fit).

Figure 7.1.5 : Enzymes lower the activation energy of a chemical reaction.

Figure 7.1.6 : According to the induced-fit model, the active site of the enzyme undergoes conformational changes upon binding
with the substrate.

Environmental Concerns
Enzymes are organic molecules and thus subject to influences by local environmental conditions such as pH, substrate
concentration, and temperature. Although increasing the environmental temperature generally increases reaction rates, enzyme
catalyzed or otherwise, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the
active site, making them less well suited to bind substrates. High temperatures will eventually cause enzymes, like other biological
molecules, to denature, losing their three-dimensional structure and function. Enzymes are also suited to function best within a
certain pH range, and, as with temperature, extreme environmental pH values (acidic or basic) can cause enzymes to denature.
Active-site amino-acid side chains have their own acidic or basic properties that are optimal for catalysis and, therefore, are
sensitive to changes in pH.
Another factor that influences enzyme activity is substrate concentration: Enzyme activity is increased at higher concentrations of
substrate until it reaches a saturation point at which the enzyme can bind no additional substrate. Overall, enzymes are optimized to
work best under the environmental conditions in which the organisms that produce them live. For example, while microbes that
inhabit hot springs have enzymes that work best at high temperatures, human pathogens have enzymes that work best at 37°C.

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Similarly, while enzymes produced by most organisms work best at a neutral pH, microbes growing in acidic environments make
enzymes optimized to low pH conditions, allowing for their growth at those conditions.

Cofactors and Coenzymes

Many enzymes do not work optimally, or even at all, unless bound to other specific nonprotein helper molecules, either temporarily
through ionic or hydrogen bonds or permanently through stronger covalent bonds. Binding to these molecules promotes optimal
conformation and function for their respective enzymes. Two types of helper molecules are cofactors and coenzymes.
Cofactors are inorganic ions such as iron (Fe2+) and magnesium (Mg2+) that help stabilize enzyme conformation and function. One
example of an enzyme that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which
requires a bound zinc ion (Zn2+) to function. Coenzymes are organic helper molecules that are required for enzyme action. Like
enzymes, they are not consumed and, hence, are reusable. The most common sources of coenzymes are dietary vitamins. Some
vitamins are precursors to coenzymes and others act directly as coenzymes.
Some cofactors and coenzymes, like coenzyme A (CoA), often bind to the enzyme’s active site, aiding in the chemistry of the
transition of a substrate to a product (Figure 7.1.7). In such cases, an enzyme lacking a necessary cofactor or coenzyme is called an
apoenzyme and is inactive. Conversely, an enzyme with the necessary associated cofactor or coenzyme is called a holoenzyme and
is active. NADH and ATP are also both examples of commonly used coenzymes that provide high-energy electrons or phosphate
groups, respectively, which bind to enzymes, thereby activating them.

Figure 7.1.7 : The binding of a coenzyme or cofactor to an apoenzyme is often required to form an active holoenzyme.

Exercise 7.1.4

What role do enzymes play in a chemical reaction?

Enzyme Inhibitors
Enzymes can be regulated in ways that either promote or reduce their activity. There are many different kinds of molecules that
inhibit or promote enzyme function, and various mechanisms exist for doing so (Figure 7.1.8). A competitive inhibitor is a
molecule similar enough to a substrate that it can compete with the substrate for binding to the active site by simply blocking the
substrate from binding. For a competitive inhibitor to be effective, the inhibitor concentration needs to be approximately equal to
the substrate concentration. Sulfa drugs provide a good example of competitive competition. They are used to treat bacterial
infections because they bind to the active site of an enzyme within the bacterial folic acid synthesis pathway. When present in a
sufficient dose, a sulfa drug prevents folic acid synthesis, and bacteria are unable to grow because they cannot synthesize DNA,
RNA, and proteins. Humans are unaffected because we obtain folic acid from our diets.

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 Figure 7.1.8 : Enzyme activity can be regulated by either competitive inhibitors, which bind to the active site, or noncompetitive
inhibitors, which bind to an allosteric site.
On the other hand, a noncompetitive (allosteric) inhibitor binds to the enzyme at an allosteric site, a location other than the active
site, and still manages to block substrate binding to the active site by inducing a conformational change that reduces the affinity of
the enzyme for its substrate (Figure 7.1.9). Because only one inhibitor molecule is needed per enzyme for effective inhibition, the
concentration of inhibitors needed for noncompetitive inhibition is typically much lower than the substrate concentration.

Figure 7.1.9: Binding of an allosteric inhibitor reduces enzyme activity, but binding of an allosteric activator increases enzyme
activity.

In addition to allosteric inhibitors, there are allosteric activators that bind to locations on an enzyme away from the active site,
inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s).
Allosteric control is an important mechanism of regulation of metabolic pathways involved in both catabolism and anabolism. In a
most efficient and elegant way, cells have evolved also to use the products of their own metabolic reactions for feedback inhibition
of enzyme activity. Feedback inhibition involves the use of a pathway product to regulate its own further production. The cell
responds to the abundance of specific products by slowing production during anabolic or catabolic reactions (Figure 7.1.10).

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 Figure 7.1.10 : Feedback inhibition, where the end product of the pathway serves as a noncompetitive inhibitor to an enzyme early
in the pathway, is an important mechanism of allosteric regulation in cells.

Exercise 7.1.5

Explain the difference between a competitive inhibitor and a noncompetitive inhibitor.

Key Concepts and Summary

Metabolism includes chemical reactions that break down complex molecules (catabolism) and those that build complex
molecules (anabolism).
Organisms may be classified according to their source of carbon. Autotrophs convert inorganic carbon dioxide into organic
carbon; heterotrophs use fixed organic carbon compounds.
Organisms may also be classified according to their energy source. Phototrophs obtain their energy from light. Chemotrophs
get their energy from chemical compounds. Organotrophs use organic molecules, and lithotrophs use inorganic chemicals.
Cellular electron carriers accept high-energy electrons from foods and later serve as electron donors in subsequent redox
reactions. FAD/FADH2, NAD+/NADH, and NADP+/NADPH are important electron carriers.
Adenosine triphosphate (ATP) serves as the energy currency of the cell, safely storing chemical energy in its two high-energy
phosphate bonds for later use to drive processes requiring energy.
Enzymes are biological catalysts that increase the rate of chemical reactions inside cells by lowering the activation energy
required for the reaction to proceed.
In nature, exergonic reactions do not require energy beyond activation energy to proceed, and they release energy. They may
proceed without enzymes, but at a slow rate. Conversely, endergonic reactions require energy beyond activation energy to
occur. In cells, endergonic reactions are coupled to exergonic reactions, making the combination energetically favorable.
Substrates bind to the enzyme’s active site. This process typically alters the structures of both the active site and the substrate,
favoring transition-state formation; this is known as induced fit.
Cofactors are inorganic ions that stabilize enzyme conformation and function. Coenzymes are organic molecules required for
proper enzyme function and are often derived from vitamins. An enzyme lacking a cofactor or coenzyme is an apoenzyme; an
enzyme with a bound cofactor or coenzyme is a holoenzyme.
Competitive inhibitors regulate enzymes by binding to an enzyme’s active site, preventing substrate binding. Noncompetitive
(allosteric) inhibitors bind to allosteric sites, inducing a conformational change in the enzyme that prevents it from
functioning. Feedback inhibition occurs when the product of a metabolic pathway noncompetitively binds to an enzyme early
on in the pathway, ultimately preventing the synthesis of the product.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 7.1: Energy, Matter, and Enzymes is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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7.2: Catabolism of Carbohydrates
Learning Objectives
Describe why glycolysis is not oxygen dependent
Define and describe the net yield of three-carbon molecules, ATP, and NADH from glycolysis
Explain how three-carbon pyruvate molecules are converted into two-carbon acetyl groups that can be funneled into the
Krebs cycle.
Define and describe the net yield of CO2, GTP/ATP, FADH2, and NADH from the Krebs cycle
Explain how intermediate carbon molecules of the Krebs cycle can be used in a cell
Compare and contrast the electron transport system location and function in a prokaryotic cell and a eukaryotic cell
Compare and contrast the differences between substrate-level and oxidative phosphorylation
Explain the relationship between chemiosmosis and proton motive force
Describe the function and location of ATP synthase in a prokaryotic versus eukaryotic cell
Compare and contrast aerobic and anaerobic respiration

Extensive enzyme pathways exist for breaking down carbohydrates to capture energy in ATP bonds. In addition, many catabolic
pathways produce intermediate molecules that are also used as building blocks for anabolic reactions. Understanding these
processes is important for several reasons. First, because the main metabolic processes involved are common to a wide range of
chemoheterotrophic organisms, we can learn a great deal about human metabolism by studying metabolism in more easily
manipulated bacteria like E. coli. Second, because animal and human pathogens are also chemoheterotrophs, learning about the
details of metabolism in these bacteria, including possible differences between bacterial and human pathways, is useful for the
diagnosis of pathogens as well as for the discovery of antimicrobial therapies targeting specific pathogens. Last, learning
specifically about the pathways involved in chemoheterotrophic metabolism also serves as a basis for comparing other more
unusual metabolic strategies used by microbes. Although the chemical source of electrons initiating electron transfer is different
between chemoheterorophs and chemoautotrophs, many similar processes are used in both types of organisms.
The typical example used to introduce concepts of metabolism to students is carbohydrate catabolism. For chemoheterotrophs, our
examples of metabolism start with the catabolism of polysaccharides such as glycogen, starch, or cellulose. Enzymes such as
amylase, which breaks down glycogen or starch, and cellulases, which break down cellulose, can cause the hydrolysis of glycosidic
bonds between the glucose monomers in these polymers, releasing glucose for further catabolism.

Glycolysis
For bacteria, eukaryotes, and most archaea, glycolysis is the most common pathway for the catabolism of glucose; it produces
energy, reduced electron carriers, and precursor molecules for cellular metabolism. Every living organism carries out some form of
glycolysis, suggesting this mechanism is an ancient universal metabolic process. The process itself does not use oxygen; however,
glycolysis can be coupled with additional metabolic processes that are either aerobic or anaerobic. Glycolysis takes place in the
cytoplasm of prokaryotic and eukaryotic cells. It begins with a single six-carbon glucose molecule and ends with two molecules of
a three-carbon sugar called pyruvate. Pyruvate may be broken down further after glycolysis to harness more energy through aerobic
or anaerobic respiration, but many organisms, including many microbes, may be unable to respire; for these organisms, glycolysis
may be their only source of generating ATP.
The type of glycolysis found in animals and that is most common in microbes is the Embden-Meyerhof-Parnas (EMP) pathway,
named after Gustav Embden (1874–1933), Otto Meyerhof (1884–1951), and Jakub Parnas (1884–1949). Glycolysis using the EMP
pathway consists of two distinct phases (Figure 7.2.1). The first part of the pathway, called the energy investment phase, uses
energy from two ATP molecules to modify a glucose molecule so that the six-carbon sugar molecule can be split evenly into two
phosphorylated three-carbon molecules called glyceraldehyde 3-phosphate (G3P). The second part of the pathway, called the
energy payoff phase, extracts energy by oxidizing G3P to pyruvate, producing four ATP molecules and reducing two molecules of
NAD+ to two molecules of NADH, using electrons that originated from glucose.
The ATP molecules produced during the energy payoff phase of glycolysis are formed by substrate-level phosphorylation (Figure
7.2.1), one of two mechanisms for producing ATP. In substrate-level phosphorylation, a phosphate group is removed from an

organic molecule and is directly transferred to an available ADP molecule, producing ATP. During glycolysis, high-energy
phosphate groups from the intermediate molecules are added to ADP to make ATP.

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Overall, in this process of glycolysis, the net gain from the breakdown of a single glucose molecule is:
two ATP molecules
two NADH molecule, and
two pyruvate molecules.

Figure 7.2.1 : The energy investment phase of the Embden-Meyerhof-Parnas glycolysis pathway uses two ATP molecules to
phosphorylate glucose, forming two glyceraldehyde 3-phosphate (G3P) molecules. The energy payoff phase harnesses the energy
in the G3P molecules, producing four ATP molecules, two NADH molecules, and two pyruvates.

Figure 7.2.2 : The ATP made during glycolysis is a result of substrate-level phosphorylation. One of the two enzymatic reactions in
the energy payoff phase of Embden Meyerhof-Parnas glycolysis that produce ATP in this way is shown here.

Other Glycolytic Pathways

When we refer to glycolysis, unless otherwise indicated, we are referring to the EMP pathway used by animals and many bacteria.
However, some prokaryotes use alternative glycolytic pathways. One important alternative is the Entner-Doudoroff (ED) pathway,
named after its discoverers Nathan Entner and Michael Doudoroff (1911–1975). Although some bacteria, including the

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opportunistic gram-negative pathogen Pseudomonas aeruginosa, contain only the ED pathway for glycolysis, other bacteria, like E.
coli, have the ability to use either the ED pathway or the EMP pathway.
A third type of glycolytic pathway that occurs in all cells, which is quite different from the previous two pathways, is the pentose
phosphate pathway (PPP) also called the phosphogluconate pathway or the hexose monophosphate shunt. Evidence suggests that
the PPP may be the most ancient universal glycolytic pathway. The intermediates from the PPP are used for the biosynthesis of
nucleotides and amino acids. Therefore, this glycolytic pathway may be favored when the cell has need for nucleic acid and/or
protein synthesis, respectively.

Exercise 7.2.1

When might an organism use the ED pathway or the PPP for glycolysis?

Transition Reaction, Coenzyme A, and the Krebs Cycle

Glycolysis produces pyruvate, which can be further oxidized to capture more energy. For pyruvate to enter the next oxidative
pathway, it must first be decarboxylated by the enzyme complex pyruvate dehydrogenase to a two-carbon acetyl group in the
transition reaction, also called the bridge reaction (Figure 7.2.3). In the transition reaction, electrons are also transferred to NAD+
to form NADH. To proceed to the next phase of this metabolic process, the comparatively tiny two-carbon acetyl must be attached
to a very large carrier compound called coenzyme A (CoA). The transition reaction occurs in the mitochondrial matrix of
eukaryotes; in prokaryotes, it occurs in the cytoplasm because prokaryotes lack membrane-enclosed organelles.
.
This illustration shows the three-step conversion of pyruvate into acetyl upper case C
lower case o upper case A. In step one, a carboxyl group is removed from pyruvate,
releasing carbon dioxide. In step two, a redox reaction forms acetate and N A D H. In
step three, the acetate is transferred coenzyme A, forming acetyl upper C lower o upper
A.
Figure 7.2.3 : Upon entering the mitochondrial matrix, a multienzyme complex converts pyruvate into acetyl CoA. In the process,
carbon dioxide is released, and one molecule of NADH is formed.
The Krebs cycle transfers remaining electrons from the acetyl group produced during the transition reaction to electron carrier
molecules, thus reducing them. The Krebs cycle also occurs in the cytoplasm of prokaryotes along with glycolysis and the
transition reaction, but it takes place in the mitochondrial matrix of eukaryotic cells where the transition reaction also occurs. The
Krebs cycle is named after its discoverer, British scientist Hans Adolf Krebs (1900–1981) and is also called the citric acid cycle, or
the tricarboxylic acid cycle (TCA) because citric acid has three carboxyl groups in its structure. Unlike glycolysis, the Krebs cycle
is a closed loop: The last part of the pathway regenerates the compound used in the first step (Figure 7.2.4). The eight steps of the
cycle are a series of chemical reactions that capture the two-carbon acetyl group (the CoA carrier does not enter the Krebs cycle)
from the transition reaction, which is added to a four-carbon intermediate in the Krebs cycle, producing the six-carbon intermediate
citric acid (giving the alternate name for this cycle). As one turn of the cycle returns to the starting point of the four-carbon
intermediate, the cycle produces two CO2 molecules, one ATP molecule (or an equivalent, such as guanosine triphosphate [GTP])
produced by substrate-level phosphorylation, and three molecules of NADH and one of FADH2.

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 Figure 7.2.4 : (a) Coenzyme A is shown here without an attached acetyl group. (b) Coenzyme A is shown here with an attached
acetyl group.
Although many organisms use the Krebs cycle as described as part of glucose metabolism, several of the intermediate compounds
in the Krebs cycle can be used in synthesizing a wide variety of important cellular molecules, including amino acids, chlorophylls,
fatty acids, and nucleotides; therefore, the cycle is both anabolic and catabolic (Figure 7.2.5).

Figure 7.2.5 : The Krebs cycle, also known as the citric acid cycle, is summarized here. Note incoming two-carbon acetyl results in
the main outputs per turn of two CO2, three NADH, one FADH2, and one ATP (or GTP) molecules made by substrate-level
phosphorylation. Two turns of the Krebs cycle are required to process all of the carbon from one glucose molecule.

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 Figure 7.2.6 : Many organisms use intermediates from the Krebs cycle, such as amino acids, fatty acids, and nucleotides, as
building blocks for biosynthesis.

The Last Step

We have just discussed two pathways in glucose catabolism—glycolysis and the Krebs cycle—that generate ATP by substrate-level
phosphorylation. Most ATP, however, is generated during a separate process called oxidative phosphorylation, which occurs during
cellular respiration. Cellular respiration begins when electrons are transferred from NADH and FADH2—made in glycolysis, the
transition reaction, and the Krebs cycle—through a series of chemical reactions to a final inorganic electron acceptor (either oxygen
in aerobic respiration or non-oxygen inorganic molecules in anaerobic respiration). These electron transfers take place on the inner
part of the cell membrane of prokaryotic cells or in specialized protein complexes in the inner membrane of the mitochondria of
eukaryotic cells. The energy of the electrons is harvested to generate an electrochemical gradient across the membrane, which is
used to make ATP by oxidative phosphorylation.

Electron Transport System

The electron transport chain (ETC) or electron transport system (ETS) is the last component involved in the process of cellular
respiration; it comprises a series of membrane-associated protein complexes and associated mobile accessory electron carriers.
Electron transport is a series of chemical reactions that resembles a bucket brigade in that electrons from NADH and FADH2 are
passed rapidly from one ETS electron carrier to the next. These carriers can pass electrons along in the ETS because of their redox
potential. For a protein or chemical to accept electrons, it must have a more positive redox potential than the electron donor.
Therefore, electrons move from electron carriers with more negative redox potential to those with more positive redox potential.
The four major classes of electron carriers involved in both eukaryotic and prokaryotic electron transport systems are the
cytochromes, flavoproteins, iron-sulfur proteins, and the quinones.
In aerobic respiration, the final electron acceptor (i.e., the one having the most positive redox potential) at the end of the ETS is an
oxygen molecule (O2) that becomes reduced to water (H2O) by the final ETS carrier. This electron carrier, cytochrome oxidase,

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differs between bacterial types and can be used to differentiate closely related bacteria for diagnoses. For example, the gram-
negative opportunist Pseudomonas aeruginosa and the gram-negative cholera-causing Vibrio cholerae use cytochrome c oxidase,
which can be detected by the oxidase test, whereas other gram-negative Enterobacteriaceae, like E. coli, are negative for this test
because they produce different cytochrome oxidase types.
There are many circumstances under which aerobic respiration is not possible, including any one or more of the following:
The cell lacks genes encoding an appropriate cytochrome oxidase for transferring electrons to oxygen at the end of the electron
transport system.
The cell lacks genes encoding enzymes to minimize the severely damaging effects of dangerous oxygen radicals produced
during aerobic respiration, such as hydrogen peroxide (H2O2) or superoxide (O2–).
The cell lacks a sufficient amount of oxygen to carry out aerobic respiration.
One possible alternative to aerobic respiration is anaerobic respiration, using an inorganic molecule other than oxygen as a final
electron acceptor. There are many types of anaerobic respiration found in bacteria and archaea. Denitrifiers are important soil
bacteria that use nitrate (NO3–) and nitrite (NO2–) as final electron acceptors, producing nitrogen gas (N2). Many aerobically
respiring bacteria, including E. coli, switch to using nitrate as a final electron acceptor and producing nitrite when oxygen levels
have been depleted.
Microbes using anaerobic respiration commonly have an intact Krebs cycle, so these organisms can access the energy of the
NADH and FADH2 molecules formed. However, anaerobic respirers use altered ETS carriers encoded by their genomes, including
distinct complexes for electron transfer to their final electron acceptors. Smaller electrochemical gradients are generated from these
electron transfer systems, so less ATP is formed through anaerobic respiration.

Exercise 7.2.2

Do both aerobic respiration and anaerobic respiration use an electron transport chain?

Chemiosmosis, Proton Motive Force, and Oxidative Phosphorylation

In each transfer of an electron through the ETS, the electron loses energy, but with some transfers, the energy is stored as potential
energy by using it to pump hydrogen ions (H+) across a membrane. In prokaryotic cells, H+ is pumped to the outside of the
cytoplasmic membrane (called the periplasmic space in gram-negative and gram-positive bacteria), and in eukaryotic cells, they are
pumped from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space. There is an uneven
distribution of H+ across the membrane that establishes an electrochemical gradient because H+ ions are positively charged
(electrical) and there is a higher concentration (chemical) on one side of the membrane. This electrochemical gradient formed by
the accumulation of H+ (also known as a proton) on one side of the membrane compared with the other is referred to as the proton
motive force (PMF). Because the ions involved are H+, a pH gradient is also established, with the side of the membrane having the
higher concentration of H+ being more acidic. Beyond the use of the PMF to make ATP, as discussed in this chapter, the PMF can
also be used to drive other energetically unfavorable processes, including nutrient transport and flagella rotation for motility.
The potential energy of this electrochemical gradient generated by the ETS causes the H+ to diffuse across a membrane (the plasma
membrane in prokaryotic cells and the inner membrane in mitochondria in eukaryotic cells). This flow of hydrogen ions across the
membrane, called chemiosmosis, must occur through a channel in the membrane via a membrane-bound enzyme complex called
ATP synthase (Figure 7.2.7). The tendency for movement in this way is much like water accumulated on one side of a dam,
moving through the dam when opened. ATP synthase (like a combination of the intake and generator of a hydroelectric dam) is a
complex protein that acts as a tiny generator, turning by the force of the H+ diffusing through the enzyme, down their
electrochemical gradient from where there are many mutually repelling H+ to where there are fewer H+. In prokaryotic cells, H+
flows from the outside of the cytoplasmic membrane into the cytoplasm, whereas in eukaryotic mitochondria, H+ flows from the
intermembrane space to the mitochondrial matrix. The turning of the parts of this molecular machine regenerates ATP from ADP
and inorganic phosphate (Pi) by oxidative phosphorylation, a second mechanism for making ATP that harvests the potential energy
stored within an electrochemical gradient.

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 The inner membrane of the mitochondria is shown. On the membrane are a series of proteins in a row and a large protein off to one side. In the inner mitochondrial matrix is the overall equation showing 2 free
hydrogen ions + 2 electrons exiting ETC + ½ of an O2 molecule produce water. This happens twice. The diagram shows 2 electrons on the first protein in the chain. These electrons come from the splitting of
NADH to NAD+. The electrons are then moved to the next protein in the chain, and down the line of 5 proteins in the electron transport chain. Electrons can also be added to the chain on the second protein from
the splitting of FADH2 into FAD+. As the electrons are passed through proteins 1, 3, and 5 protons (H+) are pumped across the membrane. These protons can then flow back to the mitochondrial matrix through
ATP synthase. As they flow through ATP synthase, they allow for the production of ATP from ADP and PO4,3-.

Figure 7.2.7: The electron transport chain is a series of electron carriers and ion pumps that are used to pump H+ ions across a
membrane. H+ then flow back through the membrane by way of ATP synthase, which catalyzes the formation of ATP. The location
of the electron transport chain is the inner mitochondrial matrix in eukaryotic cells and cytoplasmic membrane in prokaryotic cells.

The number of ATP molecules generated from the catabolism of glucose varies. For example, the number of hydrogen ions that the
electron transport system complexes can pump through the membrane varies between different species of organisms. In aerobic
respiration in mitochondria, the passage of electrons from one molecule of NADH generates enough proton motive force to make
three ATP molecules by oxidative phosphorylation, whereas the passage of electrons from one molecule of FADH2 generates
enough proton motive force to make only two ATP molecules. Thus, the 10 NADH molecules made per glucose during glycolysis,
the transition reaction, and the Krebs cycle carry enough energy to make 30 ATP molecules, whereas the two FADH2 molecules
made per glucose during these processes provide enough energy to make four ATP molecules. Overall, the theoretical maximum
yield of ATP made during the complete aerobic respiration of glucose is 38 molecules, with four being made by substrate-level
phosphorylation and 34 being made by oxidative phosphorylation (Figure 7.2.8). In reality, the total ATP yield is usually less,
ranging from one to 34 ATP molecules, depending on whether the cell is using aerobic respiration or anaerobic respiration; in
eukaryotic cells, some energy is expended to transport intermediates from the cytoplasm into the mitochondria, affecting ATP
yield. Figure 7.2.8 summarizes the theoretical maximum yields of ATP from various processes during the complete aerobic
respiration of one glucose molecule.

Figure 7.2.8 : Theoretical maximum yields of ATP from various processes during the complete aerobic respiration of one glucose
molecule.

Exercise 7.2.3

What are the functions of the proton motive force?

Clinical Focus: part 2

Many of Hannah’s symptoms are consistent with several different infections, including influenza and pneumonia. However, her
sluggish reflexes along with her light sensitivity and stiff neck suggest some possible involvement of the central nervous
system, perhaps indicating meningitis. Meningitis is an infection of the cerebrospinal fluid (CSF) around the brain and spinal
cord that causes inflammation of the meninges, the protective layers covering the brain. Meningitis can be caused by viruses,

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 bacteria, or fungi. Although all forms of meningitis are serious, bacterial meningitis is particularly serious. Bacterial meningitis
may be caused by several different bacteria, but the bacterium Neisseria meningitidis, a gram-negative, bean-shaped
diplococcus, is a common cause and leads to death within 1 to 2 days in 5% to 10% of patients.
Given the potential seriousness of Hannah’s conditions, her physician advised her parents to take her to the hospital in the
Gambian capital of Banjul and there have her tested and treated for possible meningitis. After a 3-hour drive to the hospital,
Hannah was immediately admitted. Physicians took a blood sample and performed a lumbar puncture to test her CSF. They
also immediately started her on a course of the antibiotic ceftriaxone, the drug of choice for treatment of meningitis caused by
N. meningitidis, without waiting for laboratory test results.

Exercise 7.2.4

1. How might biochemical testing be used to confirm the identity of N. meningitidis?

2. Why did Hannah’s doctors decide to administer antibiotics without waiting for the test results?

Key Concepts and Summary

Glycolysis is the first step in the breakdown of glucose, resulting in the formation of ATP, which is produced by substrate-level
phosphorylation; NADH; and two pyruvate molecules. Glycolysis does not use oxygen and is not oxygen dependent.
After glycolysis, a three-carbon pyruvate is decarboxylated to form a two-carbon acetyl group, coupled with the formation of
NADH. The acetyl group is attached to a large carrier compound called coenzyme A.
After the transition step, coenzyme A transports the two-carbon acetyl to the Krebs cycle, where the two carbons enter the
cycle. Per turn of the cycle, one acetyl group derived from glycolysis is further oxidized, producing three NADH molecules,
one FADH2, and one ATP by substrate-level phosphorylation, and releasing two CO2molecules.
The Krebs cycle may be used for other purposes. Many of the intermediates are used to synthesize important cellular molecules,
including amino acids, chlorophylls, fatty acids, and nucleotides.
Most ATP generated during the cellular respiration of glucose is made by oxidative phosphorylation.
An electron transport system (ETS) is composed of a series of membrane-associated protein complexes and associated mobile
accessory electron carriers. The ETS is embedded in the cytoplasmic membrane of prokaryotes and the inner mitochondrial
membrane of eukaryotes.
Each ETS complex has a different redox potential, and electrons move from electron carriers with more negative redox
potential to those with more positive redox potential.
To carry out aerobic respiration, a cell requires oxygen as the final electron acceptor. A cell also needs a complete Krebs
cycle, an appropriate cytochrome oxidase, and oxygen detoxification enzymes to prevent the harmful effects of oxygen radicals
produced during aerobic respiration.
Organisms performing anaerobic respiration use alternative electron transport system carriers for the ultimate transfer of
electrons to the final non-oxygen electron acceptors.
Microbes show great variation in the composition of their electron transport systems, which can be used for diagnostic purposes
to help identify certain pathogens.
As electrons are passed from NADH and FADH2 through an ETS, the electron loses energy. This energy is stored through the
pumping of H+ across the membrane, generating a proton motive force.
The energy of this proton motive force can be harnessed by allowing hydrogen ions to diffuse back through the membrane by
chemiosmosis using ATP synthase. As hydrogen ions diffuse through down their electrochemical gradient, components of
ATP synthase spin, making ATP from ADP and Pi by oxidative phosphorylation.
Aerobic respiration forms more ATP (a maximum of 34 ATP molecules) during oxidative phosphorylation than does anaerobic
respiration (between one and 32 ATP molecules).

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

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This page titled 7.2: Catabolism of Carbohydrates is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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7.3: Alternate Forms of Catabolism: Fermentation, Lipids and Proteins
Learning Objectives
Define fermentation and explain why it does not require oxygen
Describe the fermentation pathways and their end products and give examples of microorganisms that use these pathways
Compare and contrast fermentation and anaerobic respiration
Describe how lipids are catabolized
Describe how lipid catabolism can be used to identify microbes
Describe how proteins are catabolized
Describe how protein catabolism can be used to identify bacteria

Fermentation
Many cells are unable to carry out respiration because of one or more of the following circumstances:
1. The cell lacks a sufficient amount of any appropriate, inorganic, final electron acceptor to carry out cellular respiration.
2. The cell lacks genes to make appropriate complexes and electron carriers in the electron transport system.
3. The cell lacks genes to make one or more enzymes in the Krebs cycle.
Whereas lack of an appropriate inorganic final electron acceptor is environmentally dependent, the other two conditions are
genetically determined. Thus, many prokaryotes, including members of the clinically important genus Streptococcus, are
permanently incapable of respiration, even in the presence of oxygen. Conversely, many prokaryotes are facultative, meaning that,
should the environmental conditions change to provide an appropriate inorganic final electron acceptor for respiration, organisms
containing all the genes required to do so will switch to cellular respiration for glucose metabolism because respiration allows for
much greater ATP production per glucose molecule.
If respiration does not occur, NADH must be reoxidized to NAD+ for reuse as an electron carrier for glycolysis, the cell’s only
mechanism for producing any ATP, to continue. Some living systems use an organic molecule (commonly pyruvate) as a final
electron acceptor through a process called fermentation. Fermentation does not involve an electron transport system and does not
directly produce any additional ATP beyond that produced during glycolysis by substrate-level phosphorylation. Organisms
carrying out fermentation, called fermenters, produce a maximum of two ATP molecules per glucose during glycolysis. Table 7.3.1
compares the final electron acceptors and methods of ATP synthesis in aerobic respiration, anaerobic respiration, and fermentation.
Note that the number of ATP molecules shown for glycolysis assumes the Embden-Meyerhof-Parnas pathway. The number of ATP
molecules made by substrate-level phosphorylation (SLP) versus oxidative phosphorylation (OP) are indicated.
Table 7.3.1 : Comparison of Respiration Versus Fermentation
Pathways Involved in ATP
Maximum Yield of ATP
Type of Metabolism Example Final Electron Acceptor Synthesis (Type of
Molecules
Phosphorylation)

EMP glycolysis (SLP)

2
Krebs cycle (SLP)
2
Aerobic respiration Pseudomonas aeruginosa Electron transport and
O
2 34
chemiosmosis (OP):

Total 38

EMP glycolysis (SLP)

2
Krebs cycle (SLP)
−
, SO , Fe , CO
−2 +3 2
Anaerobic respiration Paracoccus denitrificans
NO
3 4 2 Electron transport and
other inorganics 1–32
chemiosmosis (OP):

Total 5–36

Organics EMP glycolysis (SLP) 2

Fermentation Candida albicans (usually pyruvate) Fermentation 0

Total 2

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Microbial fermentation processes have been manipulated by humans and are used extensively in the production of various foods
and other commercial products, including pharmaceuticals. Microbial fermentation can also be useful for identifying microbes for
diagnostic purposes.

Lactic Acid Fermentation

Fermentation by some bacteria, like those in yogurt and other soured food products, and by animals in muscles during oxygen
depletion, is lactic acid fermentation. (Figure 7.3.1). The chemical reaction of lactic acid fermentation is as follows:
Pyruvate + NADH↔lactic acid + NAD+Pyruvate + NADH ↔ lactic acid + NAD+
This type of fermentation is used routinely in mammalian red blood cells and in skeletal muscle that has an insufficient oxygen
supply to allow aerobic respiration to continue (that is, in muscles used to the point of fatigue). In muscles, lactic acid accumulation
must be removed by the blood circulation and the lactate brought to the liver for further metabolism.The enzyme used in this
reaction is often lactate dehydrogenase (LDH). The reaction can proceed in either direction, but the reaction is inhibited by acidic
conditions. Such lactic acid accumulation was once believed to cause muscle stiffness, fatigue, and soreness, although more recent
research disputes this hypothesis. Once the lactic acid has been removed from the muscle and circulated to the liver, it can be
reconverted into pyruvic acid and further catabolized for energy.
Bacteria of several gram-positive genera, including Lactobacillus, Leuconostoc, and Streptococcus, are collectively known as the
lactic acid bacteria (LAB), and various strains are important in food production. During yogurt and cheese production, the highly
acidic environment generated by lactic acid fermentation denatures proteins contained in milk, causing it to solidify. When lactic
acid is the only fermentation product, the process is said to be homolactic fermentation; such is the case for Lactobacillus
delbrueckii and S. thermophiles used in yogurt production. However, many bacteria perform heterolactic fermentation, producing a
mixture of lactic acid, ethanol and/or acetic acid, and CO2 as a result, because of their use of the branched pentose phosphate
pathway instead of the EMP pathway for glycolysis. One important heterolactic fermenter is Leuconostoc mesenteroides, which is
used for souring vegetables like cucumbers and cabbage, producing pickles and sauerkraut, respectively.
Lactic acid bacteria are also important medically. The production of low pH environments within the body inhibits the
establishment and growth of pathogens in these areas. For example, the vaginal microbiota is composed largely of lactic acid
bacteria, but when these bacteria are reduced, yeast can proliferate, causing a yeast infection. Additionally, lactic acid bacteria are
important in maintaining the health of the gastrointestinal tract and, as such, are the primary component of probiotics.

Figure 7.3.1 : Lactic acid fermentation is common in muscle cells that have run out of oxygen

Alcohol Fermentation
Another familiar fermentation process is alcohol fermentation, which produces ethanol. The ethanol fermentation reaction is shown
in Figure 7.3.2. In the first reaction, the enzyme pyruvate decarboxylase removes a carboxyl group from pyruvate, releasing CO2
gas while producing the two-carbon molecule acetaldehyde. The second reaction, catalyzed by the enzyme alcohol dehydrogenase,

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transfers an electron from NADH to acetaldehyde, producing ethanol and NAD+. The ethanol fermentation of pyruvate by the yeast
Saccharomyces cerevisiae is used in the production of alcoholic beverages and also makes bread products rise due to CO2
production. Ethanol tolerance of yeast is variable, ranging from about 5 percent to 21 percent, depending on the yeast strain and
environmental conditions. Outside of the food industry, ethanol fermentation of plant products is important in biofuel production.

Figure 7.3.2 : The chemical reactions of alcohol fermentation are shown here. Ethanol fermentation is important in the production
of alcoholic beverages and bread.

Other Fermentation Pathways

Beyond lactic acid fermentation and alcohol fermentation, many other fermentation methods occur in prokaryotes, all for the
purpose of ensuring an adequate supply of NAD+ for glycolysis (Table 7.3.2). Without these pathways, glycolysis would not occur
and no ATP would be harvested from the breakdown of glucose. It should be noted that most forms of fermentation besides
homolactic fermentation produce gas, commonly CO2 and/or hydrogen gas. Many of these different types of fermentation
pathways are also used in food production and each results in the production of different organic acids, contributing to the unique
flavor of a particular fermented food product. The propionic acid produced during propionic acid fermentation contributes to the
distinctive flavor of Swiss cheese, for example.
Several fermentation products are important commercially outside of the food industry. For example, chemical solvents such as
acetone and butanol are produced during acetone-butanol-ethanol fermentation. Complex organic pharmaceutical compounds used
in antibiotics (e.g., penicillin), vaccines, and vitamins are produced through mixed acid fermentation. Fermentation products are
used in the laboratory to differentiate various bacteria for diagnostic purposes. For example, enteric bacteria are known for their
ability to perform mixed acid fermentation, reducing the pH, which can be detected using a pH indicator. Similarly, the bacterial
production of acetoin during butanediol fermentation can also be detected. Gas production from fermentation can also be seen in an
inverted Durham tube that traps produced gas in a broth culture.
Microbes can also be differentiated according to the substrates they can ferment. For example, E. coli can ferment lactose, forming
gas, whereas some of its close gram-negative relatives cannot. The ability to ferment the sugar alcohol sorbitol is used to identify
the pathogenic enterohemorrhagic O157:H7 strain of E. coli because, unlike other E. coli strains, it is unable to ferment sorbitol.
Last, mannitol fermentation differentiates the mannitol-fermenting Staphylococcus aureus from other non–mannitol-fermenting
staphylococci.
Table 7.3.2 : Common Fermentation Pathways
Pathway End Products Example Microbes Commercial Products

Commercial solvents, gasoline

Acetone-butanol-ethanol Acetone, butanol, ethanol, CO2 Clostridium acetobutylicum
alternative

Alcohol Ethanol, CO2 Candida, Saccharomyces Beer, bread

Formic and lactic acid; ethanol;

Butanediol acetoin; 2,3 butanediol; CO2; Klebsiella, Enterobacter Chardonnay wine
hydrogen gas

Butyric acid Butyric acid, CO2, hydrogen gas Clostridium butyricum Butter

Lactic acid Lactic acid Streptococcus, Lactobacillus Sauerkraut, yogurt, cheese

Acetic, formic, lactic, and succinic Vinegar, cosmetics,

Mixed acid Escherichia, Shigella
acids; ethanol, CO2, hydrogen gas pharmaceuticals

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 Pathway End Products Example Microbes Commercial Products

Propionibacterium,
Propionic acid Acetic acid, propionic acid, CO2 Swiss cheese
Bifidobacterium

Exercise 7.3.5

When would a metabolically versatile microbe perform fermentation rather than cellular respiration?

No Carbohydrate Available
Previous sections have discussed the catabolism of glucose, which provides energy to living cells, as well as how polysaccharides
like glycogen, starch, and cellulose are degraded to glucose monomers. But microbes consume more than just carbohydrates for
food. In fact, the microbial world is known for its ability to degrade a wide range of molecules, both naturally occurring and those
made by human processes, for use as carbon sources. In this section, we will see that the pathways for both lipid and protein
catabolism connect to those used for carbohydrate catabolism, eventually leading into glycolysis, the transition reaction, and the
Krebs cycle pathways. Metabolic pathways should be considered to be porous—that is, substances enter from other pathways, and
intermediates leave for other pathways. These pathways are not closed systems. Many of the substrates, intermediates, and products
in a particular pathway are reactants in other pathways.

Lipid Catabolism
Triglycerides are a form of long-term energy storage in animals. They are made of glycerol and three fatty acids. Phospholipids
compose the cell and organelle membranes of all organisms except the archaea. Phospholipid structure is similar to triglycerides
except that one of the fatty acids is replaced by a phosphorylated head group. Triglycerides and phospholipids are broken down
first by releasing fatty acid chains (and/or the phosphorylated head group, in the case of phospholipids) from the three-carbon
glycerol backbone. The reactions breaking down triglycerides are catalyzed by lipases and those involving phospholipids are
catalyzed by phospholipases. These enzymes contribute to the virulence of certain microbes, such as the bacterium Staphylococcus
aureus and the fungus Cryptococcus neoformans. These microbes use phospholipases to destroy lipids and phospholipids in host
cells and then use the catabolic products for energy.
The resulting products of lipid catabolism, glycerol and fatty acids, can be further degraded. Glycerol can be phosphorylated to
glycerol-3-phosphate and easily converted to glyceraldehyde 3-phosphate, which continues through glycolysis. The released fatty
acids are catabolized in a process called β-oxidation, which sequentially removes two-carbon acetyl groups from the ends of fatty
acid chains, reducing NAD+ and FAD to produce NADH and FADH2, respectively, whose electrons can be used to make ATP by
oxidative phosphorylation. The acetyl groups produced during β-oxidation are carried by coenzyme A to the Krebs cycle, and their
movement through this cycle results in their degradation to CO2, producing ATP by substrate-level phosphorylation and additional
NADH and FADH2 molecules.
Other types of lipids can also be degraded by certain microbes. For example, the ability of certain pathogens, like Mycobacterium
tuberculosis, to degrade cholesterol contributes to their virulence. The side chains of cholesterol can be easily removed
enzymatically, but degradation of the remaining fused rings is more problematic. The four fused rings are sequentially broken in a
multistep process facilitated by specific enzymes, and the resulting products, including pyruvate, can be further catabolized in the
Krebs cycle.

Exercise 7.3.1

How can lipases and phospholipases contribute to virulence in microbes?

Protein Catabolism
Proteins are degraded through the concerted action of a variety of microbial protease enzymes. Extracellular proteases cut proteins
internally at specific amino acid sequences, breaking them down into smaller peptides that can then be taken up by cells. Some
clinically important pathogens can be identified by their ability to produce a specific type of extracellular protease. For example,
the production of the extracellular protease gelatinase by members of the genera Proteus and Serratia can be used to distinguish
them from other gram-negative enteric bacteria. Following inoculation and growth of microbes in gelatin broth, degradation of the
gelatin protein due to gelatinase production prevents solidification of gelatin when refrigerated. Other pathogens can be

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distinguished by their ability to degrade casein, the main protein found in milk. When grown on skim milk agar, production of the
extracellular protease caseinase causes degradation of casein, which appears as a zone of clearing around the microbial growth.
Caseinase production by the opportunist pathogen Pseudomonas aeruginosa can be used to distinguish it from other related gram-
negative bacteria.
After extracellular protease degradation and uptake of peptides in the cell, the peptides can then be broken down further into
individual amino acids by additional intracellular proteases, and each amino acid can be enzymatically deaminated to remove the
amino group. The remaining molecules can then enter the transition reaction or the Krebs cycle.

Exercise 7.3.2

How can protein catabolism help identify microbes?

Clinical Focus: part 3

Because bacterial meningitis progresses so rapidly, Hannah’s doctors had decided to treat her aggressively with antibiotics,
based on empirical observation of her symptoms. However, laboratory testing to confirm the cause of Hannah’s meningitis was
still important for several reasons. N. meningitidis is an infectious pathogen that can be spread from person to person through
close contact; therefore, if tests confirm N. meningitidis as the cause of Hannah’s symptoms, Hannah’s parents and others who
came into close contact with her might need to be vaccinated or receive prophylactic antibiotics to lower their risk of
contracting the disease. On the other hand, if it turns out that N. meningitidis is not the cause, Hannah’s doctors might need to
change her treatment.
The clinical laboratory performed a Gram stain on Hannah’s blood and CSF samples. The Gram stain showed the presence of a
bean-shaped gram-negative diplococcus. The technician in the hospital lab cultured Hannah’s blood sample on both blood agar
and chocolate agar, and the bacterium that grew on both media formed gray, nonhemolytic colonies. Next, he performed an
oxidase test on this bacterium and determined that it was oxidase positive. Last, he examined the repertoire of sugars that the
bacterium could use as a carbon source and found that the bacterium was positive for glucose and maltose use but negative for
lactose and sucrose use. All of these test results are consistent with characteristics of N. meningitidis.

Exercise 7.3.3

1. What do these test results tell us about the metabolic pathways of N. meningitidis?
2. Why do you think that the hospital used these biochemical tests for identification in lieu of molecular analysis by DNA
testing?

Key Concepts and Summary

Fermentation uses an organic molecule as a final electron acceptor to regenerate NAD+ from NADH so that glycolysis can
continue.
Fermentation does not involve an electron transport system, and no ATP is made by the fermentation process directly.
Fermenters make very little ATP—only two ATP molecules per glucose molecule during glycolysis.
Microbial fermentation processes have been used for the production of foods and pharmaceuticals, and for the identification of
microbes.
During lactic acid fermentation, pyruvate accepts electrons from NADH and is reduced to lactic acid. Microbes performing
homolactic fermentation produce only lactic acid as the fermentation product; microbes performing heterolactic
fermentation produce a mixture of lactic acid, ethanol and/or acetic acid, and CO2.
Lactic acid production by the normal microbiota prevents growth of pathogens in certain body regions and is important for the
health of the gastrointestinal tract.
During ethanol fermentation, pyruvate is first decarboxylated (releasing CO2) to acetaldehyde, which then accepts electrons
from NADH, reducing acetaldehyde to ethanol. Ethanol fermentation is used for the production of alcoholic beverages, for
making bread products rise, and for biofuel production.
Fermentation products of pathways (e.g., propionic acid fermentation) provide distinctive flavors to food products.
Fermentation is used to produce chemical solvents (acetone-butanol-ethanol fermentation) and pharmaceuticals (mixed acid
fermentation).

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 Specific types of microbes may be distinguished by their fermentation pathways and products. Microbes may also be
differentiated according to the substrates they are able to ferment.
Collectively, microbes have the ability to degrade a wide variety of carbon sources besides carbohydrates, including lipids and
proteins. The catabolic pathways for all of these molecules eventually connect into glycolysis and the Krebs cycle.
Several types of lipids can be microbially degraded. Triglycerides are degraded by extracellular lipases, releasing fatty acids
from the glycerol backbone. Phospholipids are degraded by phospholipases, releasing fatty acids and the phosphorylated head
group from the glycerol backbone. Lipases and phospholipases act as virulence factors for certain pathogenic microbes.
Fatty acids can be further degraded inside the cell through β-oxidation, which sequentially removes two-carbon acetyl groups
from the ends of fatty acid chains.
Protein degradation involves extracellular proteases that degrade large proteins into smaller peptides. Detection of the
extracellular proteases gelatinase and caseinase can be used to differentiate clinically relevant bacteria.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 7.3: Alternate Forms of Catabolism: Fermentation, Lipids and Proteins is shared under a CC BY license and was authored,
remixed, and/or curated by OpenStax.

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7.4: Photosynthesis and the Importance of Light
Learning Objectives
Describe the function and locations of photosynthetic pigments in eukaryotes and prokaryotes
Describe the major products of the light-dependent and light-independent reactions
Describe the reactions that produce glucose in a photosynthetic cell
Compare and contrast cyclic and noncyclic photophosphorylation

Heterotrophic organisms ranging from E. coli to humans rely on the chemical energy found mainly in carbohydrate molecules.
Many of these carbohydrates are produced by photosynthesis, the biochemical process by which phototrophic organisms convert
solar energy (sunlight) into chemical energy. Although photosynthesis is most commonly associated with plants, microbial
photosynthesis is also a significant supplier of chemical energy, fueling many diverse ecosystems. In this section, we will focus on
microbial photosynthesis.
Photosynthesis takes place in two sequential stages: the light-dependent reactions and the light-independent reactions (Figure
7.4.1). In the light-dependent reactions, energy from sunlight is absorbed by pigment molecules in photosynthetic membranes and

converted into stored chemical energy. In the light-independent reactions, the chemical energy produced by the light-dependent
reactions is used to drive the assembly of sugar molecules using CO2; however, these reactions are still light dependent because the
products of the light-dependent reactions necessary for driving them are short-lived. The light-dependent reactions produce ATP
and either NADPH or NADH to temporarily store energy. These energy carriers are used in the light-independent reactions to drive
the energetically unfavorable process of “fixing” inorganic CO2 in an organic form, sugar.

Figure 7.4.1 : The light-dependent reactions of photosynthesis (left) convert light energy into chemical energy, forming ATP and
NADPH. These products are used by the light-independent reactions to fix CO2, producing organic carbon molecules.

Photosynthetic Structures in Eukaryotes and Prokaryotes

In all phototrophic eukaryotes, photosynthesis takes place inside a chloroplast, an organelle that arose in eukaryotes by
endosymbiosis of a photosynthetic bacterium. These chloroplasts are enclosed by a double membrane with inner and outer layers.
Within the chloroplast is a third membrane that forms stacked, disc-shaped photosynthetic structures called thylakoids (Figure
7.4.2). A stack of thylakoids is called a granum, and the space surrounding the granum within the chloroplast is called stroma.

Photosynthetic membranes in prokaryotes, by contrast, are not organized into distinct membrane-enclosed organelles; rather, they
are infolded regions of the plasma membrane. In cyanobacteria, for example, these infolded regions are also referred to as
thylakoids. In either case, embedded within the thylakoid membranes or other photosynthetic bacterial membranes are

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photosynthetic pigment molecules organized into one or more photosystems, where light energy is actually converted into chemical
energy.
Photosynthetic pigments within the photosynthetic membranes are organized into photosystems, each of which is composed of a
light-harvesting (antennae) complex and a reaction center. The light-harvesting complex consists of multiple proteins and
associated pigments that each may absorb light energy and, thus, become excited. This energy is transferred from one pigment
molecule to another until eventually (after about a millionth of a second) it is delivered to the reaction center. Up to this point, only
energy—not electrons—has been transferred between molecules. The reaction center contains a pigment molecule that can undergo
oxidation upon excitation, actually giving up an electron. It is at this step in photosynthesis that light energy is converted into an
excited electron.
Different kinds of light-harvesting pigments absorb unique patterns of wavelengths (colors) of visible light. Pigments reflect or
transmit the wavelengths they cannot absorb, making them appear the corresponding color. Examples of photosynthetic pigments
(molecules used to absorb solar energy) are bacteriochlorophylls (green, purple, or red), carotenoids (orange, red, or yellow),
chlorophylls (green), phycocyanins (blue), and phycoerythrins (red). By having mixtures of pigments, an organism can absorb
energy from more wavelengths. Because photosynthetic bacteria commonly grow in competition for sunlight, each type of
photosynthetic bacteria is optimized for harvesting the wavelengths of light to which it is commonly exposed, leading to
stratification of microbial communities in aquatic and soil ecosystems by light quality and penetration.
Once the light harvesting complex transfers the energy to the reaction center, the reaction center delivers its high-energy electrons,
one by one, to an electron carrier in an electron transport system, and electron transfer through the ETS is initiated. The ETS is
similar to that used in cellular respiration and is embedded within the photosynthetic membrane. Ultimately, the electron is used to
produce NADH or NADPH. The electrochemical gradient that forms across the photosynthetic membrane is used to generate ATP
by chemiosmosis through the process of photophosphorylation, another example of oxidative phosphorylation (Figure 7.4.3).

Figure 7.4.2 : (a) Photosynthesis in eukaryotes takes place in chloroplasts, which contain thylakoids stacked into grana. (b) A
photosynthetic prokaryote has infolded regions of the plasma membrane that function like thylakoids. (credit: scale bar data from
Matt Russell.)

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 Figure 7.4.3 : This figure summarizes how a photosystem works. Light harvesting (LH) pigments absorb light energy, converting it
to chemical energy. The energy is passed from one LH pigment to another until it reaches a reaction center (RC) pigment, exciting
an electron. This high-energy electron is lost from the RC pigment and passed through an electron transport system (ETS),
ultimately producing NADH or NADPH and ATP. A reduced molecule (H2A) donates an electron, replacing electrons to the
electron-deficient RC pigment.

Exercise 7.4.1

In a phototrophic eukaryote, where does photosynthesis take place?

Oxygenic and Anoxygenic Photosynthesis

For photosynthesis to continue, the electron lost from the reaction center pigment must be replaced. The source of this electron
(H2A) differentiates the oxygenic photosynthesis of plants and cyanobacteria from anoxygenic photosynthesis carried out by other
types of bacterial phototrophs (Figure 7.4.4). In oxygenic photosynthesis, H2O is split and supplies the electron to the reaction
center. Because oxygen is generated as a byproduct and is released, this type of photosynthesis is referred to as oxygenic
photosynthesis. However, when other reduced compounds serve as the electron donor, oxygen is not generated; these types of
photosynthesis are called anoxygenic photosynthesis. Hydrogen sulfide (H2S) or thiosulfate (S2O2−3) can serve as the electron
donor, generating elemental sulfur and sulfate (SO2−4) ions, respectively, as a result.

Figure 7.4.4 : Eukaryotes and cyanobacteria carry out oxygenic photosynthesis, producing oxygen, whereas other bacteria carry out
anoxygenic photosynthesis, which does not produce oxygen.
Photosystems have been classified into two types: photosystem I (PSI) and photosystem II (PSII) (Figure 7.4.5). Cyanobacteria and
plant chloroplasts have both photosystems, whereas anoxygenic photosynthetic bacteria use only one of the photosystems. Both
photosystems are excited by light energy simultaneously. If the cell requires both ATP and NADPH for biosynthesis, then it will
carry out noncyclic photophosphorylation. Upon passing of the PSII reaction center electron to the ETS that connects PSII and PSI,
the lost electron from the PSII reaction center is replaced by the splitting of water. The excited PSI reaction center electron is used

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to reduce NADP+ to NADPH and is replaced by the electron exiting the ETS. The flow of electrons in this way is called the Z-
scheme.If a cell’s need for ATP is significantly greater than its need for NADPH, it may bypass the production of reducing power
through cyclic photophosphorylation. Only PSI is used during cyclic photophosphorylation; the high-energy electron of the PSI
reaction center is passed to an ETS carrier and then ultimately returns to the oxidized PSI reaction center pigment, thereby reducing
it.

Figure 7.4.5: PSI and PSII are found on the thylakoid membrane. The high-energy electron from PSII is passed to an ETS, which
generates a proton motive force for ATP synthesis by chemiosmosis, and ultimately replaces the electron lost by the PSI reaction
center. The PSI reaction center electron is used to make NADPH.

Figure 7.4.6 : When both ATP and NADPH are required, noncyclic photophosphorylation (in cyanobacteria and plants) provides
both. The electron flow described here is referred to as the Z-scheme (shown in yellow in [a]). When the cell’s ATP needs outweigh
those for NADPH, cyanobacteria and plants will use only PSI, and its reaction center electron is passed to the ETS to generate a
proton motive force used for ATP synthesis.

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 Exercise 7.4.2

Why would a photosynthetic bacterium have different pigments?

Light-Independent Reactions
After the energy from the sun is converted into chemical energy and temporarily stored in ATP and NADPH molecules (having
lifespans of millionths of a second), photoautotrophs have the fuel needed to build multicarbon carbohydrate molecules, which can
survive for hundreds of millions of years, for long-term energy storage. The carbon comes from CO2, the gas that is a waste
product of cellular respiration.
The Calvin-Benson cycle (named for Melvin Calvin [1911–1997] and Andrew Benson [1917–2015]), the biochemical pathway
used for fixation of CO2, is located within the cytoplasm of photosynthetic bacteria and in the stroma of eukaryotic chloroplasts.
The light-independent reactions of the Calvin cycle can be organized into three basic stages: fixation, reduction, and regeneration.
Fixation: The enzyme ribulose bisphosphate carboxylase (RuBisCO) catalyzes the addition of a CO2 to ribulose bisphosphate
(RuBP). This results in the production of 3-phosphoglycerate (3-PGA).
Reduction: Six molecules of both ATP and NADPH (from the light-dependent reactions) are used to convert 3-PGA into
glyceraldehyde 3-phosphate (G3P). Some G3P is then used to build glucose.
Regeneration: The remaining G3P not used to synthesize glucose is used to regenerate RuBP, enabling the system to continue
CO2 fixation. Three more molecules of ATP are used in these regeneration reactions.
The Calvin cycle is used extensively by plants and photoautotrophic bacteria, and the enzyme RuBisCO is said to be the most
plentiful enzyme on earth, composing 30%–50% of the total soluble protein in plant chloroplasts.1 However, besides its prevalent
use in photoautotrophs, the Calvin cycle is also used by many nonphotosynthetic chemoautotrophs to fix CO2. Additionally, other
bacteria and archaea use alternative systems for CO2 fixation. Although most bacteria using Calvin cycle alternatives are
chemoautotrophic, certain green sulfur photoautotrophic bacteria have been also shown to use an alternative CO2 fixation pathway.

Exercise 7.4.3

Describe the three stages of the Calvin cycle.

Clinical Focus: Resolution

Although there is a DNA test specific for Neisseria meningitidis, it is not practical for use in some developing countries
because it requires expensive equipment and a high level of expertise to perform. The hospital in Banjul was not equipped to
perform DNA testing. Biochemical testing, however, is much less expensive and is still effective for microbial identification.
Fortunately for Hannah, her symptoms began to resolve with antibiotic therapy. Patients who survive bacterial meningitis often
suffer from long-term complications such as brain damage, hearing loss, and seizures, but after several weeks of recovery,
Hannah did not seem to be exhibiting any long-term effects and her behavior returned to normal. Because of her age, her
parents were advised to monitor her closely for any signs of developmental issues and have her regularly evaluated by her
pediatrician.
N. meningitidis is found in the normal respiratory microbiota in 10%–20% of the human population.[2] In most cases, it does
not cause disease, but for reasons not fully understood, the bacterium can sometimes invade the bloodstream and cause
infections in other areas of the body, including the brain. The disease is more common in infants and children, like Hannah.
The prevalence of meningitis caused by N. meningitidis is particularly high in the so-called meningitis belt, a region of sub-
Saharan African that includes 26 countries stretching from Senegal to Ethiopia (Figure 8.27). The reasons for this high
prevalence are not clear, but several factors may contribute to higher rates of transmission, such as the dry, dusty climate;
overcrowding and low standards of living; and the relatively low immunocompetence and nutritional status of the population.
[3]
A vaccine against four bacterial strains of N. meningitidis is available. Vaccination is recommended for 11- and 12-year-old
children, with a booster at age 16 years. Vaccination is also recommended for young people who live in close quarters with
others (e.g., college dormitories, military barracks), where the disease is more easily transmitted. Travelers visiting the

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 “meningitis belt” should also be vaccinated, especially during the dry season (December through June) when the prevalence is
highest.[4][5]

Figure 7.4.7 (a) Neisseria meningitidis is a gram-negative diplococcus, as shown in this gram-stained sample. (b) The
“meningitis belt” is the area of sub-Saharan Africa with high prevalence of meningitis caused by N. meningitidis. Control and
Prevention)

Key Concepts and Summary

Heterotrophs depend on the carbohydrates produced by autotrophs, many of which are photosynthetic, converting solar energy
into chemical energy.
Different photosynthetic organisms use different mixtures of photosynthetic pigments, which increase the range of the
wavelengths of light an organism can absorb.
Photosystems (PSI and PSII) each contain a light-harvesting complex, composed of multiple proteins and associated pigments
that absorb light energy. The light-dependent reactions of photosynthesis convert solar energy into chemical energy, producing
ATP and NADPH or NADH to temporarily store this energy.
In oxygenic photosynthesis, H2O serves as the electron donor to replace the reaction center electron, and oxygen is formed as a
byproduct. In anoxygenic photosynthesis, other reduced molecules like H2S or thiosulfate may be used as the electron donor;
as such, oxygen is not formed as a byproduct.
Noncyclic photophosphorylation is used in oxygenic photosynthesis when there is a need for both ATP and NADPH
production. If a cell’s needs for ATP outweigh its needs for NADPH, then it may carry out cyclic photophosphorylation
instead, producing only ATP.
The light-independent reactions of photosynthesis use the ATP and NADPH from the light-dependent reactions to fix CO2
into organic sugar molecules.

Footnotes
1. A. Dhingra et al. “Enhanced Translation of a Chloroplast-Expressed RbcS Gene Restores Small Subunit Levels and
Photosynthesis in Nuclear RbcS Antisense Plants.” Proceedings of the National Academy of Sciences of the United States of
America 101 no. 16 (2004):6315–6320.
2. Centers for Disease Control and Prevention. “Meningococcal Disease: Causes and Transmission.”
http://www.cdc.gov/meningococcal/abo...nsmission.html. Accessed September 12, 2016.
3. Centers for Disease Control and Prevention. “Meningococcal Disease in Other Countries.”
http://www.cdc.gov/meningococcal/global.html. Accessed September 12, 2016.
4. Centers for Disease Control and Prevention. “Health Information for Travelers to the Gambia: Traveler View.”
http://wwwnc.cdc.gov/travel/destinat...one/the-gambia. Accessed September 12, 2016.

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 5. Centers for Disease Control and Prevention. “Meningococcal: Who Needs to Be Vaccinated?”
http://www.cdc.gov/vaccines/vpd-vac/
mening/who-vaccinate.htm. Accessed September 12, 2016.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 7.4: Photosynthesis and the Importance of Light is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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Chapter 7 Exercises
Review Questions for Chapter 7
Multiple Choice
1) Which of the following is an organism that obtains its energy from the transfer of electrons originating from chemical
compounds and its carbon from an inorganic source?
A. chemoautotroph
B. chemoheterotroph
C. photoheterotroph
D. photoautotroph

2) Which of the following molecules is reduced?

A. NAD+
B. FAD
C. O2
D. NADPH

3) Enzymes work by which of the following?

A. increasing the activation energy
B. reducing the activation energy
C. making exergonic reactions endergonic
D. making endergonic reactions exergonic

4) To which of the following does a competitive inhibitor most structurally resemble?

A. the active site
B. the allosteric site
C. the substrate
D. a coenzyme

5) Which of the following are organic molecules that help enzymes work correctly?
A. cofactors
B. coenzymes
C. holoenzymes
D. apoenzymes

6) During which of the following is ATP not made by substrate-level phosphorylation?

A. Embden-Meyerhof pathway
B. Transition reaction
C. Krebs cycle
D. Entner-Doudoroff pathway

7) Which of the following products is made during Embden-Meyerhof glycolysis?

A. NAD+
B. pyruvate

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C. CO2
D. two-carbon acetyl

8) During the catabolism of glucose, which of the following is produced only in the Krebs cycle?
A. ATP
B. NADH
C. NADPH
D. FADH2

9) Which of the following is not a name for the cycle resulting in the conversion of a two-carbon acetyl to one ATP, two CO2, one
FADH2, and three NADH molecules?
A. Krebs cycle
B. tricarboxylic acid cycle
C. Calvin cycle
D. citric acid cycle

10) Which is the location of electron transports systems in prokaryotes?

A. the outer mitochondrial membrane
B. the cytoplasm
C. the inner mitochondrial membrane
D. the cytoplasmic membrane

11) Which is the source of the energy used to make ATP by oxidative phosphorylation?
A. oxygen
B. high-energy phosphate bonds
C. the proton motive force
D. Pi

12) A cell might perform anaerobic respiration for which of the following reasons?
A. It lacks glucose for degradation.
B. It lacks the transition reaction to convert pyruvate to acetyl-CoA.
C. It lacks Krebs cycle enzymes for processing acetyl-CoA to CO2.
D. It lacks a cytochrome oxidase for passing electrons to oxygen.

13) In prokaryotes, which of the following is true?

A. As electrons are transferred through an ETS, H+ is pumped out of the cell.
B. As electrons are transferred through an ETS, H+ is pumped into the cell.
C. As protons are transferred through an ETS, electrons are pumped out of the cell.
D. As protons are transferred through an ETS, electrons are pumped into the cell.

14) Which of the following is not an electron carrier within an electron transport system?
A. flavoprotein
B. ATP synthase
C. ubiquinone
D. cytochrome oxidase

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15) Which of the following is the purpose of fermentation?
A. to make ATP
B. to make carbon molecule intermediates for anabolism
C. to make NADH
D. to make NAD+

16) Which molecule typically serves as the final electron acceptor during fermentation?
A. oxygen
B. NAD+
C. pyruvate
D. CO2

17) Which fermentation product is important for making bread rise?

A. ethanol
B. CO2
C. lactic acid
D. hydrogen gas

18) Which of the following is not a commercially important fermentation product?

A. ethanol
B. pyruvate
C. butanol
D. penicillin

19) Which of the following molecules is not produced during the breakdown of phospholipids?
A. glucose
B. glycerol
C. acetyl groups
D. fatty acids

20) Caseinase is which type of enzyme?

A. phospholipase
B. lipase
C. extracellular protease
D. intracellular protease

21) Which of the following is the first step in triglyceride degradation?

A. removal of fatty acids
B. β-oxidation
C. breakage of fused rings
D. formation of smaller peptides

22) During the light-dependent reactions, which molecule loses an electron?

A. a light-harvesting pigment molecule

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B. a reaction center pigment molecule
C. NADPH
D. 3-phosphoglycerate

23) In prokaryotes, in which direction are hydrogen ions pumped by the electron transport system of photosynthetic membranes?
A. to the outside of the plasma membrane
B. to the inside (cytoplasm) of the cell
C. to the stroma
D. to the intermembrane space of the chloroplast

24) Which of the following does not occur during cyclic photophosphorylation in cyanobacteria?
A. electron transport through an ETS
B. photosystem I use
C. ATP synthesis
D. NADPH formation

25) Which of the following are two products of the light-dependent reactions?
A. glucose and NADPH
B. NADPH and ATP
C. glyceraldehyde 3-phosphate and CO2
D. glucose and oxygen

Fill-in-the-Blanks
26) Processes in which cellular energy is used to make complex molecules from simpler ones are described as ________.

27) The loss of an electron from a molecule is called ________.

28) The part of an enzyme to which a substrate binds is called the ________.

29) Per turn of the Krebs cycle, one acetyl is oxidized, forming ____ CO2, ____ ATP, ____ NADH, and ____ FADH2 molecules.

30) Most commonly, glycolysis occurs by the ________ pathway.

31) The final ETS complex used in aerobic respiration that transfers energy-depleted electrons to oxygen to form H2O is called
________.

32) The passage of hydrogen ions through ________ down their electrochemical gradient harnesses the energy needed for ATP
synthesis by oxidative phosphorylation.

33) The microbe responsible for ethanol fermentation for the purpose of producing alcoholic beverages is ________.

34) ________ results in the production of a mixture of fermentation products, including lactic acid, ethanol and/or acetic acid, and
CO2.

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35) Fermenting organisms make ATP through the process of ________.

36) The process by which two-carbon units are sequentially removed from fatty acids, producing acetyl-CoA, FADH2, and NADH
is called ________.

37) The NADH and FADH2 produced during β-oxidation are used to make ________.

38) ________ is a type of medium used to detect the production of an extracellular protease called caseinase.

39) The enzyme responsible for CO2 fixation during the Calvin cycle is called ________.

40) The types of pigment molecules found in plants, algae, and cyanobacteria are ________ and ________.

Short Answers

41) In cells, can an oxidation reaction happen in the absence of a reduction reaction? Explain.

42) What is the function of molecules like NAD+/NADH and FAD/FADH2 in cells?

43) What is substrate-level phosphorylation? When does it occur during the breakdown of glucose to CO2?

44) Why is the Krebs cycle important in both catabolism and anabolism?

45) What is the relationship between chemiosmosis and the proton motive force?

46) How does oxidative phosphorylation differ from substrate-level phosphorylation?

47) How does the location of ATP synthase differ between prokaryotes and eukaryotes? Where do protons accumulate as a result of
the ETS in each cell type?

48) Why are some microbes, including Streptococcus spp., unable to perform aerobic respiration, even in the presence of oxygen?

49) How can fermentation be used to differentiate various types of microbes?

50) How are the products of lipid and protein degradation connected to glucose metabolism pathways?

51) What is the general strategy used by microbes for the degradation of macromolecules?

52) Why would an organism perform cyclic phosphorylation instead of noncyclic phosphorylation?

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53) What is the function of photosynthetic pigments in the light-harvesting complex?

54) Why must autotrophic organisms also respire or ferment in addition to fixing CO2?

Critical Thinking

55) What would be the consequences to a cell of having a mutation that knocks out coenzyme A synthesis?

56) The bacterium E. coli is capable of performing aerobic respiration, anaerobic respiration, and fermentation. When would it
perform each process and why? How is ATP made in each case?

57) Do you think that β-oxidation can occur in an organism incapable of cellular respiration? Why or why not?

58) Is life dependent on the carbon fixation that occurs during the light-independent reactions of photosynthesis? Explain.

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 CHAPTER OVERVIEW

8: Microbial Growth
8.1: How Microbes Grow
8.2: Oxygen Requirements for Microbial Growth
8.3: The Effects of pH and Temperature on Microbial Growth
8.4: Other Environmental Conditions that Affect Growth
8.5: Microbial Relationships
Chapter 8 Exercises

Thumbnail: Heavy rains cause runoff of fertilizers into Lake Erie, triggering extensive algal blooms, which can be observed along
the shoreline. Notice the brown unplanted and green planted agricultural land on the shore. (credit: NASA)

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1
8.1: How Microbes Grow
Learning Objectives
Define the generation time for growth based on binary fission
Identify and describe the activities of microorganisms undergoing typical phases of binary fission (simple cell division) in a
growth curve
Explain several laboratory methods used to determine viable and total cell counts in populations undergoing exponential
growth
Describe examples of cell division not involving binary fission, such as budding or fragmentation
Describe the formation and characteristics of biofilms
Identify health risks associated with biofilms and how they are addressed
Describe quorum sensing and its role in cell-to-cell communication and coordination of cellular activities

Clinical Focus: part 1

Jeni, a 24-year-old pregnant woman in her second trimester, visits a clinic with complaints of high fever, 38.9 °C (102 °F),
fatigue, and muscle aches—typical flu-like signs and symptoms. Jeni exercises regularly and follows a nutritious diet with
emphasis on organic foods, including raw milk that she purchases from a local farmer’s market. All of her immunizations are
up to date. However, the health-care provider who sees Jeni is concerned and orders a blood sample to be sent for testing by the
microbiology laboratory.

Exercise 8.1.1

Why is the health-care provider concerned about Jeni’s signs and symptoms?

The bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components
into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of
horizontal gene transfer takes place. Most bacteria have a single circular chromosome; however, some exceptions exist. For
example, Borrelia burgdorferi, the causative agent of Lyme disease, has a linear chromosome.

Binary Fission
The most common mechanism of cell replication in bacteria is a process called binary fission, which is depicted in Figure 8.1.1:.
Before dividing, the cell grows and increases its number of cellular components. Next, the replication of DNA starts at a location
on the circular chromosome called the origin of replication, where the chromosome is attached to the inner cell membrane.
Replication continues in opposite directions along the chromosome until the terminus is reached.

Figure 8.1.1 : (a) The electron micrograph depicts two cells of Salmonella typhimurium after a binary fission event. (b) Binary
fission in bacteria starts with the replication of DNA as the cell elongates. A division septum forms in the center of the cell. Two
daughter cells of similar size form and separate, each receiving a copy of the original chromosome. (credit a: modification of work
by Centers for Disease Control and Prevention).
The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a complete copy of the
parental genome and a division of the cytoplasm (cytokinesis). This process of cytokinesis and cell division is directed by a protein
called FtsZ. FtsZ assembles into a Z ring on the cytoplasmic membrane (Figure 8.1.2). The Z ring is anchored by FtsZ-binding

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proteins and defines the division plane between the two daughter cells. Additional proteins required for cell division are added to
the Z ring to form a structure called the divisome. The divisome activates to produce a peptidoglycan cell wall and build a septum
that divides the two daughter cells. The daughter cells are separated by the division septum, where all of the cells’ outer layers (the
cell wall and outer membranes, if present) must be remodeled to complete division. For example, we know that specific enzymes
break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.

Figure 8.1.2 : FtsZ proteins assemble to form a Z ring that is anchored to the plasma membrane. The Z ring pinches the cell
envelope to separate the cytoplasm of the new cells.

Exercise 8.1.2

What is the name of the protein that assembles into a Z ring to initiate cytokinesis and cell division?

Generation Time
In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations.
For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which
may reproduce rapidly or remain dormant for thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also
called the doubling time and is defined as the time it takes for the population to double through one round of binary fission.
Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth
conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments. Most
pathogens grow rapidly, like E. coli, but there are exceptions. For example, Mycobacterium tuberculosis, the causative agent of
tuberculosis, has a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen’s disease
(leprosy), grows much more slowly, with a doubling time of 14 days.

Calculating Number of Cells

It is possible to predict the number of cells in a population when they divide by binary fission at a constant rate. As an
example, consider what happens if a single cell divides every 30 minutes for 24 hours. The diagram in Figure 8.1.3 shows the
increase in cell numbers for the first three generations.

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 The number of cells increases exponentially and can be expressed as 2n, where n is the number of generations. If cells divide
every 30 minutes, after 24 hours, 48 divisions would have taken place. If we apply the formula 2n, where n is equal to 48, the
single cell would give rise to 248 or 281,474,976,710,656 cells at 48 generations (24 hours). When dealing with such huge
numbers, it is more practical to use scientific notation. Therefore, we express the number of cells as 2.8 × 1014 cells.
In our example, we used one cell as the initial number of cells. For any number of starting cells, the formula is adapted as
follows:
n
Nn = N0 2 (8.1.1)

Nn is the number of cells at any generation n, N0 is the initial number of cells, and n is the number of generations.

Figure 8.1.3 : The parental cell divides and gives rise to two daughter cells. Each of the daughter cells, in turn, divides, giving a
total of four cells in the second generation and eight cells in the third generation. Each division doubles the number of cells.

Exercise 8.1.3

With a doubling time of 30 minutes and a starting population size of 1 × 105 cells, how many cells will be present after 2 hours,
assuming no cell death?

The Growth Curve

Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are added and most waste is not
removed, follow a reproducible growth pattern referred to as the growth curve. An example of a batch culture in nature is a pond in
which a small number of cells grow in a closed environment. The culture density is defined as the number of cells per unit volume.
In a closed environment, the culture density is also a measure of the number of cells in the population. Infections of the body do not
always follow the growth curve, but correlations can exist depending upon the site and type of infection. When the number of live
cells is plotted against time, distinct phases can be observed in the curve (Figure 8.1.4).

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 Figure 8.1.4 : The growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a
function of time. The graph can be divided into four phases according to the slope, each of which matches events in the cell. The
four phases are lag, log, stationary, and death.
The Lag Phase
The beginning of the growth curve represents a small number of cells, referred to as an inoculum, that are added to a fresh culture
medium, a nutritional broth that supports growth. The initial phase of the growth curve is called the lag phase, during which cells
are gearing up for the next phase of growth. The number of cells does not change during the lag phase; however, cells grow larger
and are metabolically active, synthesizing proteins needed to grow within the medium. If any cells were damaged or shocked
during the transfer to the new medium, repair takes place during the lag phase. The duration of the lag phase is determined by many
factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original
inoculum.
The Log Phase
In the logarithmic (log) growth phase, sometimes called exponential growth phase, the cells are actively dividing by binary fission
and their number increases exponentially. For any given bacterial species, the generation time under specific growth conditions
(nutrients, temperature, pH, and so forth) is genetically determined, and this generation time is called the intrinsic growth rate.
During the log phase, the relationship between time and number of cells is not linear but exponential; however, the growth curve is
often plotted on a semilogarithmic graph, as shown in Figure 8.1.5, which gives the appearance of a linear relationship.

Figure 8.1.5 : Both graphs illustrate population growth during the log phase for a bacterial sample with an initial population of one
cell and a doubling time of 1 hour. (a) When plotted on an arithmetic scale, the growth rate resembles a curve. (b) When plotted on
a semilogarithmic scale (meaning the values on the y-axis are logarithmic), the growth rate appears linear.

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Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, cells in the log phase are
preferentially used for industrial applications and research work. The log phase is also the stage where bacteria are the most
susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.
Stationary Phase
As the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate. Waste products
accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This
combination of unfavorable conditions slows and finally stalls population growth. The total number of live cells reaches a plateau
referred to as the stationary phase (Figure 8.1.4). In this phase, the number of new cells created by cell division is now equivalent
to the number of cells dying; thus, the total population of living cells is relatively stagnant. The culture density in a stationary
culture is constant. The culture’s carrying capacity, or maximum culture density, depends on the types of microorganisms in the
culture and the specific conditions of the culture; however, carrying capacity is constant for a given organism grown under the
same conditions.
During the stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the synthesis of
peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes.
In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. Secondary metabolites,
including antibiotics, are synthesized in the stationary phase. In certain pathogenic bacteria, the stationary phase is also associated
with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a
host organism. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down
human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.
The Death Phase
As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers. Soon, the
number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells (Figure
8.1.4). This is the aptly named death phase, sometimes called the decline phase. Many cells lyse and release nutrients into the

medium, allowing surviving cells to maintain viability and form endospores. A few cells, the so-called persisters, are characterized
by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as
tuberculosis, that do not respond to antibiotic treatment.

Sustaining Microbial Growth

The growth pattern shown in Figure 8.1.4 takes place in a closed environment; nutrients are not added and waste and dead cells are
not removed. In many cases, though, it is advantageous to maintain cells in the logarithmic phase of growth. One example is in
industries that harvest microbial products. A chemostat (Figure 8.1.6) is used to maintain a continuous culture in which nutrients
are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the
same rate as nutrients flow in to maintain an optimal growth environment.

Figure 8.1.6 : A chemostat is a culture vessel fitted with an opening to add nutrients (feed) and an outlet to remove contents
(effluent), effectively diluting toxic wastes and dead cells. The addition and removal of fluids is adjusted to maintain the culture in
the logarithmic phase of growth. If aerobic bacteria are grown, suitable oxygen levels are maintained.

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 Exercise 8.1.4

1. During which phase does growth occur at the fastest rate?

2. Name two factors that limit microbial growth.

Measurement of Bacterial Growth

Estimating the number of bacterial cells in a sample, known as a bacterial count, is a common task performed by microbiologists.
The number of bacteria in a clinical sample serves as an indication of the extent of an infection. Quality control of drinking water,
food, medication, and even cosmetics relies on estimates of bacterial counts to detect contamination and prevent the spread of
disease. Two major approaches are used to measure cell number. The direct methods involve counting cells, whereas the indirect
methods depend on the measurement of cell presence or activity without actually counting individual cells. Both direct and indirect
methods have advantages and disadvantages for specific applications.

Direct Cell Count

Direct cell count refers to counting the cells in a liquid culture or colonies on a plate. It is a direct way of estimating how many
organisms are present in a sample. Let’s look first at a simple and fast method that requires only a specialized slide and a compound
microscope.
The simplest way to count bacteria is called the direct microscopic cell count, which involves transferring a known volume of a
culture to a calibrated slide and counting the cells under a light microscope. The calibrated slide is called a Petroff-Hausser
chamber (Figure 8.1.7) and is similar to a hemocytometer used to count red blood cells. The central area of the counting chamber is
etched into squares of various sizes. A sample of the culture suspension is added to the chamber under a coverslip that is placed at a
specific height from the surface of the grid. It is possible to estimate the concentration of cells in the original sample by counting
individual cells in a number of squares and determining the volume of the sample observed. The area of the squares and the height
at which the coverslip is positioned are specified for the chamber. The concentration must be corrected for dilution if the sample
was diluted before enumeration.

Figure 8.1.7 : (a) A Petroff-Hausser chamber is a special slide designed for counting the bacterial cells in a measured volume of a
sample. A grid is etched on the slide to facilitate precision in counting. (b) This diagram illustrates the grid of a Petroff-Hausser
chamber, which is made up of squares of known areas. The enlarged view shows the square within which bacteria (red cells) are
counted. If the coverslip is 0.2 mm above the grid and the square has an area of 0.04 mm2, then the volume is 0.008 mm3, or
0.000008 mL. Since there are 10 cells inside the square, the density of bacteria is 10 cells/0.000008 mL, which equates to
1,250,000 cells/mL. (credit a: modification of work by Jeffrey M. Vinocur)
Cells in several small squares must be counted and the average taken to obtain a reliable measurement. The advantages of the
chamber are that the method is easy to use, relatively fast, and inexpensive. On the downside, the counting chamber does not work
well with dilute cultures because there may not be enough cells to count.
Using a counting chamber does not necessarily yield an accurate count of the number of live cells because it is not always possible
to distinguish between live cells, dead cells, and debris of the same size under the microscope. However, newly developed
fluorescence staining techniques make it possible to distinguish viable and dead bacteria. These viability stains (or live stains) bind
to nucleic acids, but the primary and secondary stains differ in their ability to cross the cytoplasmic membrane. The primary stain,
which fluoresces green, can penetrate intact cytoplasmic membranes, staining both live and dead cells. The secondary stain, which
fluoresces red, can stain a cell only if the cytoplasmic membrane is considerably damaged. Thus, live cells fluoresce green because

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they only absorb the green stain, whereas dead cells appear red because the red stain displaces the green stain on their nucleic acids
(Figure 8.1.8).

Figure 8.1.8 : Fluorescence staining can be used to differentiate between viable and dead bacterial cells in a sample for purposes of
counting. Viable cells are stained green, whereas dead cells are stained red. (credit: modification of work by Panseri S, Cunha C,
D’Alessandro T, Sandri M, Giavaresi G, Maracci M, Hung CT, Tampieri A)
Another technique uses an electronic cell counting device (Coulter counter) to detect and count the changes in electrical resistance
in a saline solution. A glass tube with a small opening is immersed in an electrolyte solution. A first electrode is suspended in the
glass tube. A second electrode is located outside of the tube. As cells are drawn through the small aperture in the glass tube, they
briefly change the resistance measured between the two electrodes and the change is recorded by an electronic sensor (Figure
8.1.9); each resistance change represents a cell. The method is rapid and accurate within a range of concentrations; however, if the

culture is too concentrated, more than one cell may pass through the aperture at any given time and skew the results. This method
also does not differentiate between live and dead cells.
Direct counts provide an estimate of the total number of cells in a sample. However, in many situations, it is important to know the
number of live, or viable, cells. Counts of live cells are needed when assessing the extent of an infection, the effectiveness of
antimicrobial compounds and medication, or contamination of food and water.

Figure 8.1.9 : A Coulter counter is an electronic device that counts cells. It measures the change in resistance in an electrolyte
solution that takes place when a cell passes through a small opening in the inside container wall. A detector automatically counts
the number of cells passing through the opening. (credit b: modification of work by National Institutes of Health)

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 Exercise 8.1.5

1. Why would you count the number of cells in more than one square in the Petroff-Hausser chamber to estimate cell
numbers?
2. In the viability staining method, why do dead cells appear red?

Plate Count
The viable plate count, or simply plate count, is a count of viable or live cells. It is based on the principle that viable cells replicate
and give rise to visible colonies when incubated under suitable conditions for the specimen. The results are usually expressed as
colony-forming units per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have landed on the
same spot to give rise to a single colony. Furthermore, samples of bacteria that grow in clusters or chains are difficult to disperse
and a single colony may represent several cells. Some cells are described as viable but nonculturable and will not form colonies on
solid media. For all these reasons, the viable plate count is considered a low estimate of the actual number of live cells. These
limitations do not detract from the usefulness of the method, which provides estimates of live bacterial numbers.
Microbiologists typically count plates with 30–300 colonies. Samples with too few colonies (<30) do not give statistically reliable
numbers, and overcrowded plates (>300 colonies) make it difficult to accurately count individual colonies. Also, counts in this
range minimize occurrences of more than one bacterial cell forming a single colony. Thus, the calculated CFU is closer to the true
number of live bacteria in the population.
There are two common approaches to inoculating plates for viable counts: the pour plate and the spread plate methods. Although
the final inoculation procedure differs between these two methods, they both start with a serial dilution of the culture.

Serial Dilution
The serial dilution of a culture is an important first step before proceeding to either the pour plate or spread plate method. The goal
of the serial dilution process is to obtain plates with CFUs in the range of 30–300, and the process usually involves several
dilutions in multiples of 10 to simplify calculation. The number of serial dilutions is chosen according to a preliminary estimate of
the culture density. Figure 8.1.10 illustrates the serial dilution method.

Figure 8.1.10 : Serial dilution involves diluting a fixed volume of cells mixed with dilution solution using the previous dilution as
an inoculum. The result is dilution of the original culture by an exponentially growing factor. (credit: modification of work by
“Leberechtc”/Wikimedia Commons)
A fixed volume of the original culture, 1.0 mL, is added to and thoroughly mixed with the first dilution tube solution, which
contains 9.0 mL of sterile broth. This step represents a dilution factor of 10, or 1:10, compared with the original culture. From this

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first dilution, the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution solution. The dilution
factor is now 1:100 compared with the original culture. This process continues until a series of dilutions is produced that will
bracket the desired cell concentration for accurate counting. From each tube, a sample is plated on solid medium using either the
pour plate method (Figure 8.1.11) or the spread plate method (Figure 8.1.12). The plates are incubated until colonies appear. Two
to three plates are usually prepared from each dilution and the numbers of colonies counted on each plate are averaged. In all cases,
thorough mixing of samples with the dilution medium (to ensure the cell distribution in the tube is random) is paramount to
obtaining reliable results.

Figure 8.1.11 : In the pour plate method of cell counting, the sample is mixed in liquid warm agar (45–50 °C) poured into a sterile
Petri dish and further mixed by swirling. This process is repeated for each serial dilution prepared. The resulting colonies are
counted and provide an estimate of the number of cells in the original volume sampled.
The dilution factor is used to calculate the number of cells in the original cell culture. In our example, an average of 50 colonies
was counted on the plates obtained from the 1:10,000 dilution. Because only 0.1 mL of suspension was pipetted on the plate, the
multiplier required to reconstitute the original concentration is 10 × 10,000. The number of CFU per mL is equal to 50 × 100 ×
10,000 = 5,000,000. The number of bacteria in the culture is estimated as 5 million cells/mL. The colony count obtained from the
1:1000 dilution was 389, well below the expected 500 for a 10-fold difference in dilutions. This highlights the issue of inaccuracy
when colony counts are greater than 300 and more than one bacterial cell grows into a single colony.

Figure 8.1.12 : In the spread plate method of cell counting, the sample is poured onto solid agar and then spread using a sterile
spreader. This process is repeated for each serial dilution prepared. The resulting colonies are counted and provide an estimate of
the number of cells in the original volume samples.
A very dilute sample—drinking water, for example—may not contain enough organisms to use either of the plate count methods
described. In such cases, the original sample must be concentrated rather than diluted before plating. This can be accomplished
using a modification of the plate count technique called the membrane filtration technique. Known volumes are vacuum-filtered

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aseptically through a membrane with a pore size small enough to trap microorganisms. The membrane is transferred to a Petri plate
containing an appropriate growth medium. Colonies are counted after incubation. Calculation of the cell density is made by
dividing the cell count by the volume of filtered liquid.
Watch this video for demonstrations of serial dilutions and spread plate techniques.

The Most Probable Number

The number of microorganisms in dilute samples is usually too low to be detected by the plate count methods described thus far.
For these specimens, microbiologists routinely use the most probable number (MPN) method, a statistical procedure for estimating
of the number of viable microorganisms in a sample. Often used for water and food samples, the MPN method evaluates detectable
growth by observing changes in turbidity or color due to metabolic activity.
A typical application of MPN method is the estimation of the number of coliforms in a sample of pond water. Coliforms are gram-
negative rod bacteria that ferment lactose. The presence of coliforms in water is considered a sign of contamination by fecal matter.
For the method illustrated in Figure 8.1.13, a series of three dilutions of the water sample is tested by inoculating five lactose broth
tubes with 10 mL of sample, five lactose broth tubes with 1 mL of sample, and five lactose broth tubes with 0.1 mL of sample. The
lactose broth tubes contain a pH indicator that changes color from red to yellow when the lactose is fermented. After inoculation
and incubation, the tubes are examined for an indication of coliform growth by a color change in media from red to yellow. The
first set of tubes (10-mL sample) showed growth in all the tubes; the second set of tubes (1 mL) showed growth in two tubes out of
five; in the third set of tubes, no growth is observed in any of the tubes (0.1-mL dilution). The numbers 5, 2, and 0 are compared
with an estimation table, which has been constructed using a probability model of the sampling procedure. From our reading of the
table, we conclude that 49 is the most probable number of bacteria per 100 mL of pond water.

Figure 8.1.13: In the most probable number method, sets of five lactose broth tubes are inoculated with three different volumes of
pond water: 10 mL, 1 mL, and 0.1mL. Bacterial growth is assessed through a change in the color of the broth from red to yellow as
lactose is fermented.

Exercise 8.1.6
1. What is a colony-forming unit?
2. What two methods are frequently used to estimate bacterial numbers in water samples?

Indirect Cell Counts

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Besides direct methods of counting cells, other methods, based on an indirect detection of cell density, are commonly used to
estimate and compare cell densities in a culture. The foremost approach is to measure the turbidity (cloudiness) of a sample of
bacteria in a liquid suspension. The laboratory instrument used to measure turbidity is called a spectrophotometer (Figure 8.1.14).
In a spectrophotometer, a light beam is transmitted through a bacterial suspension, the light passing through the suspension is
measured by a detector, and the amount of light passing through the sample and reaching the detector is converted to either percent
transmission or a logarithmic value called absorbance (optical density). As the numbers of bacteria in a suspension increase, the
turbidity also increases and causes less light to reach the detector. The decrease in light passing through the sample and reaching
the detector is associated with a decrease in percent transmission and increase in absorbance measured by the spectrophotometer.
Measuring turbidity is a fast method to estimate cell density as long as there are enough cells in a sample to produce turbidity. It is
possible to correlate turbidity readings to the actual number of cells by performing a viable plate count of samples taken from
cultures having a range of absorbance values. Using these values, a calibration curve is generated by plotting turbidity as a function
of cell density. Once the calibration curve has been produced, it can be used to estimate cell counts for all samples obtained or
cultured under similar conditions and with densities within the range of values used to construct the curve.

Figure 8.1.14 : (a) A spectrophotometer is commonly used to measure the turbidity of a bacterial cell suspension as an indirect
measure of cell density. (b) A spectrophotometer works by splitting white light from a source into a spectrum. The
spectrophotometer allows choice of the wavelength of light to use for the measurement. The optical density (turbidity) of the
sample will depend on the wavelength, so once one wavelength is chosen, it must be used consistently. The filtered light passes
through the sample (or a control with only medium) and the light intensity is measured by a detector. The light passing into a
suspension of bacteria is scattered by the cells in such a way that some fraction of it never reaches the detector. This scattering
happens to a far lesser degree in the control tube with only the medium. (credit a: modification of work by Hwang HS, Kim MS;
credit b “test tube photos”: modification of work by Suzanne Wakim)
Measuring dry weight of a culture sample is another indirect method of evaluating culture density without directly measuring cell
counts. The cell suspension used for weighing must be concentrated by filtration or centrifugation, washed, and then dried before
the measurements are taken. The degree of drying must be standardized to account for residual water content. This method is
especially useful for filamentous microorganisms, which are difficult to enumerate by direct or viable plate count.
As we have seen, methods to estimate viable cell numbers can be labor intensive and take time because cells must be grown.
Recently, indirect ways of measuring live cells have been developed that are both fast and easy to implement. These methods
measure cell activity by following the production of metabolic products or disappearance of reactants. Adenosine triphosphate
(ATP) formation, biosynthesis of proteins and nucleic acids, and consumption of oxygen can all be monitored to estimate the
number of cells.

Exercise 8.1.7
1. What is the purpose of a calibration curve when estimating cell count from turbidity measurements?
2. What are the newer indirect methods of counting live cells?

Alternative Patterns of Cell Division

Binary fission is the most common pattern of cell division in prokaryotes, but it is not the only one. Other mechanisms usually
involve asymmetrical division (as in budding) or production of spores in aerial filaments.

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In some cyanobacteria, many nucleoids may accumulate in an enlarged round cell or along a filament, leading to the generation of
many new cells at once. The new cells often split from the parent filament and float away in a process called fragmentation (Figure
8.1.15). Fragmentation is commonly observed in the Actinomycetes, a group of gram-positive, anaerobic bacteria commonly found

in soil. Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by the giant
bacterium Epulopiscium. Several daughter cells grow fully in the parent cell, which eventually disintegrates, releasing the new cells
to the environment. Other species may form a long narrow extension at one pole in a process called budding. The tip of the
extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell. Budding is most common in yeast
(Figure 8.1.15), but it is also observed in prosthecate bacteria and some cyanobacteria.

Figure 8.1.15 : (a) Filamentous cyanobacteria, like those pictured here, replicate by fragmentation. (b) In this electron micrograph,
cells of the bacterium Gemmata obscuriglobus are budding. The larger cell is the mother cell. Labels indicate the nucleoids (N) and
the still-forming nuclear envelope (NE) of the daughter cell. (credit a: modification of work by CSIRO; credit b: modification of
work by Kuo-Chang Lee, Rick I Webb and John A Fuerst)
The soil bacteria Actinomyces grow in long filaments divided by septa, similar to the mycelia seen in fungi, resulting in long cells
with multiple nucleoids. Environmental signals, probably related to low nutrient availability, lead to the formation of aerial
filaments. Within these aerial filaments, elongated cells divide simultaneously. The new cells, which contain a single nucleoid,
develop into spores that give rise to new colonies.

Exercise 8.1.8

Identify at least one difference between fragmentation and budding.

Biofilms
In nature, microorganisms grow mainly in biofilms, complex and dynamic ecosystems that form on a variety of environmental
surfaces, from industrial conduits and water treatment pipelines to rocks in river beds. Biofilms are not restricted to solid surface
substrates, however. Almost any surface in a liquid environment containing some minimal nutrients will eventually develop a
biofilm. Microbial mats that float on water, for example, are biofilms that contain large populations of photosynthetic
microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species. Regardless of the environment
where they occur, biofilms are not random collections of microorganisms; rather, they are highly structured communities that
provide a selective advantage to their constituent microorganisms.

Biofilm Structure
Observations using confocal microscopy have shown that environmental conditions influence the overall structure of biofilms.
Filamentous biofilms called streamers form in rapidly flowing water, such as freshwater streams, eddies, and specially designed
laboratory flow cells that replicate growth conditions in fast-moving fluids. The streamers are anchored to the substrate by a “head”
and the “tail” floats downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-like shape. The
structure of biofilms may also change with other environmental conditions such as nutrient availability.
Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal clusters of microorganisms
embedded in a matrix interspersed with open water channels. The extracellular matrix consists of extracellular polymeric
substances (EPS) secreted by the organisms in the biofilm. The extracellular matrix represents a large fraction of the biofilm,
accounting for 50%–90% of the total dry mass. The properties of the EPS vary according to the resident organisms and
environmental conditions.

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EPS is a hydrated gel composed primarily of polysaccharides and containing other macromolecules such as proteins, nucleic acids,
and lipids. It plays a key role in maintaining the integrity and function of the biofilm. Channels in the EPS allow movement of
nutrients, waste, and gases throughout the biofilm. This keeps the cells hydrated, preventing desiccation. EPS also shelters
organisms in the biofilm from predation by other microbes or cells (e.g., protozoans, white blood cells in the human body).

Biofilm Formation
Free-floating microbial cells that live in an aquatic environment are called planktonic cells. The formation of a biofilm essentially
involves the attachment of planktonic cells to a substrate, where they become sessile (attached to a surface). This occurs in stages,
as depicted in Figure 8.1.16. The first stage involves the attachment of planktonic cells to a surface coated with a conditioning film
of organic material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes that facilitate the
formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures, including
an extensive matrix and water channels. Appendages such as fimbriae, pili, and flagella interact with the EPS, and microscopy and
genetic analysis suggest that such structures are required for the establishment of a mature biofilm. In the last stage of the biofilm
life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new
sites. This stage is referred to as dispersal.

Figure 8.1.16 : Stages in the formation and life cycle of a biofilm. (credit: modification of work by Public Library of Science and
American Society for Microbiology)
Within a biofilm, different species of microorganisms establish metabolic collaborations in which the waste product of one
organism becomes the nutrient for another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that
promote the growth of anaerobes. This occurs in many polymicrobial infections that involve both aerobic and anaerobic pathogens.
The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called quorum
sensing. Quorum sensing—which can occur between cells of different species within a biofilm—enables microorganisms to detect
their cell density through the release and binding of small, diffusible molecules called autoinducers. When the cell population
reaches a critical threshold (a quorum), these autoinducers initiate a cascade of reactions that activate genes associated with cellular
functions that are beneficial only when the population reaches a critical density. For example, in some pathogens, synthesis of
virulence factors only begins when enough cells are present to overwhelm the immune defenses of the host. Although mostly
studied in bacterial populations, quorum sensing takes place between bacteria and eukaryotes and between eukaryotic cells such as
the fungus Candida albicans, a common member of the human microbiota that can cause infections in immunocompromised
individuals.
The signaling molecules in quorum sensing belong to two major classes. Gram-negative bacteria communicate mainly using N-
acylated homoserine lactones, whereas gram-positive bacteria mostly use small peptides (Figure 8.1.17). In all cases, the first step

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in quorum sensing consists of the binding of the autoinducer to its specific receptor only when a threshold concentration of
signaling molecules is reached. Once binding to the receptor takes place, a cascade of signaling events leads to changes in gene
expression. The result is the activation of biological responses linked to quorum sensing, notably an increase in the production of
signaling molecules themselves, hence the term autoinducer.

Figure 8.1.17 : Short peptides in gram-positive bacteria and N-acetylated homoserine lactones in gram-negative bacteria act as
autoinducers in quorum sensing and mediate the coordinated response of bacterial cells. The R side chain of the N-acetylated
homoserine lactone is specific for the species of gram-negative bacteria. Some secreted homoserine lactones are recognized by
more than one species.

Biofilms and Human Health

The human body harbors many types of biofilms, some beneficial and some harmful. For example, the layers of normal microbiota
lining the intestinal and respiratory mucosa play a role in warding off infections by pathogens. However, other biofilms in the body
can have a detrimental effect on health. For example, the plaque that forms on teeth is a biofilm that can contribute to dental and
periodontal disease. Biofilms can also form in wounds, sometimes causing serious infections that can spread. The bacterium
Pseudomonas aeruginosa often colonizes biofilms in the airways of patients with cystic fibrosis, causing chronic and sometimes
fatal infections of the lungs. Biofilms can also form on medical devices used in or on the body, causing infections in patients with
in-dwelling catheters, artificial joints, or contact lenses.
Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-floating counterparts. Several
hypotheses have been proposed to explain why. Cells in the deep layers of a biofilm are metabolically inactive and may be less
susceptible to the action of antibiotics that disrupt metabolic activities. The EPS may also slow the diffusion of antibiotics and
antiseptics, preventing them from reaching cells in the deeper layers of the biofilm. Phenotypic changes may also contribute to the
increased resistance exhibited by bacterial cells in biofilms. For example, the increased production of efflux pumps, membrane-
embedded proteins that actively extrude antibiotics out of bacterial cells, have been shown to be an important mechanism of
antibiotic resistance among biofilm-associated bacteria. Finally, biofilms provide an ideal environment for the exchange of
extrachromosomal DNA, which often includes genes that confer antibiotic resistance.

Exercise 8.1.9
1. What is the matrix of a biofilm composed of?
2. What is the role of quorum sensing in a biofilm?

Key Concepts and Summary

Most bacterial cells divide by binary fission. Generation time in bacterial growth is defined as the doubling timeof the
population.
Cells in a closed system follow a pattern of growth with four phases: lag, logarithmic (exponential), stationary, and death.
Cells can be counted by direct viable cell count. The pour plate and spread plate methods are used to plate serial dilutions
into or onto, respectively, agar to allow counting of viable cells that give rise to colony-forming units. Membrane filtration is
used to count live cells in dilute solutions. The most probable cell number (MPN)method allows estimation of cell numbers in
cultures without using solid media.
Indirect methods can be used to estimate culture density by measuring turbidity of a culture or live cell density by measuring
metabolic activity.

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 Other patterns of cell division include multiple nucleoid formation in cells; asymmetric division, as in budding; and the
formation of hyphae and terminal spores.
Biofilms are communities of microorganisms enmeshed in a matrix of extracellular polymeric substance. The formation of a
biofilm occurs when planktonic cells attach to a substrate and become sessile. Cells in biofilms coordinate their activity by
communicating through quorum sensing.
Biofilms are commonly found on surfaces in nature and in the human body, where they may be beneficial or cause severe
infections. Pathogens associated with biofilms are often more resistant to antibiotics and disinfectants.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 8.1: How Microbes Grow is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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8.2: Oxygen Requirements for Microbial Growth
Learning Objectives
Interpret visual data demonstrating minimum, optimum, and maximum oxygen or carbon dioxide requirements for growth
Identify and describe different categories of microbes with requirements for growth with or without oxygen: obligate
aerobe, obligate anaerobe, facultative anaerobe, aerotolerant anaerobe, microaerophile, and capnophile
Give examples of microorganisms for each category of growth requirements

Ask most people “What are the major requirements for life?” and the answers are likely to include water and oxygen. Few would
argue about the need for water, but what about oxygen? Can there be life without oxygen?
The answer is that molecular oxygen (O2) is not always needed. The earliest signs of life are dated to a period when conditions on
earth were highly reducing and free oxygen gas was essentially nonexistent. Only after cyanobacteria started releasing oxygen as a
byproduct of photosynthesis and the capacity of iron in the oceans for taking up oxygen was exhausted did oxygen levels increase
in the atmosphere. This event, often referred to as the Great Oxygenation Event or the Oxygen Revolution, caused a massive
extinction. Most organisms could not survive the powerful oxidative properties of reactive oxygen species (ROS), highly unstable
ions and molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure with which
they come in contact. Singlet oxygen (O2•), superoxide (O2−), peroxides (H2O2), hydroxyl radical (OH•), and hypochlorite ion
(OCl−), the active ingredient of household bleach, are all examples of ROS. The organisms that were able to detoxify reactive
oxygen species harnessed the high electronegativity of oxygen to produce free energy for their metabolism and thrived in the new
environment.

Oxygen Requirements of Microorganisms

Many ecosystems are still free of molecular oxygen. Some are found in extreme locations, such as deep in the ocean or in earth’s
crust; others are part of our everyday landscape, such as marshes, bogs, and sewers. Within the bodies of humans and other
animals, regions with little or no oxygen provide an anaerobic environment for microorganisms. (Figure 8.2.1).

Figure 8.2.1 : Anaerobic environments are still common on earth. They include environments like (a) a bog where undisturbed
dense sediments are virtually devoid of oxygen, and (b) the rumen (the first compartment of a cow’s stomach), which provides an
oxygen-free incubator for methanogens and other obligate anaerobic bacteria. (credit a: modification of work by National Park
Service; credit b: modification of work by US Department of Agriculture)
We can easily observe different requirements for molecular oxygen by growing bacteria in thioglycolate tube cultures. A test-tube
culture starts with autoclaved thioglycolate medium containing a low percentage of agar to allow motile bacteria to move
throughout the medium. Thioglycolate has strong reducing properties and autoclaving flushes out most of the oxygen. The tubes
are inoculated with the bacterial cultures to be tested and incubated at an appropriate temperature. Over time, oxygen slowly
diffuses throughout the thioglycolate tube culture from the top. Bacterial density increases in the area where oxygen concentration
is best suited for the growth of that particular organism.
The growth of bacteria with varying oxygen requirements in thioglycolate tubes is illustrated in Figure 8.2.2. In tube A, all the
growth is seen at the top of the tube. The bacteria are obligate (strict) aerobes that cannot grow without an abundant supply of
oxygen. Tube B looks like the opposite of tube A. Bacteria grow at the bottom of tube B. Those are obligate anaerobes, which are
killed by oxygen. Tube C shows heavy growth at the top of the tube and growth throughout the tube, a typical result with

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facultative anaerobes. Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence by
relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able
to perform anaerobic respiration. The aerotolerant anaerobes in tube D are indifferent to the presence of oxygen. They do not use
oxygen because they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen as obligate
anaerobes are. Tube E on the right shows a “Goldilocks” culture. The oxygen level has to be just right for growth, not too much and
not too little. These microaerophiles are bacteria that require a minimum level of oxygen for growth, about 1%–10%, well below
the 21% found in the atmosphere.

Figure 8.2.2 : Diagram of bacterial cell distribution in thioglycolate tubes.

Examples of obligate aerobes are Mycobacterium tuberculosis, the causative agent of tuberculosis and Micrococcus luteus, a gram-
positive bacterium that colonizes the skin. Neisseria meningitidis, the causative agent of severe bacterial meningitis, and N.
gonorrheae, the causative agent of sexually transmitted gonorrhea, are also obligate aerobes.
Many obligate anaerobes are found in the environment where anaerobic conditions exist, such as in deep sediments of soil, still
waters, and at the bottom of the deep ocean where there is no photosynthetic life. Anaerobic conditions also exist naturally in the
intestinal tract of animals. Obligate anaerobes, mainly Bacteroidetes, represent a large fraction of the microbes in the human gut.
Transient anaerobic conditions exist when tissues are not supplied with blood circulation; they die and become an ideal breeding
ground for obligate anaerobes. Another type of obligate anaerobe encountered in the human body is the gram-positive, rod-shaped
Clostridium spp. Their ability to form endospores allows them to survive in the presence of oxygen. One of the major causes of
health-acquired infections is C. difficile, known as C. diff. Prolonged use of antibiotics for other infections increases the probability
of a patient developing a secondary C. difficile infection. Antibiotic treatment disrupts the balance of microorganisms in the
intestine and allows the colonization of the gut by C. difficile, causing a significant inflammation of the colon.
Other clostridia responsible for serious infections include C. tetani, the agent of tetanus, and C. perfringens, which causes gas
gangrene. In both cases, the infection starts in necrotic tissue (dead tissue that is not supplied with oxygen by blood circulation).
This is the reason that deep puncture wounds are associated with tetanus. When tissue death is accompanied by lack of circulation,
gangrene is always a danger.
The study of obligate anaerobes requires special equipment. Obligate anaerobic bacteria must be grown under conditions devoid of
oxygen. The most common approach is culture in an anaerobic jar (Figure 8.2.3). Anaerobic jars include chemical packs that
remove oxygen and release carbon dioxide (CO2). An anaerobic chamber is an enclosed box from which all oxygen is removed.
Gloves sealed to openings in the box allow handling of the cultures without exposing the culture to air (Figure 8.2.3).

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 Figure 8.2.3 : (a) An anaerobic jar is pictured that is holding nine Petri plates supporting cultures. (b) Openings in the side of an
anaerobic box are sealed by glove-like sleeves that allow for the handling of cultures inside the box. (credit a: modification of work
by Centers for Disease Control and Prevention; credit b: modification of work by NIST)
Staphylococci and Enterobacteriaceae are examples of facultative anaerobes. Staphylococci are found on the skin and upper
respiratory tract. Enterobacteriaceae are found primarily in the gut and upper respiratory tract but can sometimes spread to the
urinary tract, where they are capable of causing infections. It is not unusual to see mixed bacterial infections in which the
facultative anaerobes use up the oxygen, creating an environment for the obligate anaerobes to flourish.
Examples of aerotolerant anaerobes include lactobacilli and streptococci, both found in the oral microbiota. Campylobacter jejuni,
which causes gastrointestinal infections, is an example of a microaerophile and is grown under low-oxygen conditions.
The optimum oxygen concentration, as the name implies, is the ideal concentration of oxygen for a particular microorganism. The
lowest concentration of oxygen that allows growth is called the minimum permissive oxygen concentration. The highest tolerated
concentration of oxygen is the maximum permissive oxygen concentration. The organism will not grow outside the range of
oxygen levels found between the minimum and maximum permissive oxygen concentrations.

Exercise 8.2.1

1. Would you expect the oldest bacterial lineages to be aerobic or anaerobic?

2. Which bacteria grow at the top of a thioglycolate tube, and which grow at the bottom of the tube?

An Unwelcome Anaerobe
Charles is a retired bus driver who developed type 2 diabetes over 10 years ago. Since his retirement, his lifestyle has become
very sedentary and he has put on a substantial amount of weight. Although he has felt tingling and numbness in his left foot for
a while, he has not been worried because he thought his foot was simply “falling asleep.” Recently, a scratch on his foot does
not seem to be healing and is becoming increasingly ugly. Because the sore did not bother him much, Charles figured it could
not be serious until his daughter noticed a purplish discoloration spreading on the skin and oozing (Figure). When he was
finally seen by his physician, Charles was rushed to the operating room. His open sore, or ulcer, is the result of a diabetic foot.
The concern here is that gas gangrene may have taken hold in the dead tissue. The most likely agent of gas gangrene is
Clostridium perfringens, an endospore-forming, gram-positive bacterium. It is an obligate anaerobe that grows in tissue devoid
of oxygen. Since dead tissue is no longer supplied with oxygen by the circulatory system, the dead tissue provides pockets of
ideal environment for the growth of C. perfringens.
A surgeon examines the ulcer and radiographs of Charles’s foot and determines that the bone is not yet infected. The wound
will have to be surgically debrided (debridement refers to the removal of dead and infected tissue) and a sample sent for
microbiological lab analysis, but Charles will not have to have his foot amputated. Many diabetic patients are not so lucky. In
2008, nearly 70,000 diabetic patients in the United States lost a foot or limb to amputation, according to statistics from the
Centers for Disease Control and Prevention.

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 Figure 8.2.4 : This clinical photo depicts ulcers on the foot of a diabetic patient. Dead tissue accumulating in ulcers can provide
an ideal growth environment for the anaerobe C. perfringens, a causative agent of gas gangrene. (credit: Shigeo Kono, Reiko
Nakagawachi, Jun Arata, Benjamin A Lipsky)

Exercise 8.2.2

Which growth conditions would you recommend for the detection of C. perfringens?

Detoxification of Reactive Oxygen Species

Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be detoxified. Even organisms that
do not use aerobic respiration need some way to break down some of the ROS that may form from atmospheric oxygen. Three
main enzymes break down those toxic byproducts: superoxide dismutase, peroxidase, and catalase. Each one catalyzes a different
reaction. Reactions of type seen in Reaction 1 are catalyzed by peroxidases.
+
X − (2 H ) + H2 O2 → oxidized − X + 2 H2 O (8.2.1)

In these reactions, an electron donor (reduced compound; e.g., reduced nicotinamide adenine dinucleotide [NADH]) oxidizes
hydrogen peroxide, or other peroxides, to water. The enzymes play an important role by limiting the damage caused by
peroxidation of membrane lipids. Reaction 2 is mediated by the enzyme superoxide dismutase (SOD) and breaks down the
powerful superoxide anions generated by aerobic metabolism:
2− +
2O + 2H → H2 O2 + O2 (8.2.2)

The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.
2 H2 O2 → 2 H2 O + O2 (8.2.3)

Obligate anaerobes usually lack all three enzymes. Aerotolerant anaerobes do have SOD but no catalase. Reaction 3, shown
occurring in Figure 8.2.5, is the basis of a useful and rapid test to distinguish streptococci, which are aerotolerant and do not
possess catalase, from staphylococci, which are facultative anaerobes. A sample of culture rapidly mixed in a drop of 3% hydrogen
peroxide will release bubbles if the culture is catalase positive.

Figure 8.2.5 : The catalase test detects the presence of the enzyme catalase by noting whether bubbles are released when hydrogen
peroxide is added to a culture sample. Compare the positive result (right) with the negative result (left). (credit: Centers for Disease
Control and Prevention)
Bacteria that grow best in a higher concentration of CO2 and a lower concentration of oxygen than present in the atmosphere are
called capnophiles. One common approach to grow capnophiles is to use a candle jar. A candle jar consists of a jar with a tight-

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fitting lid that can accommodate the cultures and a candle. After the cultures are added to the jar, the candle is lit and the lid closed.
As the candle burns, it consumes most of the oxygen present and releases CO2.

Exercise 8.2.3

1. What substance is added to a sample to detect catalase?

2. What is the function of the candle in a candle jar?

Clinical Focus: part 2

The health-care provider who saw Jeni was concerned primarily because of her pregnancy. Her condition enhances the risk for
infections and makes her more vulnerable to those infections. The immune system is downregulated during pregnancy, and
pathogens that cross the placenta can be very dangerous for the fetus. A note on the provider’s order to the microbiology lab
mentions a suspicion of infection by Listeria monocytogenes, based on the signs and symptoms exhibited by the patient.
Jeni’s blood samples are streaked directly on sheep blood agar, a medium containing tryptic soy agar enriched with 5% sheep
blood. (Blood is considered sterile; therefore, competing microorganisms are not expected in the medium.) The inoculated
plates are incubated at 37 °C for 24 to 48 hours. Small grayish colonies surrounded by a clear zone emerge. Such colonies are
typical of Listeria and other pathogens such as streptococci; the clear zone surrounding the colonies indicates complete lysis of
blood in the medium, referred to as beta-hemolysis (Figure 8.2.6). When tested for the presence of catalase, the colonies give a
positive response, eliminating Streptococcus as a possible cause. Furthermore, a Gram stain shows short gram-positive bacilli.
Cells from a broth culture grown at room temperature displayed the tumbling motility characteristic of Listeria (Figure 8.2.6).
All of these clues lead the lab to positively confirm the presence of Listeria in Jeni’s blood samples.

Figure 8.2.6 : (a) A sample blood agar test showing beta-hemolysis. (b) A sample motility test showing both positive and
negative results. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work
by “VeeDunn”/Flickr)

Exercise 8.2.4

How serious is Jeni’s condition and what is the appropriate treatment?

Key Concepts and Summary

Aerobic and anaerobic environments can be found in diverse niches throughout nature, including different sites within and on
the human body.
Microorganisms vary in their requirements for molecular oxygen. Obligate aerobes depend on aerobic respiration and use
oxygen as a terminal electron acceptor. They cannot grow without oxygen.
Obligate anaerobes cannot grow in the presence of oxygen. They depend on fermentation and anaerobic respiration using a
final electron acceptor other than oxygen.
Facultative anaerobes show better growth in the presence of oxygen but will also grow without it.
Although aerotolerant anaerobes do not perform aerobic respiration, they can grow in the presence of oxygen. Most
aerotolerant anaerobes test negative for the enzyme catalase.
Microaerophiles need oxygen to grow, albeit at a lower concentration than 21% oxygen in air.

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 Optimum oxygen concentration for an organism is the oxygen level that promotes the fastest growth rate. The minimum
permissive oxygen concentration and the maximum permissive oxygen concentration are, respectively, the lowest and the
highest oxygen levels that the organism will tolerate.
Peroxidase, superoxide dismutase, and catalase are the main enzymes involved in the detoxification of the reactive oxygen
species. Superoxide dismutase is usually present in a cell that can tolerate oxygen. All three enzymes are usually detectable in
cells that perform aerobic respiration and produce more ROS.
A capnophile is an organism that requires a higher than atmospheric concentration of CO2 to grow.

Footnotes
1. 1 Centers for Disease Control and Prevention. “Living With Diabetes: Keep Your Feet Healthy.”
http://www.cdc.gov/Features/DiabetesFootHealth/

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 8.2: Oxygen Requirements for Microbial Growth is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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8.3: The Effects of pH and Temperature on Microbial Growth
Learning Objectives
Illustrate and briefly describe minimum, optimum, and maximum pH requirements for growth
Identify and describe the different categories of microbes with pH requirements for growth: acidophiles, neutrophiles, and
alkaliphiles
Illustrate and briefly describe minimum, optimum, and maximum temperature requirements for growth
Identify and describe different categories of microbes with temperature requirements for growth: psychrophile,
psychrotrophs, mesophile, thermophile, hyperthermophile

Effects of pH
Yogurt, pickles, sauerkraut, and lime-seasoned dishes all owe their tangy taste to a high acid content (Figure 8.3.1). Recall that
acidity is a function of the concentration of hydrogen ions [H+] and is measured as pH. Environments with pH values below 7.0 are
considered acidic, whereas those with pH values above 7.0 are considered basic. Extreme pH affects the structure of all
macromolecules. The hydrogen bonds holding together strands of DNA break up at high pH. Lipids are hydrolyzed by an
extremely basic pH. The proton motive force responsible for production of ATP in cellular respiration depends on the concentration
gradient of H+ across the plasma membrane. If H+ ions are neutralized by hydroxide ions, the concentration gradient collapses and
impairs energy production. But the component most sensitive to pH in the cell is its workhorse, the protein. Moderate changes in
pH modify the ionization of amino-acid functional groups and disrupt hydrogen bonding, which, in turn, promotes changes in the
folding of the molecule, promoting denaturation and destroying activity.

Figure 8.3.1 : Lactic acid bacteria that ferment milk into yogurt or transform vegetables in pickles thrive at a pH close to 4.0.
Sauerkraut and dishes such as pico de gallo owe their tangy flavor to their acidity. Acidic foods have been a mainstay of the human
diet for centuries, partly because most microbes that cause food spoilage grow best at a near neutral pH and do not tolerate acidity
well. (credit “yogurt”: modification of work by “nina.jsc”/Flickr; credit “pickles”: modification of work by Noah Sussman; credit
“sauerkraut”: modification of work by Jesse LaBuff; credit “pico de gallo”: modification of work by “regan76”/Flickr)
The optimum growth pH is the most favorable pH for the growth of an organism. The lowest pH value that an organism can
tolerate is called the minimum growth pH and the highest pH is the maximum growth pH. These values can cover a wide range,
which is important for the preservation of food and to microorganisms’ survival in the stomach. For example, the optimum growth
pH of Salmonella spp. is 7.0–7.5, but the minimum growth pH is closer to 4.2.
Most bacteria are neutrophiles, meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7 (see Figure
8.3.2). Most familiar bacteria, like Escherichia coli, staphylococci, and Salmonella spp. are neutrophiles and do not fare well in the

acidic pH of the stomach. However, there are pathogenic strains of E. coli, S. typhi, and other species of intestinal pathogens that
are much more resistant to stomach acid. In comparison, fungi thrive at slightly acidic pH values of 5.0–6.0.
Microorganisms that grow optimally at pH less than 5.55 are called acidophiles. For example, the sulfur-oxidizing Sulfolobus spp.
isolated from sulfur mud fields and hot springs in Yellowstone National Park are extreme acidophiles. These archaea survive at pH
values of 2.5–3.5. Species of the archaean genus Ferroplasma live in acid mine drainage at pH values of 0–2.9. Lactobacillus
bacteria, which are an important part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5–6.8
and also contribute to the acidity of the vagina (pH of 4, except at the onset of menstruation) through their metabolic production of
lactic acid. The vagina’s acidity plays an important role in inhibiting other microbes that are less tolerant of acidity. Acidophilic
microorganisms display a number of adaptations to survive in strong acidic environments. For example, proteins show increased
negative surface charge that stabilizes them at low pH. Pumps actively eject H+ ions out of the cells. The changes in the
composition of membrane phospholipids probably reflect the need to maintain membrane fluidity at low pH.

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 Figure 8.3.2 : The curves show the approximate pH ranges for the growth of the different classes of pH-specific prokaryotes. Each
curve has an optimal pH and extreme pH values at which growth is much reduced. Most bacteria are neutrophiles and grow best at
near-neutral pH (center curve). Acidophiles have optimal growth at pH values near 3 and alkaliphiles have optimal growth at pH
values above 9.

At the other end of the spectrum are alkaliphiles, microorganisms that grow best at pH between 8.0 and 10.5. Vibrio cholerae, the
pathogenic agent of cholera, grows best at the slightly basic pH of 8.0; it can survive pH values of 11.0 but is inactivated by the
acid of the stomach. When it comes to survival at high pH, the bright pink archaean Natronobacterium, found in the soda lakes of
the African Rift Valley, may hold the record at a pH of 10.5 (Figure 8.3.3). Extreme alkaliphiles have adapted to their harsh
environment through evolutionary modification of lipid and protein structure and compensatory mechanisms to maintain the proton
motive force in an alkaline environment. For example, the alkaliphile Bacillus firmus derives the energy for transport reactions and
motility from a Na+ ion gradient rather than a proton motive force. Many enzymes from alkaliphiles have a higher isoelectric point,
due to an increase in the number of basic amino acids, than homologous enzymes from neutrophiles.

Figure 8.3.3 : View from space of Lake Natron in Tanzania. The pink color is due to the pigmentation of the extreme alkaliphilic
and halophilic microbes that colonize the lake. (credit: NASA)

Survival at the Low pH of the Stomach

Peptic ulcers (or stomach ulcers) are painful sores on the stomach lining. Until the 1980s, they were believed to be caused by
spicy foods, stress, or a combination of both. Patients were typically advised to eat bland foods, take anti-acid medications, and
avoid stress. These remedies were not particularly effective, and the condition often recurred. This all changed dramatically
when the real cause of most peptic ulcers was discovered to be a slim, corkscrew-shaped bacterium, Helicobacter pylori. This
organism was identified and isolated by Barry Marshall and Robin Warren, whose discovery earned them the Nobel Prize in
Medicine in 2005.
The ability of H. pylori to survive the low pH of the stomach would seem to suggest that it is an extreme acidophile. As it turns
out, this is not the case. In fact, H. pylori is a neutrophile. So, how does it survive in the stomach? Remarkably, H. pylori

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 creates a microenvironment in which the pH is nearly neutral. It achieves this by producing large amounts of the enzyme
urease, which breaks down urea to form NH4+ and CO2. The ammonium ion raises the pH of the immediate environment.
This metabolic capability of H. pylori is the basis of an accurate, noninvasive test for infection. The patient is given a solution
of urea containing radioactively labeled carbon atoms. If H. pylori is present in the stomach, it will rapidly break down the
urea, producing radioactive CO2 that can be detected in the patient’s breath. Because peptic ulcers may lead to gastric cancer,
patients who are determined to have H. pylori infections are treated with antibiotics.

Exercise 8.3.1

1. What effect do extremes of pH have on proteins?

2. What pH-adaptive type of bacteria would most human pathogens be?

Effects of Temperature
When the exploration of Lake Whillans started in Antarctica, researchers did not expect to find much life. Constant subzero
temperatures and lack of obvious sources of nutrients did not seem to be conditions that would support a thriving ecosystem. To
their surprise, the samples retrieved from the lake showed abundant microbial life. In a different but equally harsh setting, bacteria
grow at the bottom of the ocean in sea vents (Figure 8.3.1), where temperatures can reach 340 °C (700 °F).
Microbes can be roughly classified according to the range of temperature at which they can grow. The growth rates are the highest
at the optimum growth temperature for the organism. The lowest temperature at which the organism can survive and replicate is its
minimum growth temperature. The highest temperature at which growth can occur is its maximum growth temperature. The
following ranges of permissive growth temperatures are approximate only and can vary according to other environmental factors.
Organisms categorized as mesophiles (“middle loving”) are adapted to moderate temperatures, with optimal growth temperatures
ranging from room temperature (about 20 °C) to about 45 °C. As would be expected from the core temperature of the human body,
37 °C (98.6 °F), normal human microbiota and pathogens (e.g., E. coli, Salmonella spp., and Lactobacillus spp.) are mesophiles.
Organisms called psychrotrophs, also known as psychrotolerant, prefer cooler environments, from a high temperature of 25 °C to
refrigeration temperature about 4 °C. They are found in many natural environments in temperate climates. They are also
responsible for the spoilage of refrigerated food.

Clinical Focus- Resolution

The presence of Listeria in Jeni’s blood suggests that her symptoms are due to listeriosis, an infection caused by L.
monocytogenes. Listeriosis is a serious infection with a 20% mortality rate and is a particular risk to Jeni’s fetus. A sample
from the amniotic fluid cultured for the presence of Listeria gave negative results. Because the absence of organisms does not
rule out the possibility of infection, a molecular test based on the nucleic acid amplification of the 16S ribosomal RNA of
Listeria was performed to confirm that no bacteria crossed the placenta. Fortunately, the results from the molecular test were
also negative.
Jeni was admitted to the hospital for treatment and recovery. She received a high dose of two antibiotics intravenously for 2
weeks. The preferred drugs for the treatment of listeriosis are ampicillin or penicillin G with an aminoglycoside antibiotic.
Resistance to common antibiotics is still rare in Listeria and antibiotic treatment is usually successful. She was released to
home care after a week and fully recovered from her infection.
L. monocytogenes is a gram-positive short rod found in soil, water, and food. It is classified as a psychrophile and is
halotolerant. Its ability to multiply at refrigeration temperatures (4–10 °C) and its tolerance for high concentrations of salt (up
to 10% sodium chloride [NaCl]) make it a frequent source of food poisoning. Because Listeria can infect animals, it often
contaminates food such as meat, fish, or dairy products. Contamination of commercial foods can often be traced to persistent
biofilms that form on manufacturing equipment that is not sufficiently cleaned.
Listeria infection is relatively common among pregnant women because the elevated levels of progesterone downregulate the
immune system, making them more vulnerable to infection. The pathogen can cross the placenta and infect the fetus, often
resulting in miscarriage, stillbirth, or fatal neonatal infection. Pregnant women are thus advised to avoid consumption of soft
cheeses, refrigerated cold cuts, smoked seafood, and unpasteurized dairy products. Because Listeria bacteria can easily be

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 confused with diphtheroids, another common group of gram-positive rods, it is important to alert the laboratory when
listeriosis is suspected.

The organisms retrieved from arctic lakes such as Lake Whillans are considered extreme psychrophiles (cold loving).
Psychrophiles are microorganisms that can grow at 0 °C and below, have an optimum growth temperature close to 15 °C, and
usually do not survive at temperatures above 20 °C. They are found in permanently cold environments such as the deep waters of
the oceans. Because they are active at low temperature, psychrophiles and psychrotrophs are important decomposers in cold
climates.

Figure 8.3.4 : A black smoker at the bottom of the ocean belches hot, chemical-rich water, and heats the surrounding waters. Sea
vents provide an extreme environment that is nonetheless teeming with macroscopic life (the red tubeworms) supported by an
abundant microbial ecosystem. (credit: NOAA)
Organisms that grow at optimum temperatures of 50 °C to a maximum of 80 °C are called thermophiles (“heat loving”). They do
not multiply at room temperature. Thermophiles are widely distributed in hot springs, geothermal soils, and manmade
environments such as garden compost piles where the microbes break down kitchen scraps and vegetal material. Examples of
thermophiles include Thermus aquaticus and Geobacillus spp. Higher up on the extreme temperature scale we find the
hyperthermophiles, which are characterized by growth ranges from 80 °C to a maximum of 110 °C, with some extreme examples
that survive temperatures above 121 °C, the average temperature of an autoclave. The hydrothermal vents at the bottom of the
ocean are a prime example of extreme environments, with temperatures reaching an estimated 340 °C (Figure 8.3.4). Microbes
isolated from the vents achieve optimal growth at temperatures higher than 100 °C. Noteworthy examples are Pyrobolus and
Pyrodictium, archaea that grow at 105 °C and survive autoclaving. Figure 8.3.5 shows the typical skewed curves of temperature-
dependent growth for the categories of microorganisms we have discussed.

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 Figure 8.3.5 : The graph shows growth rate of bacteria as a function of temperature. Notice that the curves are skewed toward the
optimum temperature. The skewing of the growth curve is thought to reflect the rapid denaturation of proteins as the temperature
rises past the optimum for growth of the microorganism.
Life in extreme environments raises fascinating questions about the adaptation of macromolecules and metabolic processes. Very
low temperatures affect cells in many ways. Membranes lose their fluidity and are damaged by ice crystal formation. Chemical
reactions and diffusion slow considerably. Proteins become too rigid to catalyze reactions and may undergo denaturation. At the
opposite end of the temperature spectrum, heat denatures proteins and nucleic acids. Increased fluidity impairs metabolic processes
in membranes. Some of the practical applications of the destructive effects of heat on microbes are sterilization by steam,
pasteurization, and incineration of inoculating loops. Proteins in psychrophiles are, in general, rich in hydrophobic residues, display
an increase in flexibility, and have a lower number of secondary stabilizing bonds when compared with homologous proteins from
mesophiles. Antifreeze proteins and solutes that decrease the freezing temperature of the cytoplasm are common. The lipids in the
membranes tend to be unsaturated to increase fluidity. Growth rates are much slower than those encountered at moderate
temperatures. Under appropriate conditions, mesophiles and even thermophiles can survive freezing. Liquid cultures of bacteria are
mixed with sterile glycerol solutions and frozen to −80 °C for long-term storage as stocks. Cultures can withstand freeze drying
(lyophilization) and then be stored as powders in sealed ampules to be reconstituted with broth when needed.
Macromolecules in thermophiles and hyperthermophiles show some notable structural differences from what is observed in the
mesophiles. The ratio of saturated to polyunsaturated lipids increases to limit the fluidity of the cell membranes. Their DNA
sequences show a higher proportion of guanine–cytosine nitrogenous bases, which are held together by three hydrogen bonds in
contrast to adenine and thymine, which are connected in the double helix by two hydrogen bonds. Additional secondary ionic and
covalent bonds, as well as the replacement of key amino acids to stabilize folding, contribute to the resistance of proteins to
denaturation. The so-called thermoenzymes purified from thermophiles have important practical applications. For example,
amplification of nucleic acids in the polymerase chain reaction (PCR) depends on the thermal stability of Taq polymerase, an
enzyme isolated from T. aquaticus. Degradation enzymes from thermophiles are added as ingredients in hot-water detergents,
increasing their effectiveness.

Exercise 8.3.1

1. What temperature requirements do most bacterial human pathogens have?

2. What DNA adaptation do thermophiles exhibit?
3. Some hyperthermophiles can survive autoclaving temperatures. Are they a concern in health care?

Feeding the World... and the World's Algae

Artificial fertilizers have become an important tool in food production around the world. They are responsible for many of the
gains of the so-called green revolution of the 20th century, which has allowed the planet to feed many of its more than 7 billion
people. Artificial fertilizers provide nitrogen and phosphorus, key limiting nutrients, to crop plants, removing the normal
barriers that would otherwise limit the rate of growth. Thus, fertilized crops grow much faster, and farms that use fertilizer
produce higher crop yields.

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 However, careless use and overuse of artificial fertilizers have been demonstrated to have significant negative impacts on
aquatic ecosystems, both freshwater and marine. Fertilizers that are applied at inappropriate times or in too-large quantities
allow nitrogen and phosphorus compounds to escape use by crop plants and enter drainage systems. Inappropriate use of
fertilizers in residential settings can also contribute to nutrient loads, which find their way to lakes and coastal marine
ecosystems. As water warms and nutrients are plentiful, microscopic algae bloom, often changing the color of the water
because of the high cell density.
Most algal blooms are not directly harmful to humans or wildlife; however, they can cause harm indirectly. As the algal
population expands and then dies, it provides a large increase in organic matter to the bacteria that live in deep water. With this
large supply of nutrients, the population of nonphotosynthetic microorganisms explodes, consuming available oxygen and
creating “dead zones” where animal life has virtually disappeared.
Depletion of oxygen in the water is not the only damaging consequence of some algal blooms. The algae that produce red tides
in the Gulf of Mexico, Karenia brevis, secrete potent toxins that can kill fish and other organisms and also accumulate in
shellfish. Consumption of contaminated shellfish can cause severe neurological and gastrointestinal symptoms in humans.
Shellfish beds must be regularly monitored for the presence of the toxins, and harvests are often shut down when it is present,
incurring economic costs to the fishery. Cyanobacteria, which can form blooms in marine and freshwater ecosystems, produce
toxins called microcystins, which can cause allergic reactions and liver damage when ingested in drinking water or during
swimming. Recurring cyanobacterial algal blooms in Lake Erie (Figure 8.3.6) have forced municipalities to issue drinking
water bans for days at a time because of unacceptable toxin levels.
This is just a small sampling of the negative consequences of algal blooms, red tides, and dead zones. Yet the benefits of crop
fertilizer—the main cause of such blooms—are difficult to dispute. There is no easy solution to this dilemma, as a ban on
fertilizers is not politically or economically feasible. In lieu of this, we must advocate for responsible use and regulation in
agricultural and residential contexts, as well as the restoration of wetlands, which can absorb excess fertilizers before they
reach lakes and oceans.

Figure 8.3.6 : Heavy rains cause runoff of fertilizers into Lake Erie, triggering extensive algal blooms, which can be observed
along the shoreline. Notice the brown unplanted and green planted agricultural land on the shore. (credit: NASA)

This video discusses algal blooms and dead zones in more depth.

Key Concepts and Summary

Bacteria are generally neutrophiles. They grow best at neutral pH close to 7.0.
Acidophiles grow optimally at a pH near 3.0. Alkaliphiles are organisms that grow optimally between a pH of 8 and 10.5.
Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH.
Microorganisms grow best at their optimum growth pH. Growth occurs slowly or not at all below the minimum growth pH
and above the maximum growth pH.

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 Microorganisms thrive at a wide range of temperatures; they have colonized different natural environments and have adapted to
extreme temperatures. Both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and
biological processes.
Psychrophiles grow best in the temperature range of 0–15 °C whereas psychrotrophs thrive between 4°C and 25 °C.
Mesophiles grow best at moderate temperatures in the range of 20 °C to about 45 °C. Pathogens are usually mesophiles.
Thermophiles and hyperthemophiles are adapted to life at temperatures above 50 °C.
Adaptations to cold and hot temperatures require changes in the composition of membrane lipids and proteins.
Contributors
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 8.3: The Effects of pH and Temperature on Microbial Growth is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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8.4: Other Environmental Conditions that Affect Growth
Learning Objectives
Identify and describe different categories of microbes with specific growth requirements such as altered barometric
pressure, osmotic pressure, humidity, and light
Describe how osmotic pressure functions and how it is affected by the different balances of solutes and water in and around
the cell
Identify and describe different types of transport across the cell membrane

Microorganisms interact with their environment along more dimensions than pH, temperature, and free oxygen levels, although
these factors require significant adaptations. We also find microorganisms adapted to varying levels of barometric pressure,
humidity, light, and salinity.

Barometric Pressure
Microorganisms that require high atmospheric pressure for growth are called barophiles. The bacteria that live at the bottom of the
ocean must be able to withstand great pressures. Because it is difficult to retrieve intact specimens and reproduce such growth
conditions in the laboratory, the characteristics of these microorganisms are largely unknown.

Light
Photoautotrophs, such as cyanobacteria or green sulfur bacteria, and photoheterotrophs, such as purple nonsulfur bacteria, depend
on sufficient light intensity at the wavelengths absorbed by their pigments to grow and multiply. Energy from light is captured by
pigments and converted into chemical energy that drives carbon fixation and other metabolic processes. The portion of the
electromagnetic spectrum that is absorbed by these organisms is defined as photosynthetically active radiation (PAR). It lies within
the visible light spectrum ranging from 400 to 700 nanometers (nm) and extends in the near infrared for some photosynthetic
bacteria. A number of accessory pigments, such as fucoxanthin in brown algae and phycobilins in cyanobacteria, widen the useful
range of wavelengths for photosynthesis and compensate for the low light levels available at greater depths of water. Other
microorganisms, such as the archaea of the class Halobacteria, use light energy to drive their proton and sodium pumps. The light is
absorbed by a pigment protein complex called bacteriorhodopsin, which is similar to the eye pigment rhodopsin. Photosynthetic
bacteria are present not only in aquatic environments but also in soil and in symbiosis with fungi in lichens. The peculiar
watermelon snow is caused by a microalga Chlamydomonas nivalis, a green alga rich in a secondary red carotenoid pigment
(astaxanthin) which gives the pink hue to the snow where the alga grows.

Exercise 8.4.1
1. Which photosynthetic pigments were described in this section?
2. What is the fundamental stress of a hypersaline environment for a cell?

Osmotic Pressure
Most natural environments tend to have lower solute concentrations than the cytoplasm of most microorganisms. Rigid cell walls
protect the cells from bursting in a dilute environment. Not much protection is available against high osmotic pressure. In this case,
water, following its concentration gradient, flows out of the cell. This results in plasmolysis (the shrinking of the protoplasm away
from the intact cell wall) and cell death. This fact explains why brines and layering meat and fish in salt are time-honored methods
of preserving food. Microorganisms called halophiles (“salt loving”) actually require high salt concentrations for growth. These
organisms are found in marine environments where salt concentrations hover at 3.5%. Extreme halophilic microorganisms, such as
the red alga Dunaliella salina and the archaeal species Halobacterium in Figure 8.4.1, grow in hypersaline lakes such as the Great
Salt Lake, which is 3.5–8 times saltier than the ocean, and the Dead Sea, which is 10 times saltier than the ocean.

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 Figure 8.4.1 : Photograph taken from space of the Great Salt Lake in Utah. The purple color is caused by high density of the alga
Dunaliella and the archaean Halobacterium spp. (credit: NASA)
Dunaliella spp. counters the tremendous osmotic pressure of the environment with a high cytoplasmic concentration of glycerol
and by actively pumping out salt ions. Halobacterium spp. accumulates large concentrations of K+ and other ions in its cytoplasm.
Its proteins are designed for high salt concentrations and lose activity at salt concentrations below 1–2 M. Although most
halotolerant organisms, for example Halomonas spp. in salt marshes, do not need high concentrations of salt for growth, they will
survive and divide in the presence of high salt. Not surprisingly, the staphylococci, micrococci, and corynebacteria that colonize
our skin tolerate salt in their environment. Halotolerant pathogens are an important cause of food-borne illnesses because they
survive and multiply in salty food. For example, the halotolerant bacteria S. aureus, Bacillus cereus, and V. cholerae produce
dangerous enterotoxins and are major causes of food poisoning.
Microorganisms depend on available water to grow. Available moisture is measured as water activity (aw), which is the ratio of the
vapor pressure of the medium of interest to the vapor pressure of pure distilled water; therefore, the aw of water is equal to 1.0.
Bacteria require high aw (0.97–0.99), whereas fungi can tolerate drier environments; for example, the range of aw for growth of
Aspergillus spp. is 0.8–0.75. Decreasing the water content of foods by drying, as in jerky, or through freeze-drying or by increasing
osmotic pressure, as in brine and jams, are common methods of preventing spoilage.

Osmotic Pressure and the Cellular Envelope

The cell wall is a structure found in most prokaryotes and some eukaryotes; it envelopes the cell membrane, protecting the cell
from changes in osmotic pressure, i.e. water activity (Figure 8.4.2). Osmotic pressure occurs because of differences in the
concentration of solutes on opposing sides of a selectively or semipermeable membrane. Water is able to pass through a selectively
or semipermeable membrane, but solutes (dissolved molecules like salts, sugars, and other compounds) cannot always. When the
concentration of solutes is greater on one side of the membrane, water diffuses across the membrane from the side with the lower
concentration of solutes (more water) to the side with the higher concentration of solutes (less water) until the concentrations on
both sides become equal. This diffusion of water is called osmosis, and it can cause extreme osmotic pressure on a cell when its
external environment changes.
The external environment of a cell can be described as an isotonic, hypertonic, or hypotonic medium. In an isotonic medium, the
solute concentrations inside and outside the cell are approximately equal, so there is no net movement of water across the cell
membrane. In a hypertonic medium, the solute concentration outside the cell exceeds that inside the cell, so water diffuses out of
the cell and into the external medium. In a hypotonic medium, the solute concentration inside the cell exceeds that outside of the
cell, so water will move by osmosis into the cell. This causes the cell to swell and potentially lyse, or burst.
The degree to which a particular cell is able to withstand changes in osmotic pressure is called tonicity. Cells that have a cell wall
are better able to withstand subtle changes in osmotic pressure and maintain their shape. In hypertonic environments, cells that lack
a cell wall can become dehydrated, causing crenation, or shriveling of the cell; the plasma membrane contracts and appears
scalloped or notched (Figure 8.4.2). By contrast, cells that possess a cell wall undergo plasmolysis rather than crenation. In

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plasmolysis, the plasma membrane contracts and detaches from the cell wall, and there is a decrease in interior volume, but the cell
wall remains intact, thus allowing the cell to maintain some shape and integrity for a period of time (Figure 8.4.3). Likewise, cells
that lack a cell wall are more prone to lysis in hypotonic environments. The presence of a cell wall allows the cell to maintain its
shape and integrity for a longer time before lysing (Figure 8.4.3).

Figure 8.4.2 : In cells that lack a cell wall, changes in osmotic pressure can lead to crenation in hypertonic environments or cell
lysis in hypotonic environments.

Figure 8.4.3 : In prokaryotic cells, the cell wall provides some protection against changes in osmotic pressure, allowing it to
maintain its shape longer. The cell membrane is typically attached to the cell wall in an isotonic medium (left). In a hypertonic
medium, the cell membrane detaches from the cell wall and contracts (plasmolysis) as water leaves the cell. In a hypotonic medium
(right), the cell wall prevents the cell membrane from expanding to the point of bursting, although lysis will eventually occur if too
much water is absorbed.

Membrane Transport Mechanisms

One of the most important functions of the plasma membrane is to control the transport of molecules into and out of the cell.
Internal conditions must be maintained within a certain range despite any changes in the external environment. The transport of
substances across the plasma membrane allows cells to do so.

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 Figure 8.4.4 : Simple diffusion down a concentration gradient directly across the phospholipid bilayer. (credit: modification of work
by Mariana Ruiz Villareal)

Passive Diffusion
Cells use various modes of transport across the plasma membrane. Collectively this ability to move molecules with or down their
concentration gradient is called passive diffusion. This allows the cell to use no energy to move the molecules. (Figure 8.4.4).
Some small molecules, like carbon dioxide, may cross the membrane bilayer directly by simple diffusion. However, charged
molecules (including ions like Mg2+), as well as large molecules (think sugars), need the help of carriers or channels in the
membrane. These structures ferry molecules across the membrane, a process known as facilitated diffusion (Figure 8.4.5). Each
molecule or group of similar molecules have their own specific carrier or channel protein to help it across the membrane and this
allows for the cell to use no energy while still being selective about the molecules coming in and out. Osmosis as discussed before
is usually a type of facilitated diffusion due to the use of a special water channel protein called an aquaporin.

Figure 8.4.5 : Facilitated diffusion down a concentration gradient through a membrane protein. (credit: modification of work by
Mariana Ruiz Villareal)

Active Transport
Another way to move molecules is via active transport. Active transport occurs when cells move molecules across the membrane
against or "up" their concentration gradients (Figure 8.4.6). A major difference between passive and active transport is that active
transport requires adenosine triphosphate (ATP) or other forms of energy to move molecules “uphill.” Therefore, active transport
structures are often called “pumps.”

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 Figure 8.4.6 : Active transport against a concentration gradient via a membrane pump that requires energy. (credit: modification of
work by Mariana Ruiz Villareal)

Group Translocation
Group translocation also transports substances into bacterial cells. In this case, as a molecule moves into a cell against its
concentration gradient, it is chemically modified so that it does not require transport against an unfavorable concentration gradient.
A common example of this is the bacterial phosphotransferase system, a series of carriers that phosphorylates (i.e., adds phosphate
ions to) glucose or other sugars upon entry into cells. Since the phosphorylation of sugars is required during the early stages of
sugar metabolism, the phosphotransferase system is considered to be an energy neutral system.

Eukaryotic Membrane Transport Mechanisms

The processes of simple diffusion, facilitated diffusion, and active transport are used in both eukaryotic and prokaryotic cells.
However, eukaryotic cells also have the unique ability to perform various types of endocytosis, the uptake of matter through plasma
membrane invagination and vacuole/vesicle formation (Figure 8.4.7). A type of endocytosis involving the engulfment of large
particles through membrane invagination is called phagocytosis, which means “cell eating.” In phagocytosis, particles (or other
cells) are enclosed in a pocket within the membrane, which then pinches off from the membrane to form a vacuole that completely
surrounds the particle. Another type of endocytosis is called pinocytosis, which means “cell drinking.” In pinocytosis, small,
dissolved materials and liquids are taken into the cell through small vesicles. Saprophytic fungi, for example, obtain their nutrients
from dead and decaying matter largely through pinocytosis.
Receptor-mediated endocytosis is a type of endocytosis that is initiated by specific molecules called ligands when they bind to cell
surface receptors on the membrane. Receptor-mediated endocytosis is the mechanism that peptide and amine-derived hormones use
to enter cells and is also used by various viruses and bacteria for entry into host cells.

Figure 8.4.7 : Three variations of endocytosis are shown. (a) In phagocytosis, the cell membrane surrounds the particle and pinches
off to form an intracellular vacuole. (b) In pinocytosis, the cell membrane surrounds a small volume of fluid and pinches off,
forming a vesicle. (c) In receptor-mediated endocytosis, the uptake of substances is targeted to a specific substance (a ligand) that
binds at the receptor on the external cell membrane. (credit: modification of work by Mariana Ruiz Villarreal)

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The process by which secretory vesicles release their contents to the cell’s exterior is called exocytosis. Vesicles move toward the
plasma membrane and then meld with the membrane, ejecting their contents out of the cell. Exocytosis is used by cells to remove
waste products and may also be used to release chemical signals that can be taken up by other cells.

Key Concepts and Summary

Halophiles require high salt concentration in the medium, whereas halotolerant organisms can grow and multiply in the
presence of high salt but do not require it for growth.
Halotolerant pathogens are an important source of foodborne illnesses because they contaminate foods preserved in salt.
Barophiles require high atmospheric pressure.
Some molecules can move across the bacterial membrane by simple diffusion. This is dependent on the concentration of the
molecule inside the cell versus outside the cell.
Water particularly is molecule that can be diffused across the membrane and its movement from out to in or in to out determines
the osmotic pressure on the organism.
Most large molecules must be actively transported through membrane structures using cellular energy.
Photosynthetic bacteria depend on visible light for energy.
Most bacteria, with few exceptions, require high moisture to grow.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 8.4: Other Environmental Conditions that Affect Growth is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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8.5: Microbial Relationships
Learning Objectives
Describe the idea of the ubiquity of prokaryotes in various habitats on earth
Describe the range of relationships prokaryote have with other organisms on earth
Identify and describe symbiotic relationships
Compare normal/commensal/resident microbiota to transient microbiota

Prokaryotes are ubiquitous. They can be found everywhere on our planet, even in hot springs, in the Antarctic ice shield, and under
extreme pressure two miles under water. One bacterium, Paracoccus denitrificans, has even been shown to survive when scientists
removed it from its native environment (soil) and used a centrifuge to subject it to forces of gravity as strong as those found on the
surface of Jupiter.
Prokaryotes also are abundant on and within the human body. According to a report by National Institutes of Health, prokaryotes,
especially bacteria, outnumber human cells 10:1.1 More recent studies suggest the ratio could be closer to 1:1, but even that ratio
means that there are a great number of bacteria within the human body.2 Bacteria thrive in the human mouth, nasal cavity, throat,
ears, gastrointestinal tract, and vagina. Large colonies of bacteria can be found on healthy human skin, especially in moist areas
(armpits, navel, and areas behind ears). However, even drier areas of the skin are not free from bacteria.
The existence of prokaryotes is very important for the stability and thriving of ecosystems. For example, they are a necessary part
of soil formation and stabilization processes through the breakdown of organic matter and development of biofilms. One gram of
soil contains up to 10 billion microorganisms (most of them prokaryotic) belonging to about 1,000 species. Many species of
bacteria use substances released from plant roots, such as acids and carbohydrates, as nutrients. The bacteria metabolize these plant
substances and release the products of bacterial metabolism back to the soil, forming humus and thus increasing the soil’s fertility.
In salty lakes such as the Dead Sea (Figure 8.5.1), salt-loving halobacteria decompose dead brine shrimp and nourish young brine
shrimp and flies with the products of bacterial metabolism.

Figure 8.5.1 : (a) Some prokaryotes, called halophiles, can thrive in extremely salty environments such as the Dead Sea, pictured
here. (b) The archaeon Halobacterium salinarum, shown here in an electron micrograph, is a halophile that lives in the Dead Sea.
(credit a: modification of work by Jullen Menichini; credit b: modification of work by NASA)
In addition to living in the ground and the water, prokaryotic microorganisms are abundant in the air, even high in the atmosphere.
There may be up to 2,000 different kinds of bacteria in the air, similar to their diversity in the soil.
Prokaryotes can be found everywhere on earth because they are extremely resilient and adaptable. They are often metabolically
flexible, which means that they might easily switch from one energy source to another, depending on the availability of the sources,
or from one metabolic pathway to another. For example, certain prokaryotic cyanobacteria can switch from a conventional type of
lipid metabolism, which includes production of fatty aldehydes, to a different type of lipid metabolism that generates biofuel, such
as fatty acids and wax esters. Groundwater bacteria store complex high-energy carbohydrates when grown in pure groundwater, but

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they metabolize these molecules when the groundwater is enriched with phosphates. Some bacteria get their energy by reducing
sulfates into sulfides, but can switch to a different metabolic pathway when necessary, producing acids and free hydrogen ions.
Prokaryotes perform functions vital to life on earth by capturing (or “fixing”) and recycling elements like carbon and nitrogen.
Organisms such as animals require organic carbon to grow, but, unlike prokaryotes, they are unable to use inorganic carbon sources
like carbon dioxide. Thus, animals rely on prokaryotes to convert carbon dioxide into organic carbon products that they can use.
This process of converting carbon dioxide to organic carbon products is called carbon fixation.
Plants and animals also rely heavily on prokaryotes for nitrogen fixation, the conversion of atmospheric nitrogen into ammonia, a
compound that some plants can use to form many different biomolecules necessary to their survival. Bacteria in the genus
Rhizobium, for example, are nitrogen-fixing bacteria; they live in the roots of legume plants such as clover, alfalfa, and peas
(Figure 8.5.2). Ammonia produced by Rhizobium helps these plants to survive by enabling them to make building blocks of nucleic
acids. In turn, these plants may be eaten by animals—sustaining their growth and survival—or they may die, in which case the
products of nitrogen fixation will enrich the soil and be used by other plants.

Figure 8.5.2 : (a) Nitrogen-fixing bacteria such as Rhizobium live in the root nodules of legumes such as clover. (b) This
micrograph of the root nodule shows bacteroids (bacterium-like cells or modified bacterial cells) within the plant cells. The
bacteroids are visible as darker ovals within the larger plant cell. (credit a: modification of work by USDA)
Another positive function of prokaryotes is in cleaning up the environment. Recently, some researchers focused on the diversity
and functions of prokaryotes in manmade environments. They found that some bacteria play a unique role in degrading toxic
chemicals that pollute water and soil.3
Despite all of the positive and helpful roles prokaryotes play, some are human pathogens that may cause illness or infection when
they enter the body. In addition, some bacteria can contaminate food, causing spoilage or foodborne illness, which makes them
subjects of concern in food preparation and safety. Less than 1% of prokaryotes (all of them bacteria) are thought to be human
pathogens, but collectively these species are responsible for a large number of the diseases that afflict humans.
Besides pathogens, which have a direct impact on human health, prokaryotes also affect humans in many indirect ways. For
example, prokaryotes are now thought to be key players in the processes of climate change. In recent years, as temperatures in the
earth’s polar regions have risen, soil that was formerly frozen year-round (permafrost) has begun to thaw. Carbon trapped in the
permafrost is gradually released and metabolized by prokaryotes. This produces massive amounts of carbon dioxide and methane,
greenhouse gases that escape into the atmosphere and contribute to the greenhouse effect.

Exercise 8.5.1

1. In what types of environments can prokaryotes be found?

2. Name some ways that plants and animals rely on prokaryotes.

Symbiotic Relationships
Prokaryotic microorganisms can associate with plants and animals. Often, this association results in unique relationships between
organisms. For example, bacteria living on the roots or leaves of a plant get nutrients from the plant and, in return, produce

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substances that protect the plant from pathogens. On the other hand, some bacteria are plant pathogens that use mechanisms of
infection similar to bacterial pathogens of animals and humans.
Prokaryotes live in a community, or a group of interacting populations of organisms. A population is a group of individual
organisms belonging to the same biological species and limited to a certain geographic area. Populations can have cooperative
interactions, which benefit the populations, or competitive interactions, in which one population competes with another for
resources. The study of these interactions between populations is called microbial ecology.
Any interaction between different species within a community is called symbiosis. Such interactions fall along a continuum
between opposition and cooperation. Interactions in a symbiotic relationship may be beneficial or harmful, or have no effect on one
or both of the species involved. Table 8.5.1 summarizes the main types of symbiotic interactions among prokaryotes.
Table 8.5.1 : Types of Symbiotic Relationships
Type Population A Population B

Mutualism Benefitted Benefitted

Amensalism Harmed Unaffected

Commensalism Benefitted Unaffected

Neutralism Unaffected Unaffected

Parasitism Benefitted Harmed

When two species benefit from each other, the symbiosis is called mutualism (or syntropy, or crossfeeding). For example, humans
have a mutualistic relationship with the bacterium Bacteroides thetaiotetraiotamicron, which lives in the intestinal tract. B.
thetaiotetraiotamicron digests complex polysaccharide plant materials that human digestive enzymes cannot break down,
converting them into monosaccharides that can be absorbed by human cells. Humans also have a mutualistic relationship with
certain strains of Escherichia coli, another bacterium found in the gut. E. coli relies on intestinal contents for nutrients, and humans
derive certain vitamins from E. coli, particularly vitamin K, which is required for the formation of blood clotting factors. (This is
only true for some strains of E. coli, however. Other strains are pathogenic and do not have a mutualistic relationship with humans.)
A type of symbiosis in which one population harms another but remains unaffected itself is called amensalism. In the case of
bacteria, some amensalist species produce bactericidal substances that kill other species of bacteria. For example, the bacterium
Lucilia sericata produces a protein that destroys Staphylococcus aureus, a bacterium commonly found on the surface of the human
skin. Too much handwashing can affect this relationship and lead to S. aureus diseases and transmission.
In another type of symbiosis, called commensalism, one organism benefits while the other is unaffected. This occurs when the
bacterium Staphylococcus epidermidis uses the dead cells of the human skin as nutrients. Billions of these bacteria live on our skin,
but in most cases (especially when our immune system is healthy), we do not react to them in any way.
If neither of the symbiotic organisms is affected in any way, we call this type of symbiosis neutralism. An example of neutralism is
the coexistence of metabolically active (vegetating) bacteria and endospores (dormant, metabolically passive bacteria). For
example, the bacterium Bacillus anthracis typically forms endospores in soil when conditions are unfavorable. If the soil is warmed
and enriched with nutrients, some endospores germinate and remain in symbiosis with other endospores that have not germinated.
A type of symbiosis in which one organism benefits while harming the other is called parasitism. The relationship between humans
and many pathogenic prokaryotes can be characterized as parasitic because these organisms invade the body, producing toxic
substances or infectious diseases that cause harm. Diseases such as tetanus, diphtheria, pertussis, tuberculosis, and leprosy all arise
from interactions between bacteria and humans.

The Microbiome
Scientists have coined the term microbiome to refer to all prokaryotic and eukaryotic microorganisms that are associated with a
certain organism. Within the human microbiome, there are resident microbiota and transient microbiota. The resident microbiota
consists of microorganisms that constantly live in or on our bodies. The term transient microbiota refers to microorganisms that are
only temporarily found in the human body, and these may include pathogenic microorganisms. Hygiene and diet can alter both the
resident and transient microbiota.
The resident microbiota is amazingly diverse, not only in terms of the variety of species but also in terms of the preference of
different microorganisms for different areas of the human body. For example, in the human mouth, there are thousands of

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commensal or mutualistic species of bacteria. Some of these bacteria prefer to inhabit the surface of the tongue, whereas others
prefer the internal surface of the cheeks, and yet others prefer the front or back teeth or gums. The inner surface of the cheek has
the least diverse microbiota because of its exposure to oxygen. By contrast, the crypts of the tongue and the spaces between teeth
are two sites with limited oxygen exposure, so these sites have more diverse microbiota, including bacteria living in the absence of
oxygen (e.g., Bacteroides, Fusobacterium). Differences in the oral microbiota between randomly chosen human individuals are
also significant. Studies have shown, for example, that the prevalence of such bacteria as Streptococcus, Haemophilus, Neisseria,
and others was dramatically different when compared between individuals.4
There are also significant differences between the microbiota of different sites of the same human body. The inner surface of the
cheek has a predominance of Streptococcus, whereas in the throat, the palatine tonsil, and saliva, there are two to three times fewer
Streptococcus, and several times more Fusobacterium. In the plaque removed from gums, the predominant bacteria belong to the
genus Fusobacterium. However, in the intestine, both Streptococcus and Fusobacterium disappear, and the genus Bacteroides
becomes predominant.
Not only can the microbiota vary from one body site to another, the microbiome can also change over time within the same
individual. Humans acquire their first inoculations of normal flora during natural birth and shortly after birth. Before birth, there is
a rapid increase in the population of Lactobacillus spp. in the vagina, and this population serves as the first colonization of
microbiota during natural birth. After birth, additional microbes are acquired from health-care providers, parents, other relatives,
and individuals who come in contact with the baby. This process establishes a microbiome that will continue to evolve over the
course of the individual’s life as new microbes colonize and are eliminated from the body. For example, it is estimated that within a
9-hour period, the microbiota of the small intestine can change so that half of the microbial inhabitants will be different.5 The
importance of the initial Lactobacillus colonization during vaginal child birth is highlighted by studies demonstrating a higher
incidence of diseases in individuals born by cesarean section, compared to those born vaginally. Studies have shown that babies
born vaginally are predominantly colonized by vaginal lactobacillus, whereas babies born by cesarean section are more frequently
colonized by microbes of the normal skin microbiota, including common hospital-acquired pathogens.
Throughout the body, resident microbiotas are important for human health because they occupy niches that might be otherwise
taken by pathogenic microorganisms. For instance, Lactobacillus spp. are the dominant bacterial species of the normal vaginal
microbiota for most women. Lactobacillus produce lactic acid, contributing to the acidity of the vagina and inhibiting the growth of
pathogenic yeasts. However, when the population of the resident microbiota is decreased for some reason (e.g., because of taking
antibiotics), the pH of the vagina increases, making it a more favorable environment for the growth of yeasts such as Candida
albicans. Antibiotic therapy can also disrupt the microbiota of the intestinal tract and respiratory tract, increasing the risk for
secondary infections and/or promoting the long-term carriage and shedding of pathogens. This will be discussed more in chapter
12.

Exercise 8.5.2
1. Explain the difference between cooperative and competitive interactions in microbial communities.
2. List the types of symbiosis and explain how each population is affected.

Human Microbiome Project

The Human Microbiome Project was launched by the National Institutes of Health (NIH) in 2008. One main goal of the project
is to create a large repository of the gene sequences of important microbes found in humans, helping biologists and clinicians
understand the dynamics of the human microbiome and the relationship between the human microbiota and diseases. A
network of labs working together has been compiling the data from swabs of several areas of the skin, gut, and mouth from
hundreds of individuals.
One of the challenges in understanding the human microbiome has been the difficulty of culturing many of the microbes that
inhabit the human body. It has been estimated that we are only able to culture 1% of the bacteria in nature and that we are
unable to grow the remaining 99%. To address this challenge, researchers have used metagenomic analysis, which studies
genetic material harvested directly from microbial communities, as opposed to that of individual species grown in a culture.
This allows researchers to study the genetic material of all microbes in the microbiome, rather than just those that can be
cultured.6
One important achievement of the Human Microbiome Project is establishing the first reference database on microorganisms
living in and on the human body. Many of the microbes in the microbiome are beneficial, but some are not. It was found,

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 somewhat unexpectedly, that all of us have some serious microbial pathogens in our microbiota. For example, the conjunctiva
of the human eye contains 24 genera of bacteria and numerous pathogenic species.7 A healthy human mouth contains a number
of species of the genus Streptococcus, including pathogenic species S. pyogenes and S. pneumoniae.8 This raises the question
of why certain prokaryotic organisms exist commensally in certain individuals but act as deadly pathogens in others. Also
unexpected was the number of organisms that had never been cultured. For example, in one metagenomic study of the human
gut microbiota, 174 new species of bacteria were identified.9
Another goal for the near future is to characterize the human microbiota in patients with different diseases and to find out
whether there are any relationships between the contents of an individual’s microbiota and risk for or susceptibility to specific
diseases. Analyzing the microbiome in a person with a specific disease may reveal new ways to fight diseases.

Key Concepts and Summary

Prokaryotes can be found everywhere on our planet, even in the most extreme environments.
Prokaryotes are very flexible metabolically, so they are able to adjust their feeding to the available natural resources.
Prokaryotes live in communities that interact among themselves and with large organisms that they use as hosts (including
humans).
Interactions are symbiotic and can be classified into several different types including mutualism, amensalism, commensalism,
neutralism and parasitism.
The totality of forms of prokaryotes (particularly bacteria) living on the human body is called the human microbiome, which
varies between regions of the body and individuals, and changes over time.
The totality of forms of prokaryotes (particularly bacteria) living in a certain region of the human body (e.g., mouth, throat, gut,
eye, vagina) is called the microbiota of this region.

Footnotes
1. Medical Press. “Mouth Bacteria Can Change Their Diet, Supercomputers Reveal.” August 12, 2014.
medicalxpress.com/news/2014-0...rs-reveal.html. Accessed February 24, 2015.
2. A. Abbott. “Scientists Bust Myth That Our Bodies Have More Bacteria Than Human Cells: Decades-Old Assumption about
Microbiota Revisited.” Nature. http://www.nature.com/news/scientist...-cells-1.19136. Accessed June 3, 2016.
3. A.M. Kravetz “Unique Bacteria Fights Man-Made Chemical Waste.” 2012. www.livescience.com/25181-bac...s-nsf-bts.html.
Accessed March 9, 2015.
4. E.M. Bik et al. “Bacterial Diversity in the Oral Cavity of 10 Healthy Individuals.” The ISME Journal 4 no. 8 (2010):962–974.
5. C.C. Booijink et al. “High Temporal and Intra-Individual Variation Detected in the Human Ileal Microbiota.” Environmental
Microbiology 12 no. 12 (2010):3213–3227.
6. National Institutes of Health. “Human Microbiome Project. Overview.” commonfund.nih.gov/hmp/overview. Accessed June 7,
2016.
7. Q. Dong et al. “Diversity of Bacteria at Healthy Human Conjunctiva.” Investigative Ophthalmology & Visual Science 52 no. 8
(2011):5408–5413.
8. F.E. Dewhirst et al. “The Human Oral Microbiome.” Journal of Bacteriology 192 no. 19 (2010):5002–5017.
9. J.C. Lagier et al. “Microbial Culturomics: Paradigm Shift in the Human Gut Microbiome Study.” Clinical Microbiology and
Infection 18 no. 12 (2012):1185–1193.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 8.5: Microbial Relationships is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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Chapter 8 Exercises
Review Questions for Chapter 8
Multiple Choice

1) Which of the following methods would be used to measure the concentration of bacterial contamination in processed peanut
butter?
A. turbidity measurement
B. total plate count
C. dry weight measurement
D. direct counting of bacteria on a calibrated slide under the microscope

2) In which phase would you expect to observe the most endospores in a Bacillus cell culture?
A. death phase
B. lag phase
C. log phase
D. log, lag, and death phases would all have roughly the same number of endospores.

3) During which phase would penicillin, an antibiotic that inhibits cell-wall synthesis, be most effective?
A. death phase
B. lag phase
C. log phase
D. stationary phase

4) Which of the following is the best definition of generation time in a bacterium?

A. the length of time it takes to reach the log phase
B. the length of time it takes for a population of cells to double
C. the time it takes to reach stationary phase
D. the length of time of the exponential phase

5) What is the function of the Z ring in binary fission?

A. It controls the replication of DNA.
B. It forms a contractile ring at the septum.
C. It separates the newly synthesized DNA molecules.
D. It mediates the addition of new peptidoglycan subunits.

6) If a culture starts with 50 cells, how many cells will be present after five generations with no cell death?
A. 200
B. 400
C. 1600
D. 3200

7) Filamentous cyanobacteria often divide by which of the following?

A. budding

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B. mitosis
C. fragmentation
D. formation of endospores

8) Which is a reason for antimicrobial resistance being higher in a biofilm than in free-floating bacterial cells?
A. The EPS allows faster diffusion of chemicals in the biofilm.
B. Cells are more metabolically active at the base of a biofilm.
C. Cells are metabolically inactive at the base of a biofilm.
D. The structure of a biofilm favors the survival of antibiotic resistant cells.

9) Quorum sensing is used by bacterial cells to determine which of the following?

A. the size of the population
B. the availability of nutrients
C. the speed of water flow
D. the density of the population

10) Which of the following statements about autoinducers is incorrect?

A. They bind directly to DNA to activate transcription.
B. They can activate the cell that secreted them.
C. N-acylated homoserine lactones are autoinducers in gram-negative cells.
D. Autoinducers may stimulate the production of virulence factors.

11) An inoculated thioglycolate medium culture tube shows dense growth at the surface and turbidity throughout the rest of the
tube. What is your conclusion?
A. The organisms die in the presence of oxygen
B. The organisms are facultative anaerobes.
C. The organisms should be grown in an anaerobic chamber.
D. The organisms are obligate aerobes.

12) An inoculated thioglycolate medium culture tube is clear throughout the tube except for dense growth at the bottom of the tube.
What is your conclusion?
A. The organisms are obligate anaerobes.
B. The organisms are facultative anaerobes.
C. The organisms are aerotolerant.
D. The organisms are obligate aerobes.

13) Pseudomonas aeruginosa is a common pathogen that infects the airways of patients with cystic fibrosis. It does not grow in the
absence of oxygen. The bacterium is probably which of the following?
A. an aerotolerant anaerobe
B. an obligate aerobe
C. an obligate anaerobe
D. a facultative anaerobe

14) Streptococcus mutans is a major cause of cavities. It resides in the gum pockets, does not have catalase activity, and can be
grown outside of an anaerobic chamber. The bacterium is probably which of the following?

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A. a facultative anaerobe
B. an obligate aerobe
C. an obligate anaerobe
D. an aerotolerant anaerobe

15) Why do the instructions for the growth of Neisseria gonorrhoeae recommend a CO2-enriched atmosphere?
A. It uses CO2 as a final electron acceptor in respiration.
B. It is an obligate anaerobe.
C. It is a capnophile.
D. It fixes CO2 through photosynthesis.

16) Bacteria that grow in mine drainage at pH 1–2 are probably which of the following?
A. alkaliphiles
B. acidophiles
C. neutrophiles
D. obligate anaerobes

17) Bacteria isolated from Lake Natron, where the water pH is close to 10, are which of the following?
A. alkaliphiles
B. facultative anaerobes
C. neutrophiles
D. obligate anaerobes

18) In which environment are you most likely to encounter an acidophile?

A. human blood at pH 7.2
B. a hot vent at pH 1.5
C. human intestine at pH 8.5
D. milk at pH 6.5

19) A soup container was forgotten in the refrigerator and shows contamination. The contaminants are probably which of the
following?
A. thermophiles
B. acidophiles
C. mesophiles
D. psychrotrophs

20) Bacteria isolated from a hot tub at 39 °C are probably which of the following?
A. thermophiles
B. psychrotrophs
C. mesophiles
D. hyperthermophiles

21) In which environment are you most likely to encounter a hyperthermophile?

A. hot tub
B. warm ocean water in Florida

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C. hydrothermal vent at the bottom of the ocean
D. human body

22) Which of the following environments would harbor psychrophiles?

A. mountain lake with a water temperature of 12 °C
B. contaminated plates left in a 35 °C incubator
C. yogurt cultured at room temperature
D. salt pond in the desert with a daytime temperature of 34 °C

23) Which of the following is the reason jams and dried meats often do not require refrigeration to prevent spoilage?
A. low pH
B. toxic alkaline chemicals
C. naturally occurring antibiotics
D. low water activity

24) Bacteria living in salt marshes are most likely which of the following?
A. acidophiles
B. barophiles
C. halotolerant
D. thermophiles

25) Which of the following refers to the type of interaction between two prokaryotic populations in which one population benefits
and the other is not affected?
A. mutualism
B. commensalism
C. parasitism
D. neutralism

Fill-in-the-Blanks

26) Direct count of total cells can be performed using a ________ or a ________.

27) The ________ method allows direct count of total cells growing on solid medium.

28) A statistical estimate of the number of live cells in a liquid is usually done by ________.

29) For this indirect method of estimating the growth of a culture, you measure ________ using a spectrophotometer.

30) Active growth of a culture may be estimated indirectly by measuring the following products of cell metabolism: ________ or
________.

31) A bacterium that thrives in a soda lake where the average pH is 10.5 can be classified as a(n) ________.

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32) Lactobacillus acidophilus grows best at pH 4.5. It is considered a(n) ________.

33) A bacterium that thrives in the Great Salt Lake but not in fresh water is probably a ________.

34) Bacteria isolated from the bottom of the ocean need high atmospheric pressures to survive. They are ________.

35) Staphylococcus aureus can be grown on multipurpose growth medium or on mannitol salt agar that contains 7.5% NaCl. The
bacterium is ________.

36) When prokaryotes live as interacting communities in which one population benefits to the harm of the other, the type of
symbiosis is called ________.

Short Answers
37) What is the direction of water flow for a bacterial cell living in a hypotonic environment? How do cell walls help bacteria
living in such environments?

38) Why is it important to measure the transmission of light through a control tube with only broth in it when making turbidity
measures of bacterial cultures?

39) In terms of counting cells, what does a plating method accomplish that an electronic cell counting method does not?

40) Order the following stages of the development of a biofilm from the earliest to the last step.
1. secretion of EPS
2. reversible attachment
3. dispersal
4. formation of water channels
5. irreversible attachment

41) Infections among hospitalized patients are often related to the presence of a medical device in the patient. Which conditions
favor the formation of biofilms on in-dwelling catheters and prostheses?

42) Why are some obligate anaerobes able to grow in tissues (e.g., gum pockets) that are not completely free of oxygen?

43) Why should Haemophilus influenzae be grown in a candle jar?

44) In terms of oxygen requirements, what type of organism would most likely be responsible for a foodborne illness associated
with canned foods?

45) Which macromolecule in the cell is most sensitive to changes in pH?

46) Which metabolic process in the bacterial cell is particularly challenging at high pH?

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47) How are hyperthermophile’s proteins adapted to the high temperatures of their environment?

48) Why would NASA be funding microbiology research in Antarctica?

49) Fish sauce is a salty condiment produced using fermentation. What type of organism is likely responsible for the fermentation
of the fish sauce?

50) Compare commensalism and amensalism.

Critical Thinking

51) A patient in the hospital has an intravenous catheter inserted to allow for the delivery of medications, fluids, and electrolytes.
Four days after the catheter is inserted, the patient develops a fever and an infection in the skin around the catheter. Blood cultures
reveal that the patient has a blood-borne infection. Tests in the clinical laboratory identify the blood-borne pathogen
as Staphylococcus epidermidis, and antibiotic susceptibility tests are performed to provide doctors with essential information for
selecting the best drug for treatment of the infection. Antibacterial chemotherapy is initiated and delivered through the intravenous
catheter that was originally inserted into the patient. Within 7 days, the skin infection is gone, blood cultures are negative for S.
epidermidis, and the antibacterial chemotherapy is discontinued. However, 2 days after discontinuing the antibacterial
chemotherapy, the patient develops another fever and skin infection and the blood cultures are positive for the same strain of S.
epidermidis that had been isolated the previous week. This time, doctors remove the intravenous catheter and administer oral
antibiotics, which successfully treat both the skin and blood-borne infection caused by S. epidermidis. Furthermore, the infection
does not return after discontinuing the oral antibacterial chemotherapy. What are some possible reasons why intravenous
chemotherapy failed to completely cure the patient despite laboratory tests showing the bacterial strain was susceptible to the
prescribed antibiotic? Why might the second round of antibiotic therapy have been more successful? Justify your answers.

52) Why are autoinducers small molecules?

53) If the results from a pond water sample were recorded as 3, 2, 1, what would be the MPN of bacteria in 100 mL of pond water?

54) Why does turbidity lose reliability at high cell concentrations when the culture reaches the stationary phase?

55) A microbiology instructor prepares cultures for a gram-staining practical laboratory by inoculating growth medium with a
gram-positive coccus (nonmotile) and a gram-negative rod (motile). The goal is to demonstrate staining of a mixed culture. The
flask is incubated at 35 °C for 24 hours without aeration. A sample is stained and reveals only gram-negative rods. Both cultures
are known facultative anaerobes. Give a likely reason for success of the gram-negative rod. Assume that the cultures have
comparable intrinsic growth rates.

56) People who use proton pumps inhibitors or antacids are more prone to infections of the gastrointestinal tract. Can you explain
the observation in light of what you have learned?

57) The bacterium that causes Hansen’s disease (leprosy), Mycobacterium leprae, infects mostly the extremities of the body: hands,
feet, and nose. Can you make an educated guess as to its optimum temperature of growth?

58) Some hyperthermophiles can survive autoclaving temperatures. Are they a concern in health care?

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59) Haemophilus, influenzae grows best at 35–37 °C with ~5% CO2 (or in a candle-jar) and requires hemin (X factor) and
nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for growth. Using the vocabulary learned in this chapter,
describe H. influenzae.1

Footnotes
1. Centers for Disease Control and Prevention, World Health Organization. “CDC Laboratory Methods for the Diagnosis of
Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenza. WHO Manual, 2nd edition.”
2011. http://www.cdc.gov/meningitis/lab-ma...ull-manual.pdf)

Chapter 8 Exercises is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

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 CHAPTER OVERVIEW

9: Acellular Pathogens
9.1: Viruses
9.2: The Viral Cycles
9.3: Isolation, Culture, and Identification of Viruses
9.4: Viroids, Virusoids, and Prions
Chapter 9 Exercises

Thumbnail: This colorized transmission electron microscopic (TEM) image revealed some of the ultrastructural morphology
displayed by an Ebola virus virion. (Public Domain; Frederick A. Murphy via CDC).

This page titled 9: Acellular Pathogens is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

1
9.1: Viruses
Learning Objectives
Describe the general characteristics of viruses as pathogens
Describe viral genomes
Describe the general characteristics of viral life cycles
Differentiate among bacteriophages, plant viruses, and animal viruses
Describe the characteristics used to identify viruses as obligate intracellular parasites

Clinical Focus - part 1

David, a 45-year-old journalist, has just returned to the U.S. from travels in Russia, China, and Africa. He is not feeling well,
so he goes to his general practitioner complaining of weakness in his arms and legs, fever, headache, noticeable agitation, and
minor discomfort. He thinks it may be related to a dog bite he suffered while interviewing a Chinese farmer. He is experiencing
some prickling and itching sensations at the site of the bite wound, but he tells the doctor that the dog seemed healthy and that
he had not been concerned until now. The doctor ordered a culture and sensitivity test to rule out bacterial infection of the
wound, and the results came back negative for any possible pathogenic bacteria.

Exercise 9.1.1
1. Based on this information, what additional tests should be performed on the patient?
2. What type of treatment should the doctor recommend?

Despite their small size, which prevented them from being seen with light microscopes, the discovery of a filterable component
smaller than a bacterium that causes tobacco mosaic disease (TMD) dates back to 1892.1 At that time, Dmitri Ivanovski, a Russian
botanist, discovered the source of TMD by using a porcelain filtering device first invented by Charles Chamberland and Louis
Pasteur in Paris in 1884. Porcelain Chamberland filters have a pore size of 0.1 µm, which is small enough to remove all bacteria
≥0.2 µm from any liquids passed through the device. An extract obtained from TMD-infected tobacco plants was made to
determine the cause of the disease. Initially, the source of the disease was thought to be bacterial. It was surprising to everyone
when Ivanovski, using a Chamberland filter, found that the cause of TMD was not removed after passing the extract through the
porcelain filter. So if a bacterium was not the cause of TMD, what could be causing the disease? Ivanovski concluded the cause of
TMD must be an extremely small bacterium or bacterial spore. Other scientists, including Martinus Beijerinck, continued
investigating the cause of TMD. It was Beijerinck, in 1899, who eventually concluded the causative agent was not a bacterium but,
instead, possibly a chemical, like a biological poison we would describe today as a toxin. As a result, the word virus, Latin for
poison, was used to describe the cause of TMD a few years after Ivanovski’s initial discovery. Even though he was not able to see
the virus that caused TMD, and did not realize the cause was not a bacterium, Ivanovski is credited as the original discoverer of
viruses and a founder of the field of virology.
Today, we can see viruses using electron microscopes (Figure 9.1.1) and we know much more about them. Viruses are distinct
biological entities; however, their evolutionary origin is still a matter of speculation. In terms of taxonomy, they are not included in
the tree of life because they are acellular (not consisting of cells). In order to survive and reproduce, viruses must infect a cellular
host, making them obligate intracellular parasites. The genome of a virus enters a host cell and directs the production of the viral
components, proteins and nucleic acids, needed to form new virus particles called virions. New virions are made in the host cell by
assembly of viral components. The new virions transport the viral genome to another host cell to carry out another round of
infection. Table 9.1.1 summarizes the properties of viruses.

Table 9.1.1 : Properties of viruses.

Characteristics of Viruses

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 Characteristics of Viruses

Infectious, acellular pathogens

Obligate intracellular parasites with host and cell-type specificity
DNA or RNA genome (never both)
Genome is surrounded by a protein capsid and, in some cases, a phospholipid membrane studded with viral glycoproteins
Lack genes for many products needed for successful reproduction, requiring exploitation of host-cell genomes to reproduce

Figure 9.1.1 : (a) Tobacco mosaic virus (TMV) viewed with transmission electron microscope. (b) Plants infected with tobacco
mosaic disease (TMD), caused by TMV. (credit a: modification of work by USDA Agricultural Research Service—scale-bar data
from Matt Russell; credit b: modification of work by USDA Forest Service, Department of Plant Pathology Archive North Carolina
State University)

Exercise 9.1.2

Why was the first virus investigated mistaken for a toxin?

Hosts and Viral Transmission

Various viruses have been known infect every type of host cell, including those of plants, animals, fungi, protists, bacteria, and
archaea. Each type of virus will only be able to infect the cells of one or a few species of living organism. This is called the host
range. However, having a wide host range is not common and viruses will typically only infect specific hosts and only specific cell
types within those hosts. The viruses that infect bacteria are called bacteriophages, or simply phages. The word phage comes from
the Greek word for devour. Other viruses are just identified by their host group, such as animal or plant viruses. Once a cell is
infected, the effects of the virus can vary depending on the type of virus. Viruses may cause abnormal growth of the cell or cell
death, alter the cell’s genome, or cause little noticeable effect in the cell.
Viruses can be transmitted through direct contact, indirect contact with fomites (inanimate objects such as handles), or through a
vector: an animal that transmits a pathogen from one host to another. Arthropods such as mosquitoes, ticks, and flies, are typical
vectors for viral diseases, and they may act as mechanical vectors or biological vectors. Mechanical transmission occurs when the
arthropod carries a viral pathogen on the outside of its body and transmits it to a new host by physical contact. Biological
transmission occurs when the arthropod carries the viral pathogen inside its body and transmits it to the new host through biting or
similar behavior.
In humans, a wide variety of viruses are capable of causing various infections and diseases. Some of the deadliest emerging
pathogens in humans are viruses, yet we have few treatments or drugs to deal with viral infections, making them difficult to
eradicate. Viruses that can be transmitted from an animal host to a human host can cause zoonoses. For example, the avian
influenza virus originates in birds, but can cause disease in humans. Reverse zoonoses are caused by infection of an animal by a
virus that originated in a human.

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 Fighting Bacteria with Viruses
The emergence of superbugs, or multidrug resistant bacteria, has become a major challenge for pharmaceutical companies and
a serious health-care problem. According to a 2013 report by the US Centers for Disease Control and Prevention (CDC), more
than 2 million people are infected with drug-resistant bacteria in the US annually, resulting in at least 23,000 deaths.2 The
continued use and overuse of antibiotics will likely lead to the evolution of even more drug-resistant strains.
One potential solution is the use of phage therapy, a procedure that uses bacteria-killing viruses (bacteriophages) to treat
bacterial infections. Phage therapy is not a new idea. The discovery of bacteriophages dates back to the early 20th century, and
phage therapy was first used in Europe in 1915 by the English bacteriologist Frederick Twort.3 However, the subsequent
discovery of penicillin and other antibiotics led to the near abandonment of this form of therapy, except in the former Soviet
Union and a few countries in Eastern Europe. Interest in phage therapy outside of the countries of the former Soviet Union is
only recently re-emerging because of the rise in antibiotic-resistant bacteria.4
Phage therapy has some advantages over antibiotics in that phages kill only one specific bacterium, whereas antibiotics kill not
only the pathogen but also beneficial bacteria of the normal microbiota. Development of new antibiotics is also expensive for
drug companies and for patients, especially for those who live in countries with high poverty rates.
Phages have also been used to prevent food spoilage. In 2006, the US Food and Drug Administration approved the use of a
solution containing six bacteriophages that can be sprayed on lunch meats such as bologna, ham, and turkey to kill Listeria
monocytogenes, a bacterium responsible for listeriosis, a form of food poisoning. Some consumers have concerns about the use
of phages on foods, however, especially given the rising popularity of organic products. Foods that have been treated with
phages must declare “bacteriophage preparation” in the list of ingredients or include a label declaring that the meat has been
“treated with antimicrobial solution to reduce microorganisms.”5

Exercise 9.1.3

1. Why do humans not have to be concerned about the presence of bacteriophages in their food?
2. What are three ways that viruses can be transmitted between hosts?

Viral Structures
In general, virions (viral particles) are small and cannot be observed using a regular light microscope. They are much smaller than
prokaryotic and eukaryotic cells; this is an adaptation allowing viruses to infect these larger cells (see Figure 9.1.2). The size of a
virion can range from 20 nm for small viruses up to 900 nm for typical, large viruses (see Figure 9.1.3). Recent discoveries,
however, have identified new giant viral species, such as Pandoravirus salinus and Pithovirus sibericum, with sizes approaching
that of a bacterial cell.6

Figure 9.1.2 : (a) In this transmission electron micrograph, a bacteriophage (a virus that infects bacteria) is dwarfed by the bacterial
cell it infects. (b) An illustration of the bacteriophage in the micrograph. (credit a: modification of work by U.S. Department of
Energy, Office of Science, LBL, PBD)

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In 1935, after the development of the electron microscope, Wendell Stanley was the first scientist to crystallize the structure of the
tobacco mosaic virus and discovered that it is composed of RNA and protein. In 1943, he isolated Influenza B virus, which
contributed to the development of an influenza (flu) vaccine. Stanley’s discoveries unlocked the mystery of the nature of viruses
that had been puzzling scientists for over 40 years and his contributions to the field of virology led to him being awarded the Nobel
Prize in 1946.

Figure 9.1.3 :The size of a virus is small relative to the size of most bacterial and eukaryotic cells and their organelles.
As a result of continuing research into the nature of viruses, we now know they consist of a nucleic acid (either RNA or DNA, but
never both) surrounded by a protein coat called a capsid (see Figure 9.1.4). The interior of the capsid is not filled with cytosol, as
in a cell, but instead it contains the bare necessities in terms of genome and enzymes needed to direct the synthesis of new virions.
Each capsid is composed of protein subunits called capsomeres made of one or more different types of capsomere proteins that
interlock to form the closely packed capsid.
There are two categories of viruses based on general composition. Viruses formed from only a nucleic acid and capsid are called
naked viruses or nonenveloped viruses. Viruses formed with a nucleic-acid packed capsid surrounded by a lipid layer are called
enveloped viruses (see Figure 9.1.4). The viral envelope is a small portion of phospholipid membrane obtained as the virion buds
from a host cell. The viral envelope may either be intracellular or cytoplasmic in origin.
Extending outward and away from the capsid on naked viruses and enveloped viruses are protein structures called spikes. At the
tips of these spikes are structures that allow the virus to attach and enter a cell, like the influenza virus hemagglutinin spikes (H) or
enzymes like the neuraminidase (N) influenza virus spikes that allow the virus to detach from the cell surface during release of new
virions. Influenza viruses are often identified by their H and N spikes. For example, H1N1 influenza viruses were responsible for
the pandemics in 1918 and 2009,7 H2N2 for the pandemic in 1957, and H3N2 for the pandemic in 1968.

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 Figure 9.1.4 : (a) The naked atadenovirus uses spikes made of glycoproteins from its capsid to bind to host cells. (b) The enveloped
human immunodeficiency virus uses spikes made of glycoproteins embedded in its envelope to bind to host cells (credit a
“micrograph”: modification of work by NIAID; credit b “micrograph”: modification of work by Centers for Disease Control and
Prevention)
Viruses vary in the shape of their capsids, which can be either helical, polyhedral, or complex. A helical capsid forms the shape of
tobacco mosaic virus (TMV), a naked helical virus, and Ebola virus, an enveloped helical virus. The capsid is cylindrical or rod
shaped, with the genome fitting just inside the length of the capsid. Polyhedral capsids form the shapes of poliovirus and
rhinovirus, and consist of a nucleic acid surrounded by a polyhedral (many-sided) capsid in the form of an icosahedron. An
icosahedral capsid is a three-dimensional, 20-sided structure with 12 vertices. These capsids somewhat resemble a soccer ball. Both
helical and polyhedral viruses can have envelopes. Viral shapes seen in certain types of bacteriophages, such as T4 phage, and
poxviruses, like vaccinia virus, may have features of both polyhedral and helical viruses so they are described as a complex viral
shape (see Figure 9.1.5). In the bacteriophage complex form, the genome is located within the polyhedral head and the sheath
connects the head to the tail fibers and tail pins that help the virus attach to receptors on the host cell’s surface. Poxviruses that have
complex shapes are often brick shaped, with intricate surface characteristics not seen in the other categories of capsid.

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 Figure 9.1.5 : Viral capsids can be (a) helical, (b) polyhedral, or (c) have a complex shape. (credit a “micrograph”: modification of
work by USDA ARS; credit b “micrograph”: modification of work by U.S. Department of Energy)

Exercise 9.1.4

Which types of viruses have spikes?

Classification and Taxonomy of Viruses

Although viruses are not classified in the three domains of life, their numbers are great enough to require classification. Since 1971,
the International Union of Microbiological Societies Virology Division has given the task of developing, refining, and maintaining
a universal virus taxonomy to the International Committee on Taxonomy of Viruses (ICTV). Since viruses can mutate so quickly, it
can be difficult to classify them into a genus and a species epithet using the binomial nomenclature system. Thus, the ICTV’s viral
nomenclature system classifies viruses into families and genera based on viral genetics, chemistry, morphology, and mechanism of
multiplication. To date, the ICTV has classified known viruses in seven orders, 96 families, and 350 genera. Viral family names
end in -viridae (e.g, Parvoviridae) and genus names end in −virus (e.g., Parvovirus). The names of viral orders, families, and
genera are all italicized. When referring to a viral species, we often use a genus and species epithet such as Pandoravirus dulcis or
Pandoravirus salinus. Explore the latest virus taxonomy at the ICTV website.
The Baltimore classification system is an alternative to ICTV nomenclature. The Baltimore system classifies viruses according to
their genomes (DNA or RNA, single versus double stranded) and mode of replication. This system thus creates seven groups of
viruses that have common genetics and biology.

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Aside from formal systems of nomenclature, viruses are often informally grouped into categories based on chemistry, morphology,
or other characteristics they share in common. Categories may include naked or enveloped structure, single-stranded (ss) or double-
stranded (ds) DNA or ss or ds RNA genomes, segmented or nonsegmented genomes, and positive-strand (+) or negative-strand (−)
RNA. For example, herpes viruses can be classified as a dsDNA enveloped virus; human immunodeficiency virus (HIV) is a
+ssRNA enveloped virus, and tobacco mosaic virus is a +ssRNA virus. Other characteristics such as host specificity, tissue
specificity, capsid shape, and special genes or enzymes may also be used to describe groups of similar viruses. Table 9.1.2 lists
some of the most common viruses that are human pathogens by genome type.
Table 9.1.2 : Common Pathogenic Viruses
Genome Family Example Virus Clinical Features

Poxviridae Orthopoxvirus Skin papules, pustules, lesions

Poxviridae Parapoxvirus Skin lesions

dsDNA, enveloped
Cold sores, genital herpes,
Herpesviridae Simplexvirus
sexually transmitted disease
Respiratory infection (common
Adenoviridae Atadenovirus
cold)
Genital warts, cervical, vulvar, or
dsDNA, naked Papillomaviridae Papillomavirus
vaginal cancer
Gastroenteritis severe diarrhea
Reoviridae Reovirus
(stomach flu)

Adeno-associated
Parvoviridae Respiratory tract infection
dependoparvovirus A
ssDNA, naked
Adeno-associated
Parvoviridae Respiratory tract infection
dependoparvovirus B

dsRNA, naked Reoviridae Rotavirus Gastroenteritis

Picornaviridae Enterovirus C Poliomyelitis

Upper respiratory tract infection

+ssRNA, naked Picornaviridae Rhinovirus
(common cold)

Picornaviridae Hepatovirus Hepatitis

Togaviridae Alphavirus Encephalitis, hemorrhagic fever

Togaviridae Rubivirus Rubella

+ssRNA, enveloped
Acquired immune deficiency
Retroviridae Lentivirus
syndrome (AIDS)

Filoviridae Zaire Ebolavirus Hemorrhagic fever

−ssRNA, enveloped Orthomyxoviridae Influenzavirus A, B, C Flu

Rhabdoviridae Lyssavirus Rabies

Exercise 9.1.5

What are the types of virus genomes?

Classification of Viral Diseases

While the ICTV has been tasked with the biological classification of viruses, it has also played an important role in the
classification of diseases caused by viruses. To facilitate the tracking of virus-related human diseases, the ICTV has created
classifications that link to the International Classification of Diseases (ICD), the standard taxonomy of disease that is maintained
and updated by the World Health Organization (WHO). The ICD assigns an alphanumeric code of up to six characters to every type
of viral infection, as well as all other types of diseases, medical conditions, and causes of death. This ICD code is used in

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conjunction with two other coding systems (the Current Procedural Terminology, and the Healthcare Common Procedure Coding
System) to categorize patient conditions for treatment and insurance reimbursement.
For example, when a patient seeks treatment for a viral infection, ICD codes are routinely used by clinicians to order laboratory
tests and prescribe treatments specific to the virus suspected of causing the illness. This ICD code is then used by medical
laboratories to identify tests that must be performed to confirm the diagnosis. The ICD code is used by the health-care management
system to verify that all treatments and laboratory work performed are appropriate for the given virus. Medical coders use ICD
codes to assign the proper code for procedures performed, and medical billers, in turn, use this information to process claims for
reimbursement by insurance companies. Vital-records keepers use ICD codes to record cause of death on death certificates, and
epidemiologists used ICD codes to calculate morbidity and mortality statistics.

Exercise 9.1.6

Identify two locations where you would likely find an ICD code.

Summary
Viruses are generally ultramicroscopic, typically from 20 nm to 900 nm in length. Some large viruses have been found.
Virions are acellular and consist of a nucleic acid, DNA or RNA, but not both, surrounded by a protein capsid. There may also
be a phospholipid membrane surrounding the capsid.
Viruses are obligate intracellular parasites.
Viruses are known to infect various types of cells found in plants, animals, fungi, protists, bacteria, and archaea. Viruses
typically have limited host ranges and infect specific cell types.
Viruses may have helical, polyhedral, or complex shapes.
Classification of viruses is based on morphology, type of nucleic acid, host range, cell specificity, and enzymes carried within
the virion.
Like other diseases, viral diseases are classified using ICD codes.

Footnotes
1. H. Lecoq. “[Discovery of the First Virus, the Tobacco Mosaic Virus: 1892 or 1898?].” Comptes Rendus de l’Academie des
Sciences – Serie III – Sciences de la Vie 324, no. 10 (2001): 929–933.
2. US Department of Health and Human Services, Centers for Disease Control and Prevention. “Antibiotic Resistance Threats in
the United States, 2013.” www.cdc.gov/drugresistance/pd...s-2013-508.pdf (accessed September 22, 2015).
3. M. Clokie et al. “Phages in Nature.” Bacteriophage 1, no. 1 (2011): 31–45.
4. A. Sulakvelidze et al. “Bacteriophage Therapy.” Antimicrobial Agents and Chemotherapy 45, no. 3 (2001): 649–659.
5. US Food and Drug Administration. “FDA Approval of Listeria-specific Bacteriophage Preparation on Ready-to-Eat (RTE)
Meat and Poultry Products.” www.fda.gov/food/ingredientsp.../ucm083572.htm (accessed September 22, 2015).
6. N. Philippe et al. “Pandoraviruses: Amoeba Viruses with Genomes up to 2.5 Mb Reaching that of Parasitic Eukaryotes.”
Science 341, no. 6143 (2013): 281–286.
7. J. Cohen. “What’s Old Is New: 1918 Virus Matches 2009 H1N1 Strain. Science 327, no. 5973 (2010): 1563–1564.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 9.1: Viruses is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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9.2: The Viral Cycles
Learning Objectives
Describe the lytic and lysogenic life cycles
Describe the replication process of animal viruses
Describe unique characteristics of retroviruses and latent viruses
Discuss human viruses and their virus-host cell interactions
Explain the process of transduction
Describe the replication process of plant viruses

All viruses depend on cells for reproduction and metabolic processes. By themselves, viruses do not encode for all of the enzymes
necessary for viral replication. But within a host cell, a virus can commandeer cellular machinery to produce more viral particles.
Bacteriophages (no matter their type of nucleic acid) replicate only in the cytoplasm, since prokaryotic cells do not have a nucleus
or organelles. In eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed in the large DNA
viruses, such as the poxviruses, that can replicate in the cytoplasm. RNA viruses that infect animal cells often replicate in the
cytoplasm.

The Life Cycle of Viruses with Prokaryote Hosts

The life cycle of bacteriophages has been a good model for understanding how viruses affect the cells they infect, since similar
processes have been observed for eukaryotic viruses, which can cause immediate death of the cell or establish a latent or chronic
infection. Virulent phages typically lead to the death of the cell through cell lysis. Temperate phages, on the other hand, can
become part of a host chromosome and are replicated with the cell genome until such time as they are induced to make newly
assembled viruses, or progeny viruses.

The Lytic Cycle

During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new phages, and destroys the cell. T-even
phage is a good example of a well-characterized class of virulent phages. There are five stages in the bacteriophage lytic cycle (see
Figure 9.2.1). Attachment is the first stage in the infection process in which the phage interacts with specific bacterial surface
receptors (e.g., lipopolysaccharides and OmpC protein on host surfaces). Most phages have a narrow host range and may infect one
species of bacteria or one strain within a species. This unique recognition can be exploited for targeted treatment of bacterial
infection by phage therapy or for phage typing to identify unique bacterial subspecies or strains. The second stage of infection is
entry or penetration. This occurs through contraction of the tail sheath, which acts like a hypodermic needle to inject the viral
genome through the cell wall and membrane. The phage head and remaining components remain outside the bacteria.

Figure 9.2.1 : A virulent phage shows only the lytic cycle pictured here. In the lytic cycle, the phage replicates and lyses the host
cell.
The third stage of infection is biosynthesis of new viral components. After entering the host cell, the virus synthesizes virus-
encoded endonucleases to degrade the bacterial chromosome. It then hijacks the host cell to replicate, transcribe, and translate the
necessary viral components (capsomeres, sheath, base plates, tail fibers, and viral enzymes) for the assembly of new viruses.

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Polymerase genes are usually expressed early in the cycle, while capsid and tail proteins are expressed later. During the maturation
phase, new virions are created. To liberate free phages, the bacterial cell wall is disrupted by phage proteins such as holin or
lysozyme. The final stage is release. Mature viruses burst out of the host cell in a process called lysis and the progeny viruses are
liberated into the environment to infect new cells.

The Lysogenic Cycle

In a lysogenic cycle, a temperate phage genome also enters the cell through attachment and penetration. A prime example of a
phage with this type of life cycle is the lambda phage. During the lysogenic cycle, instead of killing the host, the phage genome
integrates into the bacterial chromosome and becomes part of the host. The integrated phage genome is called a prophage. A
bacterial host with a prophage is called a lysogen. The process in which a bacterium is infected by a temperate phage is called
lysogeny. It is typical of temperate phages to be latent or inactive within the cell. As the bacterium replicates its chromosome, it
also replicates the phage’s DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter
the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase bacterial virulence). This
change in the host phenotype is called lysogenic conversion or phage conversion. Some bacteria, such as Vibrio cholerae and
Clostridium botulinum, are less virulent in the absence of the prophage. The phages infecting these bacteria carry the toxin genes in
their genome and enhance the virulence of the host when the toxin genes are expressed. In the case of V. cholera, phage encoded
toxin can cause severe diarrhea; in C. botulinum, the toxin can cause paralysis. During lysogeny, the prophage will persist in the
host chromosome until induction, which results in the excision of the viral genome from the host chromosome. After induction has
occurred the temperate phage can proceed through a lytic cycle and then undergo lysogeny in a newly infected cell (see Figure
9.2.2).

Figure 9.2.2 : A temperate bacteriophage has both lytic and lysogenic cycles. In the lysogenic cycle, phage DNA is incorporated
into the host genome, forming a prophage, which is passed on to subsequent generations of cells. Environmental stressors such as
starvation or exposure to toxic chemicals may cause the prophage to be excised and enter the lytic cycle.
This video illustrates the stages of the lysogenic life cycle of a bacteriophage and the transition to a lytic phase.

Exercise 9.2.1

Is a latent phage undetectable in a bacterium?

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Transduction
Transduction occurs when a bacteriophage transfers bacterial DNA from one bacterium to another during sequential infections.
There are two types of transduction: generalized and specialized transduction. During the lytic cycle of viral replication, the virus
hijacks the host cell, degrades the host chromosome, and makes more viral genomes. As it assembles and packages DNA into the
phage head, packaging occasionally makes a mistake. Instead of packaging viral DNA, it takes a random piece of host DNA and
inserts it into the capsid. Once released, this virion will then inject the former host’s DNA into a newly infected host. The asexual
transfer of genetic information can allow for DNA recombination to occur, thus providing the new host with new genes (e.g., an
antibiotic-resistance gene, or a sugar-metabolizing gene). Generalized transduction occurs when a random piece of bacterial
chromosomal DNA is transferred by the phage during the lytic cycle. Specialized transduction occurs at the end of the lysogenic
cycle, when the prophage is excised and the bacteriophage enters the lytic cycle. Since the phage is integrated into the host genome,
the prophage can replicate as part of the host. However, some conditions (e.g., ultraviolet light exposure or chemical exposure)
stimulate the prophage to undergo induction, causing the phage to excise from the genome, enter the lytic cycle, and produce new
phages to leave host cells. During the process of excision from the host chromosome, a phage may occasionally remove some
bacterial DNA near the site of viral integration. The phage and host DNA from one end or both ends of the integration site are
packaged within the capsid and are transferred to the new, infected host. Since the DNA transferred by the phage is not randomly
packaged but is instead a specific piece of DNA near the site of integration, this mechanism of gene transfer is referred to as
specialized transduction (see Figure 9.2.3). The DNA can then recombine with host chromosome, giving the latter new
characteristics. Transduction seems to play an important role in the evolutionary process of bacteria, giving them a mechanism for
asexual exchange of genetic information.

Figure 9.2.3 : This flowchart illustrates the mechanism of specialized transduction. An integrated phage excises, bringing with it a
piece of the DNA adjacent to its insertion point. On reinfection of a new bacterium, the phage DNA integrates along with the
genetic material acquired from the previous host.

Exercise 9.2.2

Which phage life cycle is associated with which forms of transduction?

Clinical Focus - part 2

David’s doctor was concerned that his symptoms included prickling and itching at the site of the dog bite; these sensations
could be early symptoms of rabies. Several tests are available to diagnose rabies in live patients, but no single antemortem test
is adequate. The doctor decided to take samples of David’s blood, saliva, and skin for testing. The skin sample was taken from
the nape of the neck (posterior side of the neck near the hairline). It was about 6-mm long and contained at least 10 hair
follicles, including the superficial cutaneous nerve. An immunofluorescent staining technique was used on the skin biopsy
specimen to detect rabies antibodies in the cutaneous nerves at the base of the hair follicles. A test was also performed on a
serum sample from David’s blood to determine whether any antibodies for the rabies virus had been produced.

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 Meanwhile, the saliva sample was used for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, a test that can
detect the presence of viral nucleic acid (RNA). The blood tests came back positive for the presence of rabies virus antigen,
prompting David’s doctor to prescribe prophylactic treatment. David is given a series of intramuscular injections of human
rabies immunoglobulin along with a series of rabies vaccines.

Exercise 9.2.3

1. Why does the immunofluorescent technique look for rabies antibodies rather than the rabies virus itself?
2. If David has contracted rabies, what is his prognosis?

Life Cycle of Viruses with Animal Hosts

Lytic animal viruses follow similar infection stages to bacteriophages: attachment, penetration, biosynthesis, maturation, and
release (see Figure 9.2.4). However, the mechanisms of penetration, nucleic-acid biosynthesis, and release differ between bacterial
and animal viruses. After binding to host receptors, animal viruses enter through endocytosis (engulfment by the host cell) or
through membrane fusion (viral envelope with the host cell membrane). As with phages, animal viruses are host specific, meaning
they only infect a certain type of host; and most viruses only infect certain types of cells or tissues. This specificity is called a tissue
tropism. Examples of this are demonstrated by the poliovirus, which exhibits tropism for the tissues of the brain and spinal cord, or
the influenza virus, which has a primary tropism for the respiratory tract.

Figure 9.2.4 : In influenza virus infection, viral glycoproteins attach the virus to a host epithelial cell. As a result, the virus is
engulfed. Viral RNA and viral proteins are made and assembled into new virions that are released by budding.

Nucleic Acids in Viruses

Viruses do not always express their genes using the normal flow of genetic information—from DNA to RNA to protein. Some
viruses have a dsDNA genome like cellular organisms and can follow the normal flow. However, others may have ssDNA, dsRNA,

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or ssRNA genomes. The nature of the genome determines how the genome is replicated and expressed as viral proteins. If a
genome is ssDNA, host enzymes will be used to synthesize a second strand that is complementary to the genome strand, thus
producing dsDNA. The dsDNA can now be replicated, transcribed, and translated similar to host DNA.
If the viral genome is RNA, a different mechanism must be used. There are three types of RNA genome: dsRNA, positive (+)
single-strand (+ssRNA) or negative (−) single-strand RNA (−ssRNA). If a virus has a +ssRNA genome, it can be translated directly
to make viral proteins. Viral genomic +ssRNA acts like cellular mRNA. However, if a virus contains a −ssRNA genome, the host
ribosomes cannot translate it until the −ssRNA is replicated into +ssRNA by viral RNA-dependent RNA polymerase (RdRP) (see
Figure 9.2.5). The RdRP is brought in by the virus and can be used to make +ssRNA from the original −ssRNA genome. The
RdRP is also an important enzyme for the replication of dsRNA viruses, because it uses the negative strand of the double-stranded
genome as a template to create +ssRNA. The newly synthesized +ssRNA copies can then be translated by cellular ribosomes.

Figure 9.2.5 : RNA viruses can contain +ssRNA that can be directly read by the ribosomes to synthesize viral proteins. Viruses
containing −ssRNA must first use the −ssRNA as a template for the synthesis of +ssRNA before viral proteins can be synthesized.
An alternative mechanism for viral nucleic acid synthesis is observed in the retroviruses, which are +ssRNA viruses (see Figure
9.2.6). Single-stranded RNA viruses such as HIV carry a special enzyme called reverse transcriptase within the capsid that

synthesizes a complementary ssDNA (cDNA) copy using the +ssRNA genome as a template. The ssDNA is then made into
dsDNA, which can integrate into the host chromosome and become a permanent part of the host. In eukaryote viruses, the
integrated viral genome is called a provirus. The virus now can remain in the host for a long time to establish a chronic infection.
The provirus stage is very similar to the prophage stage in a bacterial infection during the lysogenic cycle. However, unlike
prophage, the provirus does not undergo excision after splicing into the genome.

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Figure 9.2.6 : HIV, an enveloped, icosahedral retrovirus, attaches to a cell surface receptor of an immune cell and fuses with the cell
membrane. Viral contents are released into the cell, where viral enzymes convert the single-stranded RNA genome into DNA and
incorporate it into the host genome. (credit: modification of work by NIAID, NIH)

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 Exercise 9.2.4

Is RNA-dependent RNA polymerase made from a viral gene or a host gene?

Persistent Infections
Persistent infection occurs when a virus is not completely cleared from the system of the host but stays in certain tissues or organs
of the infected person. The virus may remain silent or undergo productive infection without seriously harming or killing the host.
Mechanisms of persistent infection may involve the regulation of the viral or host gene expressions or the alteration of the host
immune response. The two primary categories of persistent infections are latent infection and chronic infection. Examples of
viruses that cause latent infections include herpes simplex virus (oral and genital herpes), varicella-zoster virus (chickenpox and
shingles), and Epstein-Barr virus (mononucleosis). Hepatitis C virus and HIV are two examples of viruses that cause long-term
chronic infections.

Latent Infection
Not all animal viruses undergo replication by the lytic cycle. There are viruses that are capable of remaining hidden or dormant
inside the cell in a process called latency. These types of viruses are known as latent viruses and may cause latent infections.
Viruses capable of latency may initially cause an acute infection before becoming dormant. Latent viruses may remain dormant by
existing as circular viral genome molecules outside of the host chromosome. Others become proviruses by integrating into the host
genome. During dormancy, viruses do not cause any symptoms of disease and may be difficult to detect. A patient may be unaware
that he or she is carrying the virus unless a viral diagnostic test has been performed.
For example, the varicella-zoster virus infects many cells throughout the body and causes chickenpox, characterized by a rash of
blisters covering the skin. About 10 to 12 days postinfection, the disease resolves and the virus goes dormant, living within nerve-
cell ganglia for years. During this time, the virus does not kill the nerve cells nor continue replicating itself. It is not clear why the
virus stops replicating within the nerve cells and expresses few viral proteins but, in some cases, typically after many years of
dormancy, the virus is reactivated and causes a new disease called shingles (Figure 9.2.7). Whereas chickenpox affects many areas
throughout the body, shingles is a nerve cell-specific disease emerging from the ganglia in which the virus was dormant.

Figure 9.2.7 : (a) Varicella-zoster, the virus that causes chickenpox, has an enveloped icosahedral capsid visible in this transmission
electron micrograph. Its double-stranded DNA genome becomes incorporated in the host DNA. (b) After a period of latency, the
virus can reactivate in the form of shingles, usually manifesting as a painful, localized rash on one side of the body. (credit a:
modification of work by Erskine Palmer and B.G. Partin—scale-bar data from Matt Russell; credit b: modification of work by
Rosmarie Voegtli)

Chronic Infection
A chronic infection is a disease with symptoms that are recurrent or persistent over a long time. Some viral infections can be
chronic if the body is unable to eliminate the virus. HIV is an example of a virus that produces a chronic infection, often after a
long period of latency. Once a person becomes infected with HIV, the virus can be detected in tissues continuously thereafter, but
untreated patients often experience no symptoms for years. However, the virus maintains chronic persistence through several
mechanisms that interfere with immune function, including preventing expression of viral antigens on the surface of infected cells,
altering immune cells themselves, restricting expression of viral genes, and rapidly changing viral antigens through mutation.

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Eventually, the damage to the immune system results in progression of the disease leading to acquired immunodeficiency syndrome
(AIDS). The various mechanisms that HIV uses to avoid being cleared by the immune system are also used by other chronically
infecting viruses, including the hepatitis C virus.

Exercise 9.2.5

In what two ways can a virus manage to maintain a persistent infection?

Life Cycle of Viruses with Plant Hosts

Plant viruses are more similar to animal viruses than they are to bacteriophages. Plant viruses may be enveloped or non-enveloped.
Like many animal viruses, plant viruses can have either a DNA or RNA genome and be single stranded or double stranded.
However, most plant viruses do not have a DNA genome; the majority have a +ssRNA genome, which acts like messenger RNA
(mRNA). Only a minority of plant viruses have other types of genomes.
Plant viruses may have a narrow or broad host range. For example, the citrus tristeza virus infects only a few plants of the Citrus
genus, whereas the cucumber mosaic virus infects thousands of plants of various plant families. Most plant viruses are transmitted
by contact between plants, or by fungi, nematodes, insects, or other arthropods that act as mechanical vectors. However, some
viruses can only be transferred by a specific type of insect vector; for example, a particular virus might be transmitted by aphids but
not whiteflies. In some cases, viruses may also enter healthy plants through wounds, as might occur due to pruning or weather
damage.
Viruses that infect plants are mainly considered biotrophic parasites, which means that they can establish an infection without
killing the host, similar to what is observed in the lysogenic life cycles of bacteriophages. Viral infection can be asymptomatic
(latent) or can lead to cell death (lytic infection). The life cycle begins with the penetration of the virus into the host cell. Next, the
virus is uncoated within the cytoplasm of the cell when the capsid is removed. Depending on the type of nucleic acid, cellular
components are used to replicate the viral genome and synthesize viral proteins for assembly of new virions. To establish a
systemic infection, the virus must enter a part of the vascular system of the plant, such as the phloem. The time required for
systemic infection may vary from a few days to a few weeks depending on the virus, the plant species, and the environmental
conditions. The virus life cycle is complete when it is transmitted from an infected plant to a healthy plant.

Exercise 9.2.6

What is the structure and genome of a typical plant virus?

Viral Growth Curve

Unlike the growth curve for a bacterial population, the growth curve for a virus population over its life cycle does not follow a
sigmoidal curve. During the initial stage, an inoculum of virus causes infection. In the eclipse phase, viruses bind and penetrate the
cells with no virions detected in the medium. The chief difference that next appears in the viral growth curve compared to a
bacterial growth curve occurs when virions are released from the lysed host cell at the same time. Such an occurrence is called a
burst, and the number of virions per bacterium released is described as the burst size. In a one-step multiplication curve for
bacteriophage, the host cells lyse, releasing many viral particles to the medium, which leads to a very steep rise in viral titer (the
number of virions per unit volume). If no viable host cells remain, the viral particles begin to degrade during the decline of the
culture (see Figure 9.2.8).

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 Figure 9.2.8 : The one-step multiplication curve for a bacteriophage population follows three steps: 1) inoculation, during which the
virions attach to host cells; 2) eclipse, during which entry of the viral genome occurs; and 3) burst, when sufficient numbers of new
virions are produced and emerge from the host cell. The burst size is the maximum number of virions produced per bacterium.

Exercise 9.2.7

What aspect of the life cycle of a virus leads to the sudden increase in the growth curve?

Unregistered Treatments
Ebola is incurable and deadly. The outbreak in West Africa in 2014 was unprecedented, dwarfing other human Ebola
epidemics in the level of mortality. Of 24,666 suspected or confirmed cases reported, 10,179 people died. 1
No approved treatments or vaccines for Ebola are available. While some drugs have shown potential in laboratory studies and
animal models, they have not been tested in humans for safety and effectiveness. Not only are these drugs untested or
unregistered but they are also in short supply.
Given the great suffering and high mortality rates, it is fair to ask whether unregistered and untested medications are better than
none at all. Should such drugs be dispensed and, if so, who should receive them, in light of their extremely limited supplies? Is
it ethical to treat untested drugs on patients with Ebola? On the other hand, is it ethical to withhold potentially life-saving drugs
from dying patients? Or should the drugs perhaps be reserved for health-care providers working to contain the disease?
In August 2014, two infected US aid workers and a Spanish priest were treated with ZMapp, an unregistered drug that had
been tested in monkeys but not in humans. The two American aid workers recovered, but the priest died. Later that month, the
WHO released a report on the ethics of treating patients with the drug. Since Ebola is often fatal, the panel reasoned that it is
ethical to give the unregistered drugs and unethical to withhold them for safety concerns. This situation is an example of
“compassionate use” outside the well-established system of regulation and governance of therapies.

Ebola in the US
On September 24, 2014, Thomas Eric Duncan arrived at the Texas Health Presbyterian Hospital in Dallas complaining of a
fever, headache, vomiting, and diarrhea—symptoms commonly observed in patients with the cold or the flu. After
examination, an emergency department doctor diagnosed him with sinusitis, prescribed some antibiotics, and sent him home.
Two days later, Duncan returned to the hospital by ambulance. His condition had deteriorated and additional blood tests
confirmed that he has been infected with the Ebola virus.

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 Further investigations revealed that Duncan had just returned from Liberia, one of the countries in the midst of a severe Ebola
epidemic. On September 15, nine days before he showed up at the hospital in Dallas, Duncan had helped transport an Ebola-
stricken neighbor to a hospital in Liberia. The hospital continued to treat Duncan, but he died several days after being admitted.
The timeline of the Duncan case is indicative of the life cycle of the Ebola virus. The incubation time for Ebola ranges from 2
days to 21 days. Nine days passed between Duncan’s exposure to the virus infection and the appearance of his symptoms. This
corresponds, in part, to the eclipse period in the growth of the virus population. During the eclipse phase, Duncan would have
been unable to transmit the disease to others. However, once an infected individual begins exhibiting symptoms, the disease
becomes very contagious. Ebola virus is transmitted through direct contact with droplets of bodily fluids such as saliva, blood,
and vomit. Duncan could conceivably have transmitted the disease to others at any time after he began having symptoms,
presumably some time before his arrival at the hospital in Dallas. Once a hospital realizes a patient like Duncan is infected with
Ebola virus, the patient is immediately quarantined, and public health officials initiate a back trace to identify everyone with
whom a patient like Duncan might have interacted during the period in which he was showing symptoms.
Public health officials were able to track down 10 high-risk individuals (family members of Duncan) and 50 low-risk
individuals to monitor them for signs of infection. None contracted the disease. However, one of the nurses charged with
Duncan’s care did become infected. This, along with Duncan’s initial misdiagnosis, made it clear that US hospitals needed to
provide additional training to medical personnel to prevent a possible Ebola outbreak in the US.

Exercise 9.2.8

1. What types of training can prepare health professionals to contain emerging epidemics like the Ebola outbreak of
2014?
2. What is the difference between a contagious pathogen and an infectious pathogen?

Figure 9.2.9 : Researchers working with Ebola virus use layers of defenses against accidental infection, including protective
clothing, breathing systems, and negative air-pressure cabinets for bench work. (credit: modification of work by Randal J.
Schoepp)

For additional information about Ebola, please visit the CDC website.

Summary
Many viruses target specific hosts or tissues. Some may have more than one host.
Many viruses follow several stages to infect host cells. These stages include attachment, penetration, uncoating,
biosynthesis, maturation, and release.
Bacteriophages have a lytic or lysogenic cycle. The lytic cycle leads to the death of the host, whereas the lysogenic cycle leads
to integration of phage into the host genome.
Bacteriophages inject DNA into the host cell, whereas animal viruses enter by endocytosis or membrane fusion.
Animal viruses can undergo latency, similar to lysogeny for a bacteriophage.
The majority of plant viruses are positive-strand ssRNA and can undergo latency, chronic, or lytic infection, as observed for
animal viruses.

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 The growth curve of bacteriophage populations is a one-step multiplication curve and not a sigmoidal curve, as compared to
the bacterial growth curve.
Bacteriophages transfer genetic information between hosts using either generalized or specialized transduction.

Footnotes
1. World Health Organization. “WHO Ebola Data and Statistics.” March 18, 2005. http://apps.who.int/gho/data/view.eb...150318?
lang=en

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 9.2: The Viral Cycles is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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9.3: Isolation, Culture, and Identification of Viruses
Learning Objectives
Discuss why viruses were originally described as filterable agents
Describe the cultivation of viruses and specimen collection and handling
Compare in vivo and in vitro techniques used to cultivate viruses

At the beginning of this chapter, we described how porcelain Chamberland filters with pores small enough to allow viruses to pass
through were used to discover TMV. Today, porcelain filters have been replaced with membrane filters and other devices used to
isolate and identify viruses.

Isolation of Viruses
Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication.
Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source
of virus. Virions in the liquid medium can be separated from the host cells by either centrifugation or filtration. Filters can
physically remove anything present in the solution that is larger than the virions; the viruses can then be collected in the filtrate
(Figure 9.3.1).

Figure 9.3.1 : Membrane filters can be used to remove cells or viruses from a solution. (a) This scanning electron micrograph
shows rod-shaped bacterial cells captured on the surface of a membrane filter. Note differences in the comparative size of the
membrane pores and bacteria. Viruses will pass through this filter. (b) The size of the pores in the filter determines what is captured
on the surface of the filter (animal [red] and bacteria [blue]) and removed from liquid passing through. Note the viruses (green)
pass through the finer filter. (credit a: modification of work by U.S. Department of Energy)

Exercise 9.3.1

What size filter pore is needed to collect a virus?

Cultivation of Viruses
Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an
artificial environment, such as a test tube, cell culture flask, or agar plate). Bacteriophages can be grown in the presence of a dense
layer of bacteria (also called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask (Figure 9.3.2a).
The agar concentration is decreased from the 1.5% usually used in culturing bacteria. The soft 0.7% agar allows the bacteriophages
to easily diffuse through the medium. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a
clear zone called a plaque is detected (Figure 9.3.1b). As the phage kills the bacteria, many plaques are observed among the cloudy
bacterial lawn.

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 Figure 9.3.2 : (a) Flasks like this may be used to culture human or animal cells for viral culturing. (b) These plates contain
bacteriophage T4 grown on an Escherichia coli lawn. Clear plaques are visible where host bacterial cells have been lysed. Viral
titers increase on the plates to the left. (credit a: modification of work by National Institutes of Health; credit b: modification of
work by American Society for Microbiology)

Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus cultivation is
important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2) production of vaccines, and 3) basic
research studies. In vivo host sources can be a developing embryo in an embryonated bird’s egg (e.g., chicken, turkey) or a whole
animal. For example, most of the influenza vaccine manufactured for annual flu vaccination programs is cultured in hens’ eggs.
The embryo or host animal serves as an incubator for viral replication (Figure 9.3.3). Location within the embryo or host animal is
important. Many viruses have a tissue tropism, and must therefore be introduced into a specific site for growth. Within an embryo,
target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may damage tissue
membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.

Figure 9.3.3 : (a) The cells within chicken eggs are used to culture different types of viruses. (b) Viruses can be replicated in various
locations within the egg, including the chorioallantoic membrane, the amniotic cavity, and the yolk sac. (credit a: modification of
work by “Chung Hoang”/YouTube)
For in vitro studies, various types of cells can be used to support the growth of viruses. A primary cell culture is freshly prepared
from animal organs or tissues. Cells are extracted from tissues by mechanical scraping or mincing to release cells or by an
enzymatic method using trypsin or collagenase to break up tissue and release single cells into suspension. Because of anchorage-
dependence requirements, primary cell cultures require a liquid culture medium in a Petri dish or tissue-culture flask so cells have a
solid surface such as glass or plastic for attachment and growth. Primary cultures usually have a limited life span. When cells in a
primary culture undergo mitosis and a sufficient density of cells is produced, cells come in contact with other cells. When this cell-
to-cell-contact occurs, mitosis is triggered to stop. This is called contact inhibition and it prevents the density of the cells from
becoming too high. To prevent contact inhibition, cells from the primary cell culture must be transferred to another vessel with
fresh growth medium. This is called a secondary cell culture. Periodically, cell density must be reduced by pouring off some cells
and adding fresh medium to provide space and nutrients to maintain cell growth. In contrast to primary cell cultures, continuous
cell lines, usually derived from transformed cells or tumors, are often able to be subcultured many times or even grown indefinitely
(in which case they are called immortal). Continuous cell lines may not exhibit anchorage dependency (they will grow in
suspension) and may have lost their contact inhibition. As a result, continuous cell lines can grow in piles or lumps resembling
small tumor growths (Figure 9.3.4).

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 Figure 9.3.4 : Cells for culture are prepared by separating them from their tissue matrix. (a) Primary cell cultures grow attached to
the surface of the culture container. Contact inhibition slows the growth of the cells once they become too dense and begin touching
each other. At this point, growth can only be sustained by making a secondary culture. (b) Continuous cell cultures are not affected
by contact inhibition. They continue to grow regardless of cell density. (credit “micrographs”: modification of work by Centers for
Disease Control and Prevention)

An example of an immortal cell line is the HeLa cell line, which was originally cultivated from tumor cells obtained from Henrietta
Lacks, a patient who died of cervical cancer in 1951. HeLa cells were the first continuous tissue-culture cell line and were used to
establish tissue culture as an important technology for research in cell biology, virology, and medicine. Prior to the discovery of
HeLa cells, scientists were not able to establish tissue cultures with any reliability or stability. More than six decades later, this cell
line is still alive and being used for medical research. See below (The Immortal Cell Line of Henrietta Lacks) to read more about
this important cell line and the controversial means by which it was obtained.

Exercise 9.3.2

What property of cells makes periodic dilutions of primary cell cultures necessary?

The Immortal Cell Line of Henrietta Lacks

In January 1951, Henrietta Lacks, a 30-year-old African American woman from Baltimore, was diagnosed with cervical cancer
at John Hopkins Hospital. We now know her cancer was caused by the human papillomavirus (HPV). Cytopathic effects of the
virus altered the characteristics of her cells in a process called transformation, which gives the cells the ability to divide
continuously. This ability, of course, resulted in a cancerous tumor that eventually killed Mrs. Lacks in October at age 31.
Before her death, samples of her cancerous cells were taken without her knowledge or permission. The samples eventually
ended up in the possession of Dr. George Gey, a biomedical researcher at Johns Hopkins University. Gey was able to grow
some of the cells from Lacks’s sample, creating what is known today as the immortal HeLa cell line. These cells have the
ability to live and grow indefinitely and, even today, are still widely used in many areas of research.
According to Lacks’s husband, neither Henrietta nor the family gave the hospital permission to collect her tissue specimen.
Indeed, the family was not aware until 20 years after Lacks’s death that her cells were still alive and actively being used for
commercial and research purposes. Yet HeLa cells have been pivotal in numerous research discoveries related to polio, cancer,
and AIDS, among other diseases. The cells have also been commercialized, although they have never themselves been
patented. Despite this, Henrietta Lacks’s estate has never benefited from the use of the cells, although, in 2013, the Lacks
family was given control over the publication of the genetic sequence of her cells.
This case raises several bioethical issues surrounding patients’ informed consent and the right to know. At the time Lacks’s
tissues were taken, there were no laws or guidelines about informed consent. Does that mean she was treated fairly at the time?
Certainly by today’s standards, the answer would be no. Harvesting tissue or organs from a dying patient without consent is not

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 only considered unethical but illegal, regardless of whether such an act could save other patients’ lives. Is it ethical, then, for
scientists to continue to use Lacks’s tissues for research, even though they were obtained illegally by today’s standards?
Ethical or not, Lacks’s cells are widely used today for so many applications that it is impossible to list them all. Is this a case in
which the ends justify the means? Would Lacks be pleased to know about her contribution to science and the millions of people
who have benefited? Would she want her family to be compensated for the commercial products that have been developed
using her cells? Or would she feel violated and exploited by the researchers who took part of her body without her consent?
Because she was never asked, we will never know.

Figure 9.3.5 : A multiphoton fluorescence image of HeLa cells in culture. Various fluorescent stains have been used to show
the DNA (cyan), microtubules (green), and Golgi apparatus (orange). (credit: modification of work by National Institutes of
Health)

Detection of a Virus
Regardless of the method of cultivation, once a virus has been introduced into a whole host organism, embryo, or tissue-culture
cell, a sample can be prepared from the infected host, embryo, or cell line for further analysis under a brightfield, electron, or
fluorescent microscope. Cytopathic effects (CPEs) are distinct observable cell abnormalities due to viral infection. CPEs can
include loss of adherence to the surface of the container, changes in cell shape from flat to round, shrinkage of the nucleus,
vacuoles in the cytoplasm, fusion of cytoplasmic membranes and the formation of multinucleated syncytia, inclusion bodies in the
nucleus or cytoplasm, and complete cell lysis (see Figure 9.3.6).
Further pathological changes include viral disruption of the host genome and altering normal cells into transformed cells, which are
the types of cells associated with carcinomas and sarcomas. The type or severity of the CPE depends on the type of virus involved.
Figure 9.3.6 lists CPEs for specific viruses.

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 Figure 9.3.6 : (credit “micrographs”: modification of work by American Society for Microbiology)
Watch this video to learn about the effects of viruses on cells.

Hemagglutination Assay
A serological assay is used to detect the presence of certain types of viruses in patient serum. Serum is the straw-colored liquid
fraction of blood plasma from which clotting factors have been removed. Serum can be used in a direct assay called a
hemagglutination assay to detect specific types of viruses in the patient’s sample. Hemagglutination is the agglutination (clumping)
together of erythrocytes (red blood cells). Many viruses produce surface proteins or spikes called hemagglutinins that can bind to
receptors on the membranes of erythrocytes and cause the cells to agglutinate. Hemagglutination is observable without using the
microscope, but this method does not always differentiate between infectious and noninfectious viral particles, since both can
agglutinate erythrocytes.
To identify a specific pathogenic virus using hemagglutination, we must use an indirect approach. Proteins called antibodies,
generated by the patient’s immune system to fight a specific virus, can be used to bind to components such as hemagglutinins that
are uniquely associated with specific types of viruses. The binding of the antibodies with the hemagglutinins found on the virus

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subsequently prevent erythrocytes from directly interacting with the virus. So when erythrocytes are added to the antibody-coated
viruses, there is no appearance of agglutination; agglutination has been inhibited. We call these types of indirect assays for virus-
specific antibodies hemagglutination inhibition (HAI) assays. HAI can be used to detect the presence of antibodies specific to many
types of viruses that may be causing or have caused an infection in a patient even months or years after infection (see Figure 9.3.7).

Figure 9.3.7 : This chart shows the possible outcomes of a hemagglutination test. Row A: Erythrocytes do not bind together and
will sink to the bottom of the well plate; this becomes visible as a red dot in the center of the well. Row B: Many viruses have
hemagglutinins that causes agglutination of erythrocytes; the resulting hemagglutination forms a lattice structure that results in red
color throughout the well. Row C: Virus-specific antibody, the viruses, and the erythrocytes are added to the well plate. The virus-
specific antibodies inhibit agglutination, as can be seen as a red dot in the bottom of the well. (credit: modification of work by
Centers for Disease Control and Prevention)

Exercise 9.3.3

What is the outcome of a positive HIA test?

Nucleic Acid Amplification Test

Nucleic acid amplification tests (NAAT) are used in molecular biology to detect unique nucleic acid sequences of viruses in patient
samples. Polymerase chain reaction (PCR) is an NAAT used to detect the presence of viral DNA in a patient’s tissue or body fluid
sample. PCR is a technique that amplifies (i.e., synthesizes many copies) of a viral DNA segment of interest. Using PCR, short
nucleotide sequences called primers bind to specific sequences of viral DNA, enabling identification of the virus.
Reverse transcriptase-PCR (RT-PCR) is an NAAT used to detect the presence of RNA viruses. RT-PCR differs from PCR in that
the enzyme reverse transcriptase (RT) is used to make a cDNA from the small amount of viral RNA in the specimen. The cDNA
can then be amplified by PCR. Both PCR and RT-PCR are used to detect and confirm the presence of the viral nucleic acid in
patient specimens.

HPV Scare
Michelle, a 21-year-old nursing student, came to the university clinic worried that she might have been exposed to a sexually
transmitted disease (STD). Her sexual partner had recently developed several bumps on the base of his penis. He had put off
going to the doctor, but Michelle suspects they are genital warts caused by HPV. She is especially concerned because she
knows that HPV not only causes warts but is a prominent cause of cervical cancer. She and her partner always use condoms for
contraception, but she is not confident that this precaution will protect her from HPV.
Michelle’s physician finds no physical signs of genital warts or any other STDs, but recommends that Michelle get a Pap smear
along with an HPV test. The Pap smear will screen for abnormal cervical cells and the CPEs associated with HPV; the HPV
test will test for the presence of the virus. If both tests are negative, Michelle can be more assured that she most likely has not

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 become infected with HPV. However, her doctor suggests it might be wise for Michelle to get vaccinated against HPV to
protect herself from possible future exposure.

Exercise 9.3.4

Why does Michelle’s physician order two different tests instead of relying on one or the other?

Enzyme Immunoassay
Enzyme immunoassays (EIAs) rely on the ability of antibodies to detect and attach to specific biomolecules called antigens. The
detecting antibody attaches to the target antigen with a high degree of specificity in what might be a complex mixture of
biomolecules. Also included in this type of assay is a colorless enzyme attached to the detecting antibody. The enzyme acts as a tag
on the detecting antibody and can interact with a colorless substrate, leading to the production of a colored end product. EIAs often
rely on layers of antibodies to capture and react with antigens, all of which are attached to a membrane filter (see Figure 9.3.8).
EIAs for viral antigens are often used as preliminary screening tests. If the results are positive, further confirmation will require
tests with even greater sensitivity, such as a western blot or an NAAT.

Figure 9.3.8 : Similar to rapid, over-the-counter pregnancy tests, EIAs for viral antigens require a few drops of diluted patient
serum or plasma applied to a membrane filter. The membrane filter has been previously modified and embedded with antibody to
viral antigen and internal controls. Antibody conjugate is added to the filter, with the targeted antibody attached to the antigen (in
the case of a positive test). Excess conjugate is washed off the filter. Substrate is added to activate the enzyme-mediated reaction to
reveal the color change of a positive test. (credit: modification of work by “Cavitri”/Wikimedia Commons)

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 Exercise 9.3.5

What typically indicates a positive EIA test?

Clinical Focus: part 3

Along with the RT/PCR analysis, David’s saliva was also collected for viral cultivation. In general, no single diagnostic test is
sufficient for antemortem diagnosis, since the results will depend on the sensitivity of the assay, the quantity of virions present
at the time of testing, and the timing of the assay, since release of virions in the saliva can vary. As it turns out, the result was
negative for viral cultivation from the saliva. This is not surprising to David’s doctor, because one negative result is not an
absolute indication of the absence of infection. It may be that the number of virions in the saliva is low at the time of sampling.
It is not unusual to repeat the test at intervals to enhance the chance of detecting higher virus loads.

Exercise 9.3.6

Should David’s doctor modify his course of treatment based on these test results?

Summary
Viral cultivation requires the presence of some form of host cell (whole organism, embryo, or cell culture).
Viruses can be isolated from samples by filtration.
Viral filtrate is a rich source of released virions.
Bacteriophages are detected by presence of clear plaques on bacterial lawn.
Animal and plant viruses are detected by cytopathic effects, molecular techniques (PCR, RT-PCR), enzyme immunoassays, and
serological assays (hemagglutination assay, hemagglutination inhibition assay).

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 9.3: Isolation, Culture, and Identification of Viruses is shared under a CC BY license and was authored, remixed, and/or curated
by OpenStax.

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9.4: Viroids, Virusoids, and Prions
Learning Objectives
Describe viroids and their unique characteristics
Describe virusoids and their unique characteristics
Describe prions and their unique characteristics

Research attempts to discover the causative agents of previously uninvestigated diseases have led to the discovery of nonliving
disease agents quite different from viruses. These include particles consisting only of RNA or only of protein that, nonetheless, are
able to self-propagate at the expense of a host—a key similarity to viruses that allows them to cause disease conditions. To date,
these discoveries include viroids, virusoids, and the proteinaceous prions.

Viroids
In 1971, Theodor Diener, a pathologist working at the Agriculture Research Service, discovered an acellular particle that he named
a viroid, meaning “virus-like.” Viroids consist only of a short strand of circular RNA capable of self-replication. The first viroid
discovered was found to cause potato tuber spindle disease, which causes slower sprouting and various deformities in potato plants
(see Figure 9.4.1). Like viruses, potato spindle tuber viroids (PSTVs) take control of the host machinery to replicate their RNA
genome. Unlike viruses, viroids do not have a protein coat to protect their genetic information.

Figure 9.4.1 : These potatoes have been infected by the potato spindle tuber viroid (PSTV), which is typically spread when infected
knives are used to cut healthy potatoes, which are then planted. (credit: Pamela Roberts, University of Florida Institute of Food and
Agricultural Sciences, USDA ARS)
Viroids can result in devastating losses of commercially important agricultural food crops grown in fields and orchards. Since the
discovery of PSTV, other viroids have been discovered that cause diseases in plants. Tomato planta macho viroid (TPMVd) infects
tomato plants, which causes loss of chlorophyll, disfigured and brittle leaves, and very small tomatoes, resulting in loss of
productivity in this field crop. Avocado sunblotch viroid (ASBVd) results in lower yields and poorer-quality fruit. ASBVd is the
smallest viroid discovered thus far that infects plants. Peach latent mosaic viroid (PLMVd) can cause necrosis of flower buds and
branches, and wounding of ripened fruit, which leads to fungal and bacterial growth in the fruit. PLMVd can also cause similar
pathological changes in plums, nectarines, apricots, and cherries, resulting in decreased productivity in these orchards, as well.
Viroids, in general, can be dispersed mechanically during crop maintenance or harvesting, vegetative reproduction, and possibly via
seeds and insects, resulting in a severe drop in food availability and devastating economic consequences.

Exercise 9.4.1

What is the genome of a viroid made of?

Virusoids
A second type of pathogenic RNA that can infect commercially important agricultural crops are the virusoids, which are subviral
particles best described as non–self-replicating ssRNAs. RNA replication of virusoids is similar to that of viroids but, unlike

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viroids, virusoids require that the cell also be infected with a specific “helper” virus. There are currently only five described types
of virusoids and their associated helper viruses. The helper viruses are all from the family of Sobemoviruses. An example of a
helper virus is the subterranean clover mottle virus, which has an associated virusoid packaged inside the viral capsid. Once the
helper virus enters the host cell, the virusoids are released and can be found free in plant cell cytoplasm, where they possess
ribozyme activity. The helper virus undergoes typical viral replication independent of the activity of the virusoid. The virusoid
genomes are small, only 220 to 388 nucleotides long. A virusoid genome does not code for any proteins, but instead serves only to
replicate virusoid RNA.
Virusoids belong to a larger group of infectious agents called satellite RNAs, which are similar pathogenic RNAs found in animals.
Unlike the plant virusoids, satellite RNAs may encode for proteins; however, like plant virusoids, satellite RNAs must coinfect
with a helper virus to replicate. One satellite RNA that infects humans and that has been described by some scientists as a virusoid
is the hepatitis delta virus (HDV), which, by some reports, is also called hepatitis delta virusoid. Much larger than a plant virusoid,
HDV has a circular, ssRNA genome of 1,700 nucleotides and can direct the biosynthesis of HDV-associated proteins. The HDV
helper virus is the hepatitis B virus (HBV). Coinfection with HBV and HDV results in more severe pathological changes in the
liver during infection, which is how HDV was first discovered.

Exercise 9.4.2

What is the main difference between a viroid and a virusoid?

Prions
At one time, scientists believed that any infectious particle must contain DNA or RNA. Then, in 1982, Stanley Prusiner, a medical
doctor studying scrapie (a fatal, degenerative disease in sheep) discovered that the disease was caused by proteinaceous infectious
particles, or prions. Because proteins are acellular and do not contain DNA or RNA, Prusiner’s findings were originally met with
resistance and skepticism; however, his research was eventually validated, and he received the Nobel Prize in Physiology or
Medicine in 1997.

Figure 9.4.2 : Endogenous normal prion protein (PrPc) is converted into the disease-causing form (PrPsc) when it encounters this
variant form of the protein. PrPsc may arise spontaneously in brain tissue, especially if a mutant form of the protein is present, or it
may originate from misfolded prions consumed in food that eventually find their way into brain tissue. (credit b: modification of
work by USDA)
A prion is a misfolded rogue form of a normal protein (PrPc) found in the cell. This rogue prion protein (PrPsc), which may be
caused by a genetic mutation or occur spontaneously, can be infectious, stimulating other endogenous normal proteins to become
misfolded, forming plaques (see Figure 9.4.2). Today, prions are known to cause various forms of transmissible spongiform
encephalopathy (TSE) in human and animals. TSE is a rare degenerative disorder that affects the brain and nervous system. The
accumulation of rogue proteins causes the brain tissue to become sponge-like, killing brain cells and forming holes in the tissue,
leading to brain damage, loss of motor coordination, and dementia (see Figure 9.4.3). Infected individuals are mentally impaired
and become unable to move or speak. There is no cure, and the disease progresses rapidly, eventually leading to death within a few
months or years.

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 Figure 9.4.3 : Creutzfeldt-Jakob disease (CJD) is a fatal disease that causes degeneration of neural tissue. (a) These brain scans
compare a normal brain to one with CJD. (b) Compared to a normal brain, the brain tissue of a CJD patient is full of sponge-like
lesions, which result from abnormal formations of prion protein. (credit a (right): modification of work by Dr. Laughlin Dawes;
credit b (top): modification of work by Suzanne Wakim; credit b (bottom): modification of work by Centers for Disease Control
and Prevention)
TSEs in humans include kuru, fatal familial insomnia, Gerstmann-Straussler-Scheinker disease, and Creutzfeldt-Jakob disease (see
Figure 9.4.3). TSEs in animals include mad cow disease, scrapie (in sheep and goats), and chronic wasting disease (in elk and
deer). TSEs can be transmitted between animals and from animals to humans by eating contaminated meat or animal feed.
Transmission between humans can occur through heredity (as is often the case with GSS and CJD) or by contact with contaminated
tissue, as might occur during a blood transfusion or organ transplant. There is no evidence for transmission via casual contact with
an infected person. Table 9.4.1 lists TSEs that affect humans and their modes of transmission.
Prions are extremely difficult to destroy because they are resistant to heat, chemicals, and radiation. Even standard sterilization
procedures do not ensure the destruction of these particles. Currently, there is no treatment or cure for TSE disease, and
contaminated meats or infected animals must be handled according to federal guidelines to prevent transmission.
Table 9.4.1 : Transmissible Spongiform Encephalopathies (TSEs) in Humans
Disease Mechanism(s) of Transmission1

Not known; possibly by alteration of normal prior protein (PrP) to rogue

Sporadic CJD (sCJD)
form due to somatic mutation
Eating contaminated cattle products and by secondary bloodborne
Variant CJD (vCJD)
transmission

Familial CJD (fCJD) Mutation in germline PrP gene

Contaminated neurosurgical instruments, corneal graft, gonadotrophic

Iatrogenic CJD (iCJD)
hormone, and, secondarily, by blood transfusion

Kuru Eating infected meat through ritualistic cannibalism

Gerstmann-Straussler-Scheinker disease (GSS) Mutation in germline PrP gene

Fatal familial insomnia (FFI) Mutation in germline PrP gene

Exercise 9.4.3

Does a prion have a genome?

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For more information on the handling of animals and prion-contaminated materials, visit the guidelines published on the CDC and
WHO websites.

Clinical Focus: Resolution

A few days later, David’s doctor receives the results of the immunofluorescence test on his skin sample. The test is negative for
rabies antigen. A second viral antigen test on his saliva sample also comes back negative. Despite these results, the doctor
decides to continue David’s current course of treatment. Given the positive RT-PCR test, it is best not to rule out a possible
rabies infection.
Near the site of the bite, David receives an injection of rabies immunoglobulin, which attaches to and inactivates any rabies
virus that may be present in his tissues. Over the next 14 days, he receives a series of four rabies-specific vaccinations in the
arm. These vaccines activate David’s immune response and help his body recognize and fight the virus. Thankfully, with
treatment, David symptoms improve and he makes a full recovery.
Not all rabies cases have such a fortunate outcome. In fact, rabies is usually fatal once the patient starts to exhibit symptoms,
and postbite treatments are mainly palliative (i.e., sedation and pain management).

Summary
Other acellular agents such as viroids, virusoids, and prions also cause diseases. Viroids consist of small, naked ssRNAs that
cause diseases in plants. Virusoids are ssRNAs that require other helper viruses to establish an infection. Prions are
proteinaceous infectious particles that cause transmissible spongiform encephalopathies.
Prions are extremely resistant to chemicals, heat, and radiation.
There are no treatments for prion infection.

Footnotes
1. National Institute of Neurological Disorders and Stroke. “Creutzfeldt-Jakob Disease Fact Sheet.”
http://www.ninds.nih.gov/disorders/cjd/detail_cjd.htm (accessed December 31, 2015).

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

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Chapter 9 Exercises
Review Questions for Chapter 9

Multiple Choice
.
1) The component(s) of a virus that is/are extended from the envelope for attachment is/are the:
1. capsomeres
2. spikes
3. nucleic acid
4. viral whiskers

2) Which of the following does a virus lack? Select all that apply.
1. ribosomes
2. metabolic processes
3. nucleic acid
4. glycoprotein

3) The envelope of a virus is derived from the host’s

1. nucleic acids
2. membrane structures
3. cytoplasm
4. genome

4) In naming viruses, the family name ends with ________ and genus name ends with _________.
1. −virus; −viridae
2. −viridae; −virus
3. −virion; virus
4. −virus; virion

5) What is another name for a nonenveloped virus?

1. enveloped virus
2. provirus
3. naked virus
4. latent virus

6) Which of the following leads to the destruction of the host cells?

1. lysogenic cycle
2. lytic cycle
3. prophage
4. temperate phage

7) A virus obtains its envelope during which of the following phases?

1. attachment

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 2. penetration
3. assembly
4. release

8) Which of the following components is brought into a cell by HIV?

1. a DNA-dependent DNA polymerase
2. RNA polymerase
3. ribosome
4. reverse transcriptase

9) A positive-strand RNA virus:

1. must first be converted to a mRNA before it can be translated.
2. can be used directly to translate viral proteins.
3. will be degraded by host enzymes.
4. is not recognized by host ribosomes.

10) What is the name for the transfer of genetic information from one bacterium to another bacterium by a phage?
1. transduction
2. penetration
3. excision
4. translation

11) Which of the followings cannot be used to culture viruses?

1. tissue culture
2. liquid medium only
3. embryo
4. animal host

12) Which of the following tests can be used to detect the presence of a specific virus?
1. EIA
2. RT-PCR
3. PCR
4. all of the above

13) Which of the following is NOT a cytopathic effect?

1. transformation
2. cell fusion
3. mononucleated cell
4. inclusion bodies

14) Which of these infectious agents do not have nucleic acid?

1. viroids
2. viruses
3. bacteria
4. prions

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15) Which of the following is true of prions?
1. They can be inactivated by boiling at 100 °C.
2. They contain a capsid.
3. They are a rogue form of protein, PrP.
4. They can be reliably inactivated by an autoclave.

Fill-in-the-Blanks

16) A virus that infects a bacterium is called a/an ___________________.

17) A/an __________ virus possesses characteristics of both a polyhedral and helical virus.

18) A virus containing only nucleic acid and a capsid is called a/an ___________________ virus or __________________ virus.

19) The ____________ _____________ on the bacteriophage allow for binding to the bacterial cell.

20) An enzyme from HIV that can make a copy of DNA from RNA is called _______________________.

21) For lytic viruses, _________________ is a phase during a viral growth curve when the virus is not detected.

22) Viruses can be diagnosed and observed using a(n) _____________ microscope.

23) Cell abnormalities resulting from a viral infection are called ____________ _____________.

24) Both viroids and virusoids have a(n) _________ genome, but virusoids require a(n) _________ to reproduce.

Short Answers

25) Discuss the geometric differences among helical, polyhedral, and complex viruses.

26) What was the meaning of the word “virus” in the 1880s and why was it used to describe the cause of tobacco mosaic disease?

27) Briefly explain the difference between the mechanism of entry of a T-even bacteriophage and an animal virus.

28) Discuss the difference between generalized and specialized transduction.

29) Differentiate between lytic and lysogenic cycles.

30) Briefly explain the various methods of culturing viruses.

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31) Describe the disease symptoms observed in animals infected with prions.

Critical Thinking

. 32) Name each labeled part of the illustrated bacteriophage.

33) In terms of evolution, which do you think arises first? The virus or the host? Explain your answer.

34) Do you think it is possible to create a virus in the lab? Imagine that you are a mad scientist. Describe how you would go about
creating a new virus.

35) Label the five stages of a bacteriophage infection in the figure:

36) Bacteriophages have lytic and lysogenic cycles. Discuss the advantages and disadvantages for the phage.

37) How does reverse transcriptase aid a retrovirus in establishing a chronic infection?

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38) Discuss some methods by which plant viruses are transmitted from a diseased plant to a healthy one.

39) Label the components indicated by arrows.

Figure 6.27 (credit: modification of work by American Society for Microbiology)

40) What are some characteristics of the viruses that are similar to a computer virus?

41) Does a prion replicate? Explain.

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 CHAPTER OVERVIEW

10: Modern Applications of Microbial Genetics

10.1: Microbes and the Tools of Genetic Engineering
10.2: Visualizing and Characterizing DNA
10.3: Whole Genome Methods and Industrial Applications
10.4: Genetic Engineering - Risks, Benefits, and Perceptions
Chapter 10 Exercises

Thumbnail: A group of Genetically modified GloFish fluorescent fish. (www.glofish.com).

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OpenStax.

1
10.1: Microbes and the Tools of Genetic Engineering
Learning Objectives
Identify tools of molecular genetics that are derived from microorganisms
Describe the methods used to create recombinant DNA molecules
Describe methods used to introduce DNA into prokaryotic cells
List the types of genomic libraries and describe their uses
Describe the methods used to introduce DNA into eukaryotic cells

Clinical Focus- part 1

Kayla, a 24-year-old electrical engineer and running enthusiast, just moved from Arizona to New Hampshire to take
a new job. On her weekends off, she loves to explore her new surroundings, going for long runs in the pine forests.
In July she spent a week hiking through the mountains. In early August, Kayla developed a low fever, headache,
and mild muscle aches, and she felt a bit fatigued. Not thinking much of it, she took some ibuprofen to combat her
symptoms and vowed to get more rest.

Exercise 10.1.1

What types of medical conditions might be responsible for Kayla’s symptoms?

The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of
plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we
associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of
genetic engineering. Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence
is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The
recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host
organism is now considered to be transgenic.
One example of a transgenic microorganism is the bacterial strain that produces human insulin (Figure 10.1.1). The insulin gene
from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these
transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and
incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction),
and taking up DNA from the environment (transformation). Recall that these mechanisms are examples of horizontal gene transfer
—the transfer of genetic material between cells of the same generation.

Figure 10.1.1 : Recombinant DNA technology is the artificial recombination of DNA from two organisms. In this example, the
human insulin gene is inserted into a bacterial plasmid. This recombinant plasmid can then be used to transform bacteria, which
gain the ability to produce the insulin protein.

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Molecular Cloning
Herbert Boyer and Stanley Cohen first demonstrated the complete molecular cloning process in 1973 when they successfully
cloned genes from the African clawed frog (Xenopus laevis) into a bacterial plasmid that was then introduced into the bacterial host
Escherichia coli. Molecular cloning is a set of methods used to construct recombinant DNA and incorporate it into a host organism;
it makes use of a number of molecular tools that are derived from microorganisms.

Restriction Enzymes and Ligases

In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria
and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases
(restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is
most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in the 1960s
from studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting
DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts
DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in
length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a
palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ
direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical
positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends)
while others cut without generating such overhangs, instead producing blunt ends (Figure 10.1.2).
Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their
sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together
of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the
lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-
phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg,
a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey
virus with E. coli bacteriophage lambda to create a hybrid.

Figure 10.1.2 : (a) In this six-nucleotide restriction enzyme site, recognized by the enzyme BamHI, notice that the sequence reads
the same in the 5ʹ to 3ʹ direction on both strands. This is known as a palindrome. The cutting of the DNA by the restriction enzyme
at the sites (indicated by the black arrows) produces DNA fragments with sticky ends. Another piece of DNA cut with the same
restriction enzyme could attach to one of these sticky ends, forming a recombinant DNA molecule. (b) This four-nucleotide
recognition site also exhibits a palindromic sequence. The cutting of the DNA by the restriction enzyme HaeIII at the indicated
sites produces DNA fragments with blunt ends. Any other piece of blunt DNA could attach to one of the blunt ends produced,
forming a recombinant DNA molecule.

Plasmids
After restriction digestion, genes of interest are commonly inserted into plasmids, small pieces of typically circular, double-
stranded DNA that replicate independently of the bacterial chromosome. In recombinant DNA technology, plasmids are often used
as vectors, DNA molecules that carry DNA fragments from one organism to another. Plasmids used as vectors can be genetically

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engineered by researchers and scientific supply companies to have specialized properties, as illustrated by the commonly used
plasmid vector pUC19 (Figure 10.1.3). Some plasmid vectors contain genes that confer antibiotic resistance; these resistance genes
allow researchers to easily find plasmid-containing colonies by plating them on media containing the corresponding antibiotic. The
antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain the vector are able to survive and
grow.
Plasmid vectors used for cloning typically have a polylinker site, or multiple cloning site (MCS). A polylinker site is a short
sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after
restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the
polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different
restriction enzymes.
This polylinker site is often found within a reporter gene, another gene sequence artificially engineered into the plasmid that
encodes a protein that allows for visualization of DNA insertion. The reporter gene allows a researcher to distinguish host cells that
contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector.
The most common reporter gene used in plasmid vectors is the bacterial lacZ gene encoding beta-galactosidase, an enzyme that
naturally degrades lactose but can also degrade a colorless synthetic analog X-gal, thereby producing blue colonies on X-gal–
containing media. The lacZ reporter gene is disabled when the recombinant DNA is spliced into the plasmid. Because the LacZ
protein is not produced when the gene is disabled, X-gal is not degraded and white colonies are produced, which can then be
isolated. This blue-white screening method is described later and shown in Figure 10.1.4. In addition to these features, some
plasmids come pre-digested and with an enzyme linked to the linearized plasmid to aid in ligation after the insertion of foreign
DNA fragments.

Figure 10.1.3 : The artificially constructed plasmid vector pUC19 is commonly used for cloning foreign DNA. Arrows indicate the
directions in which the genes are transcribed. Note the polylinker site, containing multiple unique restriction enzyme recognition
sites, found within the lacZ reporter gene. Also note the ampicillin (amp) resistance gene encoded on the plasmid.

Molecular Cloning using Transformation

The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is transformation, a process in which
bacteria take up free DNA from their surroundings. In nature, free DNA typically comes from other lysed bacterial cells; in the
laboratory, free DNA in the form of recombinant plasmids is introduced to the cell’s surroundings.
Some bacteria, such as Bacillus spp., are naturally competent, meaning they are able to take up foreign DNA. However, not all
bacteria are naturally competent. In most cases, bacteria must be made artificially competent in the laboratory by increasing the
permeability of the cell membrane. This can be achieved through chemical treatments that neutralize charges on the cell membrane
or by exposing the bacteria to an electric field that creates microscopic pores in the cell membrane. These methods yield chemically
competent or electrocompetent bacteria, respectively.
Following the transformation protocol, bacterial cells are plated onto an antibiotic-containing medium to inhibit the growth of the
many host cells that were not transformed by the plasmid conferring antibiotic resistance. A technique called blue-white screening

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is then used for lacZ-encoding plasmid vectors such as pUC19. Blue colonies have a functional beta-galactosidase enzyme because
the lacZ gene is uninterrupted, with no foreign DNA inserted into the polylinker site. These colonies typically result from the
digested, linearized plasmid religating to itself. However, white colonies lack a functional beta-galactosidase enzyme, indicating
the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene. Thus, white colonies
resulting from this blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign
DNA. To be sure the correct DNA was incorporated into the plasmid, the DNA insert can then be sequenced.

Figure 10.1.4 : The steps involved in molecular cloning using bacterial transformation are outlined in this graphic flowchart.
View an animation of molecular cloning from the DNA Learning Center.

Molecular Cloning Using Conjugation or Transduction

The bacterial process of conjugation can also be manipulated for molecular cloning. F plasmids, or fertility plasmids, are
transferred between bacterial cells through the process of conjugation. Recombinant DNA can be transferred by conjugation when
bacterial cells containing a recombinant F plasmid are mixed with compatible bacterial cells lacking the plasmid. F plasmids
encode a surface structure called an F pilus that facilitates contact between a cell containing an F plasmid and one without an F
plasmid. On contact, a cytoplasmic bridge forms between the two cells and the F-plasmid-containing cell replicates its plasmid,
transferring a copy of the recombinant F plasmid to the recipient cell. Once it has received the recombinant F plasmid, the recipient
cell can produce its own F pilus and facilitate transfer of the recombinant F plasmid to an additional cell. The use of conjugation to
transfer recombinant F plasmids to recipient cells is another effective way to introduce recombinant DNA molecules into host cells.
Alternatively, bacteriophages can be used to introduce recombinant DNA into host bacterial cells through a manipulation of the

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transduction process. In the laboratory, DNA fragments of interest can be engineered into phagemids, which are plasmids that have
phage sequences that allow them to be packaged into bacteriophages. Bacterial cells can then be infected with these bacteriophages
so that the recombinant phagemids can be introduced into the bacterial cells. Depending on the type of phage, the recombinant
DNA may be integrated into the host bacterial genome (lysogeny), or it may exist as a plasmid in the host’s cytoplasm.

Exercise 10.1.2

1. What is the original function of a restriction enzyme?

2. What two processes are exploited to get recombinant DNA into a bacterial host cell?
3. Distinguish the uses of an antibiotic resistance gene and a reporter gene in a plasmid vector.

Creating a Genomic Library

Molecular cloning may also be used to generate a genomic library. The library is a complete (or nearly complete) copy of an
organism’s genome contained as recombinant DNA plasmids engineered into unique clones of bacteria. Having such a library
allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment. These fragments can
be used to determine the sequence of the DNA and the function of any genes present.
One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid
vectors cut with the same restriction enzyme (Figure 10.1.5). After transformation into a bacterial host, each transformed bacterial
cell takes up a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and
carry the same recombinant plasmid. The resulting library is a collection of colonies, each of which contains a fragment of the
original organism’s genome, that are each separate and distinct and can each be used for further study. This makes it possible for
researchers to screen these different clones to discover the one containing a gene of interest from the original organism’s genome.

Figure 10.1.5 : The generation of a genomic library facilitates the discovery of the genomic DNA fragment that contains a gene of
interest. (credit “micrograph”: modification of work by National Institutes of Health)
To construct a genomic library using larger fragments of genomic DNA, an E. coli bacteriophage, such as lambda, can be used as a
host (Figure 10.1.6). Genomic DNA can be sheared or enzymatically digested and ligated into a pre-digested bacteriophage lambda
DNA vector. Then, these recombinant phage DNA molecules can be packaged into phage particles and used to infect E. coli host
cells on a plate. During infection within each cell, each recombinant phage will make many copies of itself and lyse the E. coli
lawn, forming a plaque. Thus, each plaque from a phage library represents a unique recombinant phage containing a distinct
genomic DNA fragment. Plaques can then be screened further to look for genes of interest. One advantage to producing a library
using phages instead of plasmids is that a phage particle holds a much larger insert of foreign DNA compared with a plasmid
vector, thus requiring a much smaller number of cultures to fully represent the entire genome of the original organism.

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 Figure 10.1.6 : Recombinant phage DNA molecules are made by ligating digested phage particles with fragmented genomic DNA
molecules. These recombinant phage DNA molecules are packaged into phage particles and allowed to infect a bacterial lawn.
Each plaque represents a unique recombinant DNA molecule that can be further screened for genes of interest.
To focus on the expressed genes in an organism or even a tissue, researchers construct libraries using the organism’s messenger
RNA (mRNA) rather than its genomic DNA. Whereas all cells in a single organism will have the same genomic DNA, different
tissues express different genes, producing different complements of mRNA. For example, all human cells’ genomic DNA contains
the gene for insulin, but only cells in the pancreas express mRNA directing the production of insulin. Because mRNA cannot be
cloned directly, in the laboratory mRNA must be used as a template by the retroviral enzyme reverse transcriptase to make
complementary DNA (cDNA). A cell’s full complement of mRNA can be reverse-transcribed into cDNA molecules, which can be
used as a template for DNA polymerase to make double-stranded DNA copies; these fragments can subsequently be ligated into
either plasmid vectors or bacteriophage to produce a cDNA library. The benefit of a cDNA library is that it contains DNA from
only the expressed genes in the cell. This means that the introns, control sequences such as promoters, and DNA not destined to be
translated into proteins are not represented in the library. The focus on translated sequences means that the library cannot be used to
study the sequence and structure of the genome in its entirety. The construction of a cDNA genomic library is shown in Figure
10.1.7.

Figure 10.1.7 : Complementary DNA (cDNA) is made from mRNA by the retroviral enzyme reverse transcriptase, converted into
double-stranded copies, and inserted into either plasmid vectors or bacteriophage, producing a cDNA library. (credit “micrograph”:
modification of work by National Institutes of Health)

Exercise 10.1.3
1. What are the hosts for the genomic libraries described?
2. What is cDNA?

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Introducing Recombinant Molecules into Eukaryotic Hosts
The use of bacterial hosts for genetic engineering laid the foundation for recombinant DNA technology; however, researchers have
also had great interest in genetically engineering eukaryotic cells, particularly those of plants and animals. The introduction of
recombinant DNA molecules into eukaryotic hosts is called transfection. Genetically engineered plants, called transgenic plants,
are of significant interest for agricultural and pharmaceutical purposes. The first transgenic plant sold commercially was the Flavr
Savr delayed-ripening tomato, which came to market in 1994. Genetically engineered livestock have also been successfully
produced, resulting, for example, in pigs with increased nutritional value1 and goats that secrete pharmaceutical products in their
milk.2

Electroporation
Compared to bacterial cells, eukaryotic cells tend to be less amenable as hosts for recombinant DNA molecules. Because
eukaryotes are typically neither competent to take up foreign DNA nor able to maintain plasmids, transfection of eukaryotic hosts
is far more challenging and requires more intrusive techniques for success. One method used for transfecting cells in cell culture is
called electroporation. A brief electric pulse induces the formation of transient pores in the phospholipid bilayers of cells through
which the gene can be introduced. At the same time, the electric pulse generates a short-lived positive charge on one side of the
cell’s interior and a negative charge on the opposite side; the charge difference draws negatively charged DNA molecules into the
cell (Figure 10.1.8).

Figure 10.1.8 : Electroporation is one laboratory technique used to introduce DNA into eukaryotic cells.

Microinjection
An alternative method of transfection is called microinjection. Because eukaryotic cells are typically larger than those of
prokaryotes, DNA fragments can sometimes be directly injected into the cytoplasm using a glass micropipette, as shown in Figure
10.1.9.

Figure 10.1.9 : Microinjection is another technique for introducing DNA into eukaryotic cells. A microinjection needle containing
recombinant DNA is able to penetrate both the cell membrane and nuclear envelope.

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Gene Guns
Transfecting plant cells can be even more difficult than animal cells because of their thick cell walls. One approach involves
treating plant cells with enzymes to remove their cell walls, producing protoplasts. Then, a gene gun is used to shoot gold or
tungsten particles coated with recombinant DNA molecules into the plant protoplasts at high speeds. Recipient protoplast cells can
then recover and be used to generate new transgenic plants (Figure 10.1.10).

Figure 10.1.10: Heavy-metal particles coated with recombinant DNA are shot into plant protoplasts using a gene gun. The resulting
transformed cells are allowed to recover and can be used to generate recombinant plants. (a) A schematic of a gene gun. (b) A
photograph of a gene gun. (credit a, b: modification of work by JA O'Brien, SC Lummis)

Shuttle Vectors
Another method of transfecting plants involves shuttle vectors, plasmids that can move between bacterial and eukaryotic cells. The
tumor-inducing (Ti) plasmids originating from the bacterium Agrobacterium tumefaciens are commonly used as shuttle vectors for
incorporating genes into plants (Figure 10.1.11). In nature, the Ti plasmids of A. tumefaciens cause plants to develop tumors when
they are transferred from bacterial cells to plant cells. Researchers have been able to manipulate these naturally occurring plasmids
to remove their tumor-causing genes and insert desirable DNA fragments. The resulting recombinant Ti plasmids can be transferred
into the plant genome through the natural transfer of Ti plasmids from the bacterium to the plant host. Once inside the plant host
cell, the gene of interest recombines into the plant cell’s genome.

Figure 10.1.11: The Ti plasmid of Agrobacterium tumefaciens is a useful shuttle vector for the uptake of genes of interest into plant
cells. The gene of interest is cloned into the Ti plasmid, which is then introduced into plant cells. The gene of interest then
recombines into the plant cell’s genome, allowing for the production of transgenic plants.

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Viral Vectors
Viral vectors can also be used to transfect eukaryotic cells. In fact, this method is often used in gene therapy to introduce healthy
genes into human patients suffering from diseases that result from genetic mutations. Viral genes can be deleted and replaced with
the gene to be delivered to the patient;3 the virus then infects the host cell and delivers the foreign DNA into the genome of the
targeted cell. Adenoviruses are often used for this purpose because they can be grown to high titer and can infect both nondividing
and dividing host cells. However, use of viral vectors for gene therapy can pose some risks for patients.

Exercise 10.1.4
1. What are the methods used to introduce recombinant DNA vectors into animal cells?
2. Compare and contrast shuttle vectors and viral vectors.

Key Concepts and Summary

Biotechology is the science of utilizing living systems to benefit humankind. In recent years, the ability to directly alter an
organism’s genome through genetic engineering has been made possible due to advances in recombinant DNA technology,
which allows researchers to create recombinant DNA molecules with new combinations of genetic material.
Molecular cloning involves methods used to construct recombinant DNA and facilitate their replication in host organisms.
These methods include the use of restriction enzymes (to cut both foreign DNA and plasmid vectors), ligation (to paste
fragments of DNA together), and the introduction of recombinant DNA into a host organism (often bacteria).
Blue-white screening allows selection of bacterial transformants that contain recombinant plasmids using the phenotype of a
reporter gene that is disabled by insertion of the DNA fragment.
Genomic libraries can be made by cloning genomic fragments from one organism into plasmid vectors or into bacteriophage.
cDNA libraries can be generated to represent the mRNA molecules expressed in a cell at a given point.
Transfection of eukaryotic hosts can be achieved through various methods using electroporation, gene guns, microinjection,
shuttle vectors, and viral vectors.

Footnotes
1. Liangxue Lai, Jing X. Kang, Rongfeng Li, Jingdong Wang, William T. Witt, Hwan Yul Yong, Yanhong Hao et al. “Generation
of Cloned Transgenic Pigs Rich in Omega-3 Fatty Acids.” Nature Biotechnology 24 no. 4 (2006): 435–436.
2. Raylene Ramos Moura, Luciana Magalhães Melo, and Vicente José de Figueirêdo Freitas. “Production of Recombinant
Proteins in Milk of Transgenic and Non-Transgenic Goats.” Brazilian Archives of Biology and Technology 54 no. 5 (2011):
927–938.
3. William S.M. Wold and Karoly Toth. “Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy.” Current
Gene Therapy 13 no. 6 (2013): 421.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 10.1: Microbes and the Tools of Genetic Engineering is shared under a CC BY license and was authored, remixed, and/or curated
by OpenStax.

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10.2: Visualizing and Characterizing DNA
Learning Objectives
Explain the use of nucleic acid probes to visualize specific DNA sequences
Explain the use of gel electrophoresis to separate DNA fragments
Explain the principle of restriction fragment length polymorphism analysis and its uses
Compare and contrast Southern and northern blots
Explain the principles and uses of microarray analysis
Describe the methods uses to separate and visualize protein variants
Explain the method and uses of polymerase chain reaction and DNA sequencing

The sequence of a DNA molecule can help us identify an organism when compared to known sequences housed in a database. The
sequence can also tell us something about the function of a particular part of the DNA, such as whether it encodes a particular
protein. Comparing protein signatures—the expression levels of specific arrays of proteins—between samples is an important
method for evaluating cellular responses to a multitude of environmental factors and stresses. Analysis of protein signatures can
reveal the identity of an organism or how a cell is responding during disease.
The DNA and proteins of interest are microscopic and typically mixed in with many other molecules including DNA or proteins
irrelevant to our interests. Many techniques have been developed to isolate and characterize molecules of interest. These methods
were originally developed for research purposes, but in many cases they have been simplified to the point that routine clinical use is
possible. For example, many pathogens, such as the bacterium Helicobacter pylori, which causes stomach ulcers, can be detected
using protein-based tests. In addition, an increasing number of highly specific and accurate DNA amplification-based identification
assays can now detect pathogens such as antibiotic-resistant enteric bacteria, herpes simplex virus, varicella-zoster virus, and many
others.

Molecular Analysis of DNA

In this subsection, we will outline some of the basic methods used for separating and visualizing specific fragments of DNA that
are of interest to a scientist. Some of these methods do not require knowledge of the complete sequence of the DNA molecule.
Before the advent of rapid DNA sequencing, these methods were the only ones available to work with DNA, but they still form the
basic arsenal of tools used by molecular geneticists to study the body’s responses to microbial and other diseases.

Nucleic Acid Probing

DNA molecules are small, and the information contained in their sequence is invisible. How does a researcher isolate a particular
stretch of DNA, or having isolated it, determine what organism it is from, what its sequence is, or what its function is? One method
to identify the presence of a certain DNA sequence uses artificially constructed pieces of DNA called probes. Probes can be used to
identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically. For
example, DNA probes are used to detect the vaginal pathogens Candida albicans, Gardnerella vaginalis, and Trichomonas
vaginalis.
To screen a genomic library for a particular gene or sequence of interest, researchers must know something about that gene. If
researchers have a portion of the sequence of DNA for the gene of interest, they can design a DNA probe, a single-stranded DNA
fragment that is complementary to part of the gene of interest and different from other DNA sequences in the sample. The DNA
probe may be synthesized chemically by commercial laboratories, or it may be created by cloning, isolating, and denaturing a DNA
fragment from a living organism. In either case, the DNA probe must be labeled with a molecular tag or beacon, such as a
radioactive phosphorus atom (as is used for autoradiography) or a fluorescent dye (as is used in fluorescent in situ hybridization, or
FISH), so that the probe and the DNA it binds to can be seen (Figure 10.2.1). The DNA sample being probed must also be
denatured to make it single-stranded so that the single-stranded DNA probe can anneal to the single-stranded DNA sample at
locations where their sequences are complementary. While these techniques are valuable for diagnosis, their direct use on sputum
and other bodily samples may be problematic due to the complex nature of these samples. DNA often must first be isolated from
bodily samples through chemical extraction methods before a DNA probe can be used to identify pathogens.

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 Figure 10.2.1 : DNA probes can be used to confirm the presence of a suspected pathogen in patient samples. This diagram
illustrates how a DNA probe can be used to search for a gene of interest associated with the suspected pathogen.

Clinical Focus- part 2

The mild, flu-like symptoms that Kayla is experiencing could be caused by any number of infectious agents. In
addition, several non-infectious autoimmune conditions, such as multiple sclerosis, systemic lupus erythematosus
(SLE), and amyotrophic lateral sclerosis (ALS), also have symptoms that are consistent with Kayla’s early
symptoms. However, over the course of several weeks, Kayla’s symptoms worsened. She began to experience
joint pain in her knees, heart palpitations, and a strange limpness in her facial muscles. In addition, she suffered
from a stiff neck and painful headaches. Reluctantly, she decided it was time to seek medical attention.

Exercise 10.2.1

1. Do Kayla’s new symptoms provide any clues as to what type of infection or other medical condition she may have?
2. What tests or tools might a health-care provider use to pinpoint the pathogen causing Kayla’s symptoms?

Agarose Gel Electrophoresis

There are a number of situations in which a researcher might want to physically separate a collection of DNA fragments of
different sizes. A researcher may also digest a DNA sample with a restriction enzyme to form fragments. The resulting size and
fragment distribution pattern can often yield useful information about the sequence of DNA bases that can be used, much like a
bar-code scan, to identify the individual or species to which the DNA belongs.
Gel electrophoresis is a technique commonly used to separate biological molecules based on size and biochemical characteristics,
such as charge and polarity. Agarose gel electrophoresis is widely used to separate DNA (or RNA) of varying sizes that may be
generated by restriction enzyme digestion or by other means, such as the PCR (Figure 10.2.2).
Due to its negatively charged backbone, DNA is strongly attracted to a positive electrode. In agarose gel electrophoresis, the gel is
oriented horizontally in a buffer solution. Samples are loaded into sample wells on the side of the gel closest to the negative
electrode, then drawn through the molecular sieve of the agarose matrix toward the positive electrode. The agarose matrix impedes
the movement of larger molecules through the gel, whereas smaller molecules pass through more readily. Thus, the distance of
migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling a longer distance through the
gel. Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run
on the same gel. To separate very large DNA fragments, such as chromosomes or viral genomes, agarose gel electrophoresis can be
modified by periodically alternating the orientation of the electric field during pulsed-field gel electrophoresis (PFGE). In PFGE,
smaller fragments can reorient themselves and migrate slightly faster than larger fragments and this technique can thus serve to
separate very large fragments that would otherwise travel together during standard agarose gel electrophoresis. In any of these

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electrophoresis techniques, the locations of the DNA or RNA fragments in the gel can be detected by various methods. One
common method is adding ethidium bromide, a stain that inserts into the nucleic acids at non-specific locations and can be
visualized when exposed to ultraviolet light. Other stains that are safer than ethidium bromide, a potential carcinogen, are now
available.

Figure 10.2.2 : (a) The process of agarose gel electrophoresis. (b) A researcher loading samples into a gel. (c) This photograph
shows a completed electrophoresis run on an agarose gel. The DNA ladder is located in lanes 1 and 9. Seven samples are located in
lanes 2 through 8. The gel was stained with ethidium bromide and photographed under ultraviolet light. (credit a: modification of
work by Magnus Manske; credit b: modification of work by U.S. Department of Agriculture; credit c: modification of work by
James Jacob)

Restriction Fragment Length Polymorphism (RFLP) Analysis

Restriction enzyme recognition sites are short (only a few nucleotides long), sequence-specific palindromes, and may be found
throughout the genome. Thus, differences in DNA sequences in the genomes of individuals will lead to differences in distribution
of restriction-enzyme recognition sites that can be visualized as distinct banding patterns on a gel after agarose gel electrophoresis.
Restriction fragment length polymorphism (RFLP) analysis compares DNA banding patterns of different DNA samples after
restriction digestion (Figure 10.2.3).

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RFLP analysis has many practical applications in both medicine and forensic science. For example, epidemiologists use RFLP
analysis to track and identify the source of specific microorganisms implicated in outbreaks of food poisoning or certain infectious
diseases. RFLP analysis can also be used on human DNA to determine inheritance patterns of chromosomes with variant genes,
including those associated with heritable diseases or to establish paternity.
Forensic scientists use RFLP analysis as a form of DNA fingerprinting, which is useful for analyzing DNA obtained from crime
scenes, suspects, and victims. DNA samples are collected, the numbers of copies of the sample DNA molecules are increased using
PCR, and then subjected to restriction enzyme digestion and agarose gel electrophoresis to generate specific banding patterns. By
comparing the banding patterns of samples collected from the crime scene against those collected from suspects or victims,
investigators can definitively determine whether DNA evidence collected at the scene was left behind by suspects or victims.

Figure 10.2.3 : RFLP analysis can be used to differentiate DNA sequences. In this example, a normal chromosome is digested into
two fragments, whereas digestion of a mutated chromosome produces only one fragment. The small red arrows pointing to the two
different chromosome segments show the locations of the restriction enzyme recognition sites. After digestion and agarose gel
electrophoresis, the banding patterns reflect the change by showing the loss of two shorter bands and the gain of a longer band.
(credit: modification of work by National Center for Biotechnology Information)

Southern Blots and Modifications

Several molecular techniques capitalize on sequence complementarity and hybridization between nucleic acids of a sample and
DNA probes. Typically, probing nucleic-acid samples within a gel is unsuccessful because as the DNA probe soaks into a gel, the
sample nucleic acids within the gel diffuse out. Thus, blotting techniques are commonly used to transfer nucleic acids to a thin,
positively charged membrane made of nitrocellulose or nylon. In the Southern blot technique, developed by Sir Edwin Southern in
1975, DNA fragments within a sample are first separated by agarose gel electrophoresis and then transferred to a membrane
through capillary action (Figure 10.2.4). The DNA fragments that bind to the surface of the membrane are then exposed to a
specific single-stranded DNA probe labeled with a radioactive or fluorescent molecular beacon to aid in detection. Southern blots
may be used to detect the presence of certain DNA sequences in a given DNA sample. Once the target DNA within the membrane
is visualized, researchers can cut out the portion of the membrane containing the fragment to recover the DNA fragment of interest.

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 Figure 10.2.4 : In the Southern blot technique, DNA fragments are first separated by agarose gel electrophoresis, then transferred
by capillary action to a nylon membrane, which is then soaked with a DNA probe tagged with a molecular beacon for easy
visualization.
Variations of the Southern blot—the dot blot, slot blot, and the spot blot—do not involve electrophoresis, but instead concentrate
DNA from a sample into a small location on a membrane. After hybridization with a DNA probe, the signal intensity detected is
measured, allowing the researcher to estimate the amount of target DNA present within the sample.
A colony blot is another variation of the Southern blot in which colonies representing different clones in a genomic library are
transferred to a membrane by pressing the membrane onto the culture plate. The cells on the membrane are lysed and the
membrane can then be probed to determine which colonies within a genomic library harbor the target gene. Because the colonies
on the plate are still growing, the cells of interest can be isolated from the plate.
In the northern blot, another variation of the Southern blot, RNA (not DNA) is immobilized on the membrane and probed. Northern
blots are typically used to detect the amount of mRNA made through gene expression within a tissue or organism sample.

Microarray Analysis
Another technique that capitalizes on the hybridization between complementary nucleic acid sequences is called microarray
analysis. Microarray analysis is useful for the comparison of gene-expression patterns between different cell types—for example,
cells infected with a virus versus uninfected cells, or cancerous cells versus healthy cells (Figure 10.2.5).
Typically, DNA or cDNA from an experimental sample is deposited on a glass slide alongside known DNA sequences. Each slide
can hold more than 30,000 different DNA fragment types. Distinct DNA fragments (encompassing an organism’s entire genomic
library) or cDNA fragments (corresponding to an organism’s full complement of expressed genes) can be individually spotted on a
glass slide.
Once deposited on the slide, genomic DNA or mRNA can be isolated from the two samples for comparison. If mRNA is isolated, it
is reverse-transcribed to cDNA using reverse transcriptase. Then the two samples of genomic DNA or cDNA are labeled with

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different fluorescent dyes (typically red and green). The labeled genomic DNA samples are then combined in equal amounts, added
to the microarray chip, and allowed to hybridize to complementary spots on the microarray.
Hybridization of sample genomic DNA molecules can be monitored by measuring the intensity of fluorescence at particular spots
on the microarray. Differences in the amount of hybridization between the samples can be readily observed. If only one sample’s
nucleic acids hybridize to a particular spot on the microarray, then that spot will appear either green or red. However, if both
samples’ nucleic acids hybridize, then the spot will appear yellow due to the combination of the red and green dyes.
Although microarray technology allows for a holistic comparison between two samples in a short time, it requires sophisticated
(and expensive) detection equipment and analysis software. Because of the expense, this technology is typically limited to research
settings. Researchers have used microarray analysis to study how gene expression is affected in organisms that are infected by
bacteria or viruses or subjected to certain chemical treatments.

Figure 10.2.5 : (a) The steps in microarray analysis are illustrated. Here, gene expression patterns are compared between cancerous
cells and healthy cells. (b) Microarray information can be expressed as a heat map. Genes are shown on the left side; different
samples are shown across the bottom. Genes expressed only in cancer cells are shown in varying shades of red; genes expressed
only in normal cells are shown in varying shades of green. Genes that are expressed in both cancerous and normal cells are shown
in yellow.
Explore microchip technology at this interactive website.

Exercise 10.2.2
1. What does a DNA probe consist of?
2. Why is a Southern blot used after gel electrophoresis of a DNA digest?

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Molecular Analysis of Proteins
In many cases it may not be desirable or possible to study DNA or RNA directly. Proteins can provide species-specific information
for identification as well as important information about how and whether a cell or tissue is responding to the presence of a
pathogenic microorganism. Various proteins require different methods for isolation and characterization.

Polyacrylamide Gel Electrophoresis

A variation of gel electrophoresis, called polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins. In
PAGE, the gel matrix is finer and composed of polyacrylamide instead of agarose. Additionally, PAGE is typically performed using
a vertical gel apparatus (Figure 10.2.6). Because of the varying charges associated with amino acid side chains, PAGE can be used
to separate intact proteins based on their net charges. Alternatively, proteins can be denatured and coated with a negatively charged
detergent called sodium dodecyl sulfate (SDS), masking the native charges and allowing separation based on size only. PAGE can
be further modified to separate proteins based on two characteristics, such as their charges at various pHs as well as their size,
through the use of two-dimensional PAGE. In any of these cases, following electrophoresis, proteins are visualized through
staining, commonly with either Coomassie blue or a silver stain.

Figure 10.2.6 : (a) SDS is a detergent that denatures proteins and masks their native charges, making them uniformly negatively
charged. (b) The process of SDS-PAGE is illustrated in these steps. (c) A photograph of an SDS-PAGE gel shows Coomassie
stained bands where proteins of different size have migrated along the gel in response to the applied voltage. A size standard lane is
visible on the right side of the gel. (credit b: modification of work by “GeneEd”/YouTube)

Exercise 10.2.3

On what basis are proteins separated in SDS-PAGE?

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Amplification-Based DNA Analysis Methods
Various methods can be used for obtaining sequences of DNA, which are useful for studying disease-causing organisms. With the
advent of rapid sequencing technology, our knowledge base of the entire genomes of pathogenic organisms has grown
phenomenally. We start with a description of the polymerase chain reaction, which is not a sequencing method but has allowed
researchers and clinicians to obtain the large quantities of DNA needed for sequencing and other studies. The polymerase chain
reaction eliminates the dependence we once had on cells to make multiple copies of DNA, achieving the same result through
relatively simple reactions outside the cell.

Polymerase Chain Reaction (PCR)

Most methods of DNA analysis, such as restriction enzyme digestion and agarose gel electrophoresis, or DNA sequencing require
large amounts of a specific DNA fragment. In the past, large amounts of DNA were produced by growing the host cells of a
genomic library. However, libraries take time and effort to prepare and DNA samples of interest often come in minute quantities.
The polymerase chain reaction (PCR) permits rapid amplification in the number of copies of specific DNA sequences for further
analysis (Figure 10.2.7). One of the most powerful techniques in molecular biology, PCR was developed in 1983 by Kary Mullis
while at Cetus Corporation. PCR has specific applications in research, forensic, and clinical laboratories, including aiding in:
near purification of a sample for determining the sequence of nucleotides in a specific region of DNA
amplifying a target region of DNA for cloning into a plasmid vector
identifying the source of a DNA sample left at a crime scene
analyzing samples to determine paternity
comparing samples of ancient DNA with modern organisms
determining the presence of difficult to culture, or unculturable, microorganisms in humans or environmental samples
certain identification tests of clinical samples
PCR is an in vitro laboratory technique that takes advantage of the natural process of DNA replication. The heat-stable DNA
polymerase enzymes used in PCR are derived from hyperthermophilic prokaryotes. Taq DNA polymerase, commonly used in PCR,
is derived from the Thermus aquaticus bacterium isolated from a hot spring in Yellowstone National Park. DNA replication
requires the use of primers for the initiation of replication to have free 3ʹ-hydroxyl groups available for the addition of nucleotides
by DNA polymerase. However, while primers composed of RNA are normally used in cells, DNA primers are used for PCR. DNA
primers are preferable due to their stability, and DNA primers with known sequences targeting a specific DNA region can be
chemically synthesized commercially. These DNA primers are functionally similar to the DNA probes used for the various
hybridization techniques described earlier, binding to specific targets due to complementarity between the target DNA sequence
and the primer.
PCR occurs over multiple cycles, each containing three steps: denaturation, annealing, and extension. Machines called thermal
cyclers are used for PCR; these machines can be programmed to automatically cycle through the temperatures required at each
step. First, double-stranded template DNA containing the target sequence is denatured at approximately 95 °C. The high
temperature required to physically (rather than enzymatically) separate the DNA strands is the reason the heat-stable DNA
polymerase is required. Next, the temperature is lowered to approximately 50 °C. This allows the DNA primers complementary to
the ends of the target sequence to anneal (stick) to the template strands, with one primer annealing to each strand. Finally, the
temperature is raised to 72 °C, the optimal temperature for the activity of the heat-stable DNA polymerase, allowing for the
addition of nucleotides to the primer using the single-stranded target as a template. Each cycle doubles the number of double-
stranded target DNA copies. Typically, PCR protocols include 25–40 cycles, allowing for the amplification of a single target
sequence by tens of millions to over a trillion.
Natural DNA replication is designed to copy the entire genome, and initiates at one or more origin sites. Primers are constructed
during replication, not before, and do not consist of a few specific sequences. PCR targets specific regions of a DNA sample using
sequence-specific primers. In recent years, a variety of isothermal PCR amplification methods that circumvent the need for thermal
cycling have been developed, taking advantage of accessory proteins that aid in the DNA replication process. As the development
of these methods continues and their use becomes more widespread in research, forensic, and clinical labs, thermal cyclers may
become obsolete.

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 Figure 10.2.7 : The polymerase chain reaction (PCR) is used to produce many copies of a specific sequence of DNA.
Deepen your understanding of the polymerase chain reaction by viewing this animation and working through an interactive
exercise.

PCR Variations
Several later modifications to PCR further increase the utility of this technique. Reverse transcriptase PCR (RT-PCR) is used for
obtaining DNA copies of a specific mRNA molecule. RT-PCR begins with the use of the reverse transcriptase enzyme to convert
mRNA molecules into cDNA. That cDNA is then used as a template for traditional PCR amplification. RT-PCR can detect whether
a specific gene has been expressed in a sample. Another recent application of PCR is real-time PCR, also known as quantitative
PCR (qPCR). Standard PCR and RT-PCR protocols are not quantitative because any one of the reagents may become limiting
before all of the cycles within the protocol are complete, and samples are only analyzed at the end. Because it is not possible to
determine when in the PCR or RT-PCR protocol a given reagent has become limiting, it is not possible to know how many cycles
were completed prior to this point, and thus it is not possible to determine how many original template molecules were present in
the sample at the start of PCR. In qPCR, however, the use of fluorescence allows one to monitor the increase in a double-stranded
template during a PCR reaction as it occurs. These kinetics data can then be used to quantify the amount of the original target
sequence. The use of qPCR in recent years has further expanded the capabilities of PCR, allowing researchers to determine the
number of DNA copies, and sometimes organisms, present in a sample. In clinical settings, qRT-PCR is used to determine viral
load in HIV-positive patients to evaluate the effectiveness of their therapy.

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STR Analysis: This section from Bio-OER by City Tech CUNY1:
PCR can also be used in DNA fingerprinting. At a crime scene, criminals don’t often leave massive amounts of tissue behind. Scant
evidence in the form of a few cells found within bodily fluids or stray hairs can be enough to use as DNA evidence. DNA is
extracted from these few cells and amplified. Analysis involves polymerase chain reaction (PCR) amplification of short tandem
repeat (STR) loci and electrophoresis to determine the length of the PCR-amplified fragment. A type of polymorphism occurs due
to these repeats expanding and contracting in non-coding regions of the chromosomes. These regions are called short tandem
repeats (STRs). Any region or location on a chromosome is referred to as locus (loci for plural). Scientists use polymorphic loci
that are known to contain STRs in order to differentiate people based on their DNA. This is often used in forensic science or in
maternity/paternity cases. Any variation of a locus is referred to as an allele. In standard genetics, we often think of an allele as a
variation of a gene that would result in a difference in a physical manifestation of that gene. In the case of STRs, these alleles are
simply a difference in the number of repeats. This means the length of DNA within this locus is either longer or shorter and gives
rise to many different alleles.

Figure 10.2.8 : DNA fingerprinting. Credit: Helixitta, The Photographer and Jeremy Seto (CC-BY-SA 3.0)
The FBI and local law enforcement agencies have developed a database called the Combined DNA Index System (CoDIS) that
gathers data on a number of STRs. By establishing the number of repeats of a given locus, law enforcement officials can
differentiate individuals based on the repeat length of these alleles. CoDIS uses a set of 20 loci that are tested together. As you
would imagine, people are bound to have the same alleles of certain loci, especially if they were related. The use of 20 different
loci makes it statistically improbable that 2 different people could be confused with each other. Think about this in terms of
physical traits. As you increase the number of physical traits used to describe someone, you are less likely to confuse that person
with someone else based on those combinations of traits. Using the CoDIS loci increases the stringency since there are many alleles
for each locus. The twentieth locus in CoDIS (called AMEL) discriminates between male and female.
Flash animation walking through what an STR is: http://www.dnalc.org/view/15981-DNA-variations.html

Figure 10.2.9 : CoDIS STRs: The FBI utilizes 20 different loci to discriminate between people. AMEL discriminates by gender and
is located on the X & Y. Credit: Jeremy Seto (CC-BY-NC-SA)

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Sanger DNA Sequencing
A basic sequencing technique is the chain termination method, also known as the dideoxy method or the Sanger DNA sequencing
method, developed by Frederick Sanger in 1972. The chain termination method involves DNA replication of a single-stranded
template with the use of a DNA primer to initiate synthesis of a complementary strand, DNA polymerase, a mix of the four regular
deoxynucleotide (dNTP) monomers, and a small proportion of dideoxynucleotides (ddNTPs), each labeled with a molecular
beacon. The ddNTPs are monomers missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to
form a chain (Figure 10.2.10). Every time a ddNTP is randomly incorporated into the growing complementary strand, it terminates
the process of DNA replication for that particular strand. This results in multiple short strands of replicated DNA that are each
terminated at a different point during replication. When the reaction mixture is subjected to gel electrophoresis, the multiple newly
replicated DNA strands form a ladder of differing sizes. Because the ddNTPs are labeled, each band on the gel reflects the size of
the DNA strand when the ddNTP terminated the reaction.
In Sanger’s day, four reactions were set up for each DNA molecule being sequenced, each reaction containing only one of the four
possible ddNTPs. Each ddNTP was labeled with a radioactive phosphorus molecule. The products of the four reactions were then
run in separate lanes side by side on long, narrow PAGE gels, and the bands of varying lengths were detected by autoradiography.
Today, this process has been simplified with the use of ddNTPs, each labeled with a different colored fluorescent dye or
fluorochrome (Figure 10.2.11), in one sequencing reaction containing all four possible ddNTPs for each DNA molecule being
sequenced (Figure 10.2.12). These fluorochromes are detected by fluorescence spectroscopy. Determining the fluorescence color of
each band as it passes by the detector produces the nucleotide sequence of the template strand.

Figure 10.2.10: A dideoxynucleotide is similar in structure to a deoxynucleotide, but is missing the 3ʹ hydroxyl group (indicated by
the shaded box). When a dideoxynucleotide is incorporated into a DNA strand, DNA synthesis stops.

Figure 10.2.11: Frederick Sanger’s dideoxy chain termination method is illustrated, using ddNTPs tagged with fluorochromes.
Using ddNTPs, a mixture of DNA fragments of every possible size, varying in length by only one nucleotide, can be generated.
The DNA is separated on the basis of size and each band can be detected with a fluorescence detector.

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 Figure 10.2.12: This diagram summarizes the Sanger sequencing method using fluorochrome-labeled ddNTPs and capillary gel
electrophoresis.
Since 2005, automated sequencing techniques used by laboratories fall under the umbrella of next generation sequencing, which is
a group of automated techniques used for rapid DNA sequencing. These methods have revolutionized the field of molecular
genetics because the low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to
600 base pairs) just in one day. Although several variants of next generation sequencing technologies are made by different
companies (for example, 454 Life Sciences’ pyrosequencing and Illumina’s Solexa technology), they all allow millions of bases to
be sequenced quickly, making the sequencing of entire genomes relatively easy, inexpensive, and commonplace. In 454 sequencing
(pyrosequencing), for example, a DNA sample is fragmented into 400–600-bp single-strand fragments, modified with the addition
of DNA adapters to both ends of each fragment. Each DNA fragment is then immobilized on a bead and amplified by PCR, using

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primers designed to anneal to the adapters, creating a bead containing many copies of that DNA fragment. Each bead is then put
into a separate well containing sequencing enzymes. To the well, each of the four nucleotides is added one after the other; when
each one is incorporated, pyrophosphate is released as a byproduct of polymerization, emitting a small flash of light that is recorded
by a detector. This provides the order of nucleotides incorporated as a new strand of DNA is made and is an example of synthesis
sequencing. Next generation sequencers use sophisticated software to get through the cumbersome process of putting all the
fragments in order. Overall, these technologies continue to advance rapidly, decreasing the cost of sequencing and increasing the
availability of sequence data from a wide variety of organisms quickly.
The National Center for Biotechnology Information houses a widely used genetic sequence database called GenBank where
researchers deposit genetic information for public use. Upon publication of sequence data, researchers upload it to GenBank, giving
other researchers access to the information. The collaboration allows researchers to compare newly discovered or unknown sample
sequence information with the vast array of sequence data that already exists.
View an animation about 454 sequencing to deepen your understanding of this method.

Using a NAAT to Diagnose a C. diff Infection

Javier, an 80-year-old patient with a history of heart disease, recently returned home from the hospital after undergoing an
angioplasty procedure to insert a stent into a cardiac artery. To minimize the possibility of infection, Javier was administered
intravenous broad-spectrum antibiotics during and shortly after his procedure. He was released four days after the procedure,
but a week later, he began to experience mild abdominal cramping and watery diarrhea several times a day. He lost his appetite,
became severely dehydrated, and developed a fever. He also noticed blood in his stool. Javier’s wife called the physician, who
instructed her to take him to the emergency room immediately.
The hospital staff ran several tests and found that Javier’s kidney creatinine levels were elevated compared with the levels in
his blood, indicating that his kidneys were not functioning well. Javier’s symptoms suggested a possible infection with
Clostridium difficile, a bacterium that is resistant to many antibiotics. The hospital collected and cultured a stool sample to look
for the production of toxins A and B by C. difficile, but the results came back negative. However, the negative results were not
enough to rule out a C. difficile infection because culturing of C. difficile and detection of its characteristic toxins can be
difficult, particularly in some types of samples. To be safe, they proceeded with a diagnostic nucleic acid amplification test
(NAAT). Currently NAATs are the clinical diagnostician’s gold standard for detecting the genetic material of a pathogen. In
Javier’s case, qPCR was used to look for the gene encoding C. difficile toxin B (tcdB). When the qPCR analysis came back
positive, the attending physician concluded that Javier was indeed suffering from a C. difficile infection and immediately
prescribed the antibiotic vancomycin, to be administered intravenously. The antibiotic cleared the infection and Javier made a
full recovery.
Because infections with C. difficile were becoming widespread in Javier’s community, his sample was further analyzed to see
whether the specific strain of C. difficile could be identified. Javier’s stool sample was subjected to ribotyping and repetitive
sequence-based PCR (rep-PCR) analysis. In ribotyping, a short sequence of DNA between the 16S rRNA and 23S rRNA genes
is amplified and subjected to restriction digestion (Figure 10.2.13). This sequence varies between strains of C. difficile, so
restriction enzymes will cut in different places. In rep-PCR, DNA primers designed to bind to short sequences commonly
found repeated within the C. difficile genome were used for PCR. Following restriction digestion, agarose gel electrophoresis
was performed in both types of analysis to examine the banding patterns that resulted from each procedure (Figure 10.2.14).
Rep-PCR can be used to further subtype various ribotypes, increasing resolution for detecting differences between strains. The
ribotype of the strain infecting Javier was found to be ribotype 27, a strain known for its increased virulence, resistance to
antibiotics, and increased prevalence in the United States, Canada, Japan, and Europe. 2

Exercise 10.2.4

1. How do banding patterns differ between strains of C. difficile?

2. Why do you think laboratory tests were unable to detect toxin production directly?

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 Figure 10.2.13: A gel showing PCR products of various Clostridium difficile strains. Javier’s sample is shown at the bottom;
note that it matches ribotype 27 in the reference set. (credit: modification of work by American Society for Microbiology)

Figure 10.2.14: Strains of infectious bacteria, such as C. difficile, can be identified by molecular analysis. PCR ribotyping is
commonly used to identify particular C. difficile strains. Rep-PCR is an alternate molecular technique that is also used to
identify particular C. difficile strains. (credit b: modification of work by American Society for Microbiology)

Exercise 10.2.5
1. How is PCR similar to the natural DNA replication process in cells? How is it different?
2. Compare RT-PCR and qPCR in terms of their respective purposes.
3. In chain-termination sequencing, how is the identity of each nucleotide in a sequence determined?

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Key Concepts and Summary
Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon
(typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample.
Agarose gel electrophoresis allows for the separation of DNA molecules based on size.
Restriction fragment length polymorphism (RFLP) analysis allows for the visualization by agarose gel electrophoresis of
distinct variants of a DNA sequence caused by differences in restriction sites.
Southern blot analysis allows researchers to find a particular DNA sequence within a sample whereas northern blot analysis
allows researchers to detect a particular mRNA sequence expressed in a sample.
Microarray technology is a nucleic acid hybridization technique that allows for the examination of many thousands of genes at
once to find differences in genes or gene expression patterns between two samples of genomic DNA or cDNA,
Polyacrylamide gel electrophoresis (PAGE) allows for the separation of proteins by size, especially if native protein charges
are masked through pretreatment with SDS.
Polymerase chain reaction allows for the rapid amplification of a specific DNA sequence. Variations of PCR can be used to
detect mRNA expression (reverse transcriptase PCR) or to quantify a particular sequence in the original sample (real-time
PCR).
STR analysis uses PCR to help amplify DNA at crime scenes. It is then analyzed by gel electrophoresis to produce a DNA
fingerprint similar to RFLP. The fingerprint can then be compared to other in databases like CoDIS.
Although the development of Sanger DNA sequencing was revolutionary, advances in next generation sequencing allow for
the rapid and inexpensive sequencing of the genomes of many organisms, accelerating the volume of new sequence data.

Footnotes
1. "Variable Number Tandem Repeats" Bio-OER. City Tech CUNY. Sept 2019. Chapter 8.3
2. Patrizia Spigaglia, Fabrizio Barbanti, Anna Maria Dionisi, and Paola Mastrantonio. “Clostridium difficile Isolates Resistant to
Fluoroquinolones in Italy: Emergence of PCR Ribotype 018.” Journal of Clinical Microbiology 48 no. 8 (2010): 2892–2896.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 10.2: Visualizing and Characterizing DNA is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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10.3: Whole Genome Methods and Industrial Applications
Learning Objectives
Explain the uses of genome-wide comparative analyses
Summarize the advantages of genetically engineered pharmaceutical products

Advances in molecular biology have led to the creation of entirely new fields of science. Among these are fields that study aspects
of whole genomes, collectively referred to as whole-genome methods. In this section, we’ll provide a brief overview of the whole-
genome fields of genomics, transcriptomics, and proteomics.

Genomics, Transcriptomics, and Proteomics

The study and comparison of entire genomes, including the complete set of genes and their nucleotide sequence and organization,
is called genomics. This field has great potential for future medical advances through the study of the human genome as well as the
genomes of infectious organisms. Analysis of microbial genomes has contributed to the development of new antibiotics, diagnostic
tools, vaccines, medical treatments, and environmental cleanup techniques.
The field of transcriptomics is the science of the entire collection of mRNA molecules produced by cells. Scientists compare gene
expression patterns between infected and uninfected host cells, gaining important information about the cellular responses to
infectious disease. Additionally, transcriptomics can be used to monitor the gene expression of virulence factors in microorganisms,
aiding scientists in better understanding pathogenic processes from this viewpoint.
When genomics and transcriptomics are applied to entire microbial communities, we use the terms metagenomics and
metatranscriptomics, respectively. Metagenomics and metatranscriptomics allow researchers to study genes and gene expression
from a collection of multiple species, many of which may not be easily cultured or cultured at all in the laboratory. A DNA
microarray (discussed in the previous section) can be used in metagenomics studies.
Another up-and-coming clinical application of genomics and transcriptomics is pharmacogenomics, also called toxicogenomics,
which involves evaluating the effectiveness and safety of drugs on the basis of information from an individual’s genomic sequence.
Genomic responses to drugs can be studied using experimental animals (such as laboratory rats or mice) or live cells in the
laboratory before embarking on studies with humans. Changes in gene expression in the presence of a drug can sometimes be an
early indicator of the potential for toxic effects. Personal genome sequence information may someday be used to prescribe
medications that will be most effective and least toxic on the basis of the individual patient’s genotype.
The study of proteomics is an extension of genomics that allows scientists to study the entire complement of proteins in an
organism, called the proteome. Even though all cells of a multicellular organism have the same set of genes, cells in various tissues
produce different sets of proteins. Thus, the genome is constant, but the proteome varies and is dynamic within an organism.
Proteomics may be used to study which proteins are expressed under various conditions within a single cell type or to compare
protein expression patterns between different organisms.
The most prominent disease being studied with proteomic approaches is cancer, but this area of study is also being applied to
infectious diseases. Research is currently underway to examine the feasibility of using proteomic approaches to diagnose various
types of hepatitis, tuberculosis, and HIV infection, which are rather difficult to diagnose using currently available techniques.1
A recent and developing proteomic analysis relies on identifying proteins called biomarkers, whose expression is affected by the
disease process. Biomarkers are currently being used to detect various forms of cancer as well as infections caused by pathogens
such as Yersinia pestis and Vaccinia virus.2
Other “-omic” sciences related to genomics and proteomics include metabolomics, glycomics, and lipidomics, which focus on the
complete set of small-molecule metabolites, sugars, and lipids, respectively, found within a cell. Through these various global
approaches, scientists continue to collect, compile, and analyze large amounts of genetic information. This emerging field of
bioinformatics can be used, among many other applications, for clues to treating diseases and understanding the workings of cells.
Additionally, researchers can use reverse genetics, a technique related to classic mutational analysis, to determine the function of
specific genes. Classic methods of studying gene function involved searching for the genes responsible for a given phenotype.
Reverse genetics uses the opposite approach, starting with a specific DNA sequence and attempting to determine what phenotype it
produces. Alternatively, scientists can attach known genes (called reporter genes) that encode easily observable characteristics to

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genes of interest, and the location of expression of such genes of interest can be easily monitored. This gives the researcher
important information about what the gene product might be doing or where it is located in the organism. Common reporter genes
include bacterial lacZ, which encodes beta-galactosidase and whose activity can be monitored by changes in colony color in the
presence of X-gal as previously described, and the gene encoding the jellyfish protein green fluorescent protein (GFP) whose
activity can be visualized in colonies under ultraviolet light exposure (Figure 10.3.1).

Figure 10.3.1 : (a) The gene encoding green fluorescence protein is a commonly used reporter gene for monitoring gene expression
patterns in organisms. Under ultraviolet light, GFP fluoresces. Here, two mice are expressing GFP, while the middle mouse is not.
(b) GFP can be used as a reporter gene in bacteria as well. Here, a plate containing bacterial colonies expressing GFP is shown. (c)
Blue-white screening in bacteria is accomplished through the use of the lacZ reporter gene, followed by plating of bacteria onto
medium containing X-gal. Cleavage of X-gal by the LacZ enzyme results in the formation of blue colonies. (credit a: modification
of work by Ingrid Moen, Charlotte Jevne, Jian Wang, Karl-Henning Kalland, Martha Chekenya, Lars A Akslen, Linda Sleire, Per Ø
Enger, Rolf K Reed, Anne M Øyan, Linda EB Stuhr; credit b: modification of work by “2.5JIGEN.com”/Flickr; credit c:
modification of work by American Society for Microbiology)

Exercise 10.3.1

1. How is genomics different from traditional genetics?

2. If you wanted to study how two different cells in the body respond to an infection, what –omics field would you apply?
3. What are the biomarkers uncovered in proteomics used for?

Use and Abuse of Genome Data

Why can some humans harbor opportunistic pathogens like Haemophilus influenzae, Staphylococcus aureus, or Streptococcus
pyogenes, in their upper respiratory tracts but remain asymptomatic carriers, while other individuals become seriously ill when
infected? There is evidence suggesting that differences in susceptibility to infection between patients may be a result, at least in
part, of genetic differences between human hosts. For example, genetic differences in human leukocyte antigens (HLAs) and
red blood cell antigens among hosts have been implicated in different immune responses and resulting disease progression
from infection with H. influenzae.
Because the genetic interplay between pathogen and host may contribute to disease outcomes, understanding differences in
genetic makeup between individuals may be an important clinical tool. Ecological genomics is a relatively new field that seeks
to understand how the genotypes of different organisms interact with each other in nature. The field answers questions about
how gene expression of one organism affects gene expression of another. Medical applications of ecological genomics will
focus on how pathogens interact with specific individuals, as opposed to humans in general. Such analyses would allow
medical professionals to use knowledge of an individual’s genotype to apply more individualized plans for treatment and
prevention of disease.
With the advent of next-generation sequencing, it is relatively easy to obtain the entire genomic sequences of pathogens; a
bacterial genome can be sequenced in as little as a day. 3 The speed and cost of sequencing the human genome has also
been greatly reduced and, already, individuals can submit samples to receive extensive reports on their personal genetic traits,
including ancestry and carrier status for various genetic diseases. As sequencing technologies progress further, such services
will continue to become less expensive, more extensive, and quicker.
However, as this day quickly approaches, there are many ethical concerns with which society must grapple. For example,
should genome sequencing be a standard practice for everybody? Should it be required by law or by employers if it will lower

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 health-care costs? If one refuses genome sequencing, does he or she forfeit his or her right to health insurance coverage? For
what purposes should the data be used? Who should oversee proper use of these data? If genome sequencing reveals
predisposition to a particular disease, do insurance companies have the right to increase rates? Will employers treat an
employee differently? Knowing that environmental influences also affect disease development, how should the data on the
presence of a particular disease-causing allele in an individual be used ethically? The Genetic Information Nondiscrimination
Act of 2008 (GINA) currently prohibits discriminatory practices based on genetic information by both health insurance
companies and employers. However, GINA does not cover life, disability, or long-term care insurance policies. Clearly, all
members of society must continue to engage in conversations about these issues so that such genomic data can be used to
improve health care while simultaneously protecting an individual’s rights.

Clinical Focus: part 3

When Kayla described her symptoms, her physician at first suspected bacterial meningitis, which is consistent with her
headaches and stiff neck. However, she soon ruled this out as a possibility because meningitis typically progresses more
quickly than what Kayla was experiencing. Many of her symptoms still paralleled those of amyotrophic lateral sclerosis (ALS)
and systemic lupus erythematosus (SLE), and the physician also considered Lyme disease a possibility given how much time
Kayla spends in the woods. Kayla did not recall any recent tick bites (the typical means by which Lyme disease is transmitted)
and she did not have the typical bull’s-eye rash associated with Lyme disease (Figure 10.3.2). However, 20–30% of patients
with Lyme disease never develop this rash, so the physician did not want to rule it out.
Kayla’s doctor ordered an MRI of her brain, a complete blood count to test for anemia, blood tests assessing liver and kidney
function, and additional tests to confirm or rule out SLE or Lyme disease. Her test results were inconsistent with both SLE and
ALS, and the result of the test looking for Lyme disease antibodies was “equivocal,” meaning inconclusive. Having ruled out
ALS and SLE, Kayla’s doctor decided to run additional tests for Lyme disease.

Exercise 10.3.2

1. Why would Kayla’s doctor still suspect Lyme disease even if the test results did not detect Lyme antibodies in the
blood?
2. What type of molecular test might be used for the detection of blood antibodies to Lyme disease

Figure 10.3.2 : A bulls-eye rash is one of the common symptoms of Lyme diseases, but up to 30% of infected individuals never
develop a rash. (credit: Centers for Disease Control and Prevention)

Recombinant DNA Technology and Pharmaceutical Production

Genetic engineering has provided a way to create new pharmaceutical products called recombinant DNA pharmaceuticals. Such
products include antibiotic drugs, vaccines, and hormones used to treat various diseases. Table 10.3.1 lists examples of
recombinant DNA products and their uses.
For example, the naturally occurring antibiotic synthesis pathways of various Streptomyces spp., long known for their antibiotic
production capabilities, can be modified to improve yields or to create new antibiotics through the introduction of genes encoding

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additional enzymes. More than 200 new antibiotics have been generated through the targeted inactivation of genes and the novel
combination of antibiotic synthesis genes in antibiotic-producing Streptomyces hosts.4
Genetic engineering is also used to manufacture subunit vaccines, which are safer than other vaccines because they contain only a
single antigenic molecule and lack any part of the genome of the pathogen. For example, a vaccine for hepatitis B is created by
inserting a gene encoding a hepatitis B surface protein into a yeast; the yeast then produces this protein, which the human immune
system recognizes as an antigen. The hepatitis B antigen is purified from yeast cultures and administered to patients as a vaccine.
Even though the vaccine does not contain the hepatitis B virus, the presence of the antigenic protein stimulates the immune system
to produce antibodies that will protect the patient against the virus in the event of exposure.5 6
Genetic engineering has also been important in the production of other therapeutic proteins, such as insulin, interferons, and human
growth hormone, to treat a variety of human medical conditions. For example, at one time, it was possible to treat diabetes only by
giving patients pig insulin, which caused allergic reactions due to small differences between the proteins expressed in human and
pig insulin. However, since 1978, recombinant DNA technology has been used to produce large-scale quantities of human insulin
using E. coli in a relatively inexpensive process that yields a more consistently effective pharmaceutical product. Scientists have
also genetically engineered E. coli capable of producing human growth hormone (HGH), which is used to treat growth disorders in
children and certain other disorders in adults. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by
cloning it into a bacterial vector. Eventually, genetic engineering will be used to produce DNA vaccines and various gene therapies,
as well as customized medicines for fighting cancer and other diseases.
Table 10.3.1 : Some Genetically Engineered Pharmaceutical Products and Applications
Recombinant DNA Product Application

Treatment of heart disease (e.g., congestive heart failure), kidney

Atrial natriuretic peptide
disease, high blood pressure

DNase Treatment of viscous lung secretions in cystic fibrosis

Erythropoietin Treatment of severe anemia with kidney damage

Factor VIII Treatment of hemophilia

Hepatitis B vaccine Prevention of hepatitis B infection

Human growth hormone Treatment of growth hormone deficiency, Turner’s syndrome, burns

Human insulin Treatment of diabetes

Treatment of multiple sclerosis, various cancers (e.g., melanoma), viral

Interferons
infections (e.g., Hepatitis B and C)

Tetracenomycins Used as antibiotics

Treatment of pulmonary embolism in ischemic stroke, myocardial

Tissue plasminogen activator
infarction

Exercise 10.3.3

1. What bacterium has been genetically engineered to produce human insulin for the treatment of diabetes?
2. Explain how microorganisms can be engineered to produce vaccines.

RNA Interference Technology

In chapter 10, we described the function of mRNA, rRNA, and tRNA. In addition to these types of RNA, cells also produce several
types of small noncoding RNA molecules that are involved in the regulation of gene expression. These include antisense RNA
molecules, which are complementary to regions of specific mRNA molecules found in both prokaryotes and eukaryotic cells. Non-
coding RNA molecules play a major role in RNA interference (RNAi), a natural regulatory mechanism by which mRNA molecules
are prevented from guiding the synthesis of proteins. RNA interference of specific genes results from the base pairing of short,
single-stranded antisense RNA molecules to regions within complementary mRNA molecules, preventing protein synthesis. Cells

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use RNA interference to protect themselves from viral invasion, which may introduce double-stranded RNA molecules as part of
the viral replication process (Figure 10.3.3).

Figure 10.3.3 : Cells like the eukaryotic cell shown in this diagram commonly make small antisense RNA molecules with
sequences complementary to specific mRNA molecules. When an antisense RNA molecule is bound to an mRNA molecule, the
mRNA can no longer be used to direct protein synthesis. (credit: modification of work by Robinson R)
Researchers are currently developing techniques to mimic the natural process of RNA interference as a way to treat viral infections
in eukaryotic cells. RNA interference technology involves using small interfering RNAs (siRNAs) or microRNAs (miRNAs)
(Figure 10.3.4). siRNAs are completely complementary to the mRNA transcript of a specific gene of interest while miRNAs are
mostly complementary. These double-stranded RNAs are bound to DICER, an endonuclease that cleaves the RNA into short
molecules (approximately 20 nucleotides long). The RNAs are then bound to RNA-induced silencing complex (RISC), a
ribonucleoprotein. The siRNA-RISC complex binds to mRNA and cleaves it. For miRNA, only one of the two strands binds to
RISC. The miRNA-RISC complex then binds to mRNA, inhibiting translation. If the miRNA is completely complementary to the
target gene, then the mRNA can be cleaved. Taken together, these mechanisms are known as gene silencing.

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 Figure 10.3.4 : This diagram illustrates the process of using siRNA or miRNA in a eukaryotic cell to silence genes involved in the
pathogenesis of various diseases. (credit: modification of work by National Center for Biotechnology Information)

Key Concepts and Summary

The science of genomics allows researchers to study organisms on a holistic level and has many applications of medical
relevance.
Transcriptomics and proteomics allow researchers to compare gene expression patterns between different cells and shows
great promise in better understanding global responses to various conditions.
The various –omics technologies complement each other and together provide a more complete picture of an organism’s or
microbial community’s (metagenomics) state.
The analysis required for large data sets produced through genomics, transcriptomics, and proteomics has led to the emergence
of bioinformatics.
Reporter genes encoding easily observable characteristics are commonly used to track gene expression patterns of genes of
unknown function.
The use of recombinant DNA technology has revolutionized the pharmaceutical industry, allowing for the rapid production of
high-quality recombinant DNA pharmaceuticals used to treat a wide variety of human conditions.
RNA interference technology has great promise as a method of treating viral infections by silencing the expression of specific
genes

Footnotes
1. E.O. List, D.E. Berryman, B. Bower, L. Sackmann-Sala, E. Gosney, J. Ding, S. Okada, and J.J. Kopchick. “The Use of
Proteomics to Study Infectious Diseases.” Infectious Disorders-Drug Targets (Formerly Current Drug Targets-Infectious
Disorders) 8 no. 1 (2008): 31–45.
2. Mohan Natesan, and Robert G. Ulrich. “Protein Microarrays and Biomarkers of Infectious Disease.” International Journal of
Molecular Sciences 11 no. 12 (2010): 5165–5183.
3. D.J. Edwards, K.E. Holt. “Beginner’s Guide to Comparative Bacterial Genome Analysis Using Next-Generation Sequence
Data.” Microbial Informatics and Experimentation 3 no. 1 (2013):2.
4. Jose-Luis Adrio and Arnold L. Demain. “Recombinant Organisms for Production of Industrial Products.” Bioengineered Bugs 1
no. 2 (2010): 116–131.

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 5. U.S. Department of Health and Human Services. “Types of Vaccines.” 2013. www.vaccines.gov/more_info/types/#subunit.
Accessed May 27, 2016.
6. The Internet Drug List. Recombivax. 2015. http://www.rxlist.com/recombivax-drug.htm. Accessed May 27, 2016.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 10.3: Whole Genome Methods and Industrial Applications is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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10.4: Genetic Engineering - Risks, Benefits, and Perceptions
Learning Objectives
Summarize the mechanisms, risks, and potential benefits of gene therapy
Identify ethical issues involving gene therapy and the regulatory agencies that provide oversight for clinical trials
Compare somatic-cell and germ-line gene therapy

Many types of genetic engineering have yielded clear benefits with few apparent risks. Few would question, for example, the value
of our now abundant supply of human insulin produced by genetically engineered bacteria. However, many emerging applications
of genetic engineering are much more controversial, often because their potential benefits are pitted against significant risks, real or
perceived. This is certainly the case for gene therapy, a clinical application of genetic engineering that may one day provide a cure
for many diseases but is still largely an experimental approach to treatment.

Mechanisms and Risks of Gene Therapy

Human diseases that result from genetic mutations are often difficult to treat with drugs or other traditional forms of therapy
because the signs and symptoms of disease result from abnormalities in a patient’s genome. For example, a patient may have a
genetic mutation that prevents the expression of a specific protein required for the normal function of a particular cell type. This is
the case in patients with Severe Combined Immunodeficiency (SCID), a genetic disease that impairs the function of certain white
blood cells essential to the immune system.
Gene therapy attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into the patient’s genome. The
nonmutated gene encodes a functional protein that the patient would otherwise be unable to produce. Viral vectors such as
adenovirus are sometimes used to introduce the functional gene; part of the viral genome is removed and replaced with the desired
gene (Figure 10.4.1). More advanced forms of gene therapy attempt to correct the mutation at the original site in the genome, such
as is the case with treatment of SCID.

Figure 10.4.1 : Gene therapy using an adenovirus vector can be used to treat or cure certain genetic diseases in which a patient has a
defective gene. (credit: modification of work by National Institutes of Health)
So far, gene therapies have proven relatively ineffective, with the possible exceptions of treatments for cystic fibrosis and
adenosine deaminase deficiency, a type of SCID. Other trials have shown the clear hazards of attempting genetic manipulation in
complex multicellular organisms like humans. In some patients, the use of an adenovirus vector can trigger an unanticipated
inflammatory response from the immune system, which may lead to organ failure. Moreover, because viruses can often target
multiple cell types, the virus vector may infect cells not targeted for the therapy, damaging these other cells and possibly leading to

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illnesses such as cancer. Another potential risk is that the modified virus could revert to being infectious and cause disease in the
patient. Lastly, there is a risk that the inserted gene could unintentionally inactivate another important gene in the patient’s genome,
disrupting normal cell cycling and possibly leading to tumor formation and cancer. Because gene therapy involves so many risks,
candidates for gene therapy need to be fully informed of these risks before providing informed consent to undergo the therapy.

Gene Therapy Gone Wrong

The risks of gene therapy were realized in the 1999 case of Jesse Gelsinger, an 18-year-old patient who received gene therapy
as part of a clinical trial at the University of Pennsylvania. Jesse received gene therapy for a condition called ornithine
transcarbamylase (OTC) deficiency, which leads to ammonia accumulation in the blood due to deficient ammonia processing.
Four days after the treatment, Jesse died after a massive immune response to the adenovirus vector. 1
Until that point, researchers had not really considered an immune response to the vector to be a legitimate risk, but on
investigation, it appears that the researchers had some evidence suggesting that this was a possible outcome. Prior to Jesse’s
treatment, several other human patients had suffered side effects of the treatment, and three monkeys used in a trial had died as
a result of inflammation and clotting disorders. Despite this information, it appears that neither Jesse nor his family were made
aware of these outcomes when they consented to the therapy. Jesse’s death was the first patient death due to a gene therapy
treatment and resulted in the immediate halting of the clinical trial in which he was involved, the subsequent halting of all other
gene therapy trials at the University of Pennsylvania, and the investigation of all other gene therapy trials in the United States.
As a result, the regulation and oversight of gene therapy overall was reexamined, resulting in new regulatory protocols that are
still in place today.

Exercise 10.4.1

1. Explain how gene therapy works in theory.

2. Identify some risks of gene therapy

Oversight of Gene Therapy

Presently, there is significant oversight of gene therapy clinical trials. At the federal level, three agencies regulate gene therapy in
parallel: the Food and Drug Administration (FDA), the Office of Human Research Protection (OHRP), and the Recombinant DNA
Advisory Committee (RAC) at the National Institutes of Health (NIH). Along with several local agencies, these federal agencies
interact with the institutional review board to ensure that protocols are in place to protect patient safety during clinical trials.
Compliance with these protocols is enforced mostly on the local level in cooperation with the federal agencies. Gene therapies are
currently under the most extensive federal and local review compared to other types of therapies, which are more typically only
under the review of the FDA. Some researchers believe that these extensive regulations actually inhibit progress in gene therapy
research. In 2013, the Institute of Medicine (now the National Academy of Medicine) called upon the NIH to relax its review of
gene therapy trials in most cases.2 However, ensuring patient safety continues to be of utmost concern.

Risky Gene Therapies

While there are currently no gene therapies on the market in the United States, many are in the pipeline and it is likely that
some will eventually be approved. With recent advances in gene therapies targeting p53, a gene whose somatic cell mutations
have been implicated in over 50% of human cancers, 3 cancer treatments through gene therapies could become much more
widespread once they reach the commercial market.
Bringing any new therapy to market poses ethical questions that pit the expected benefits against the risks. How quickly should
new therapies be brought to the market? How can we ensure that new therapies have been sufficiently tested for safety and
effectiveness before they are marketed to the public? The process by which new therapies are developed and approved
complicates such questions, as those involved in the approval process are often under significant pressure to get a new therapy
approved even in the face of significant risks.
To receive FDA approval for a new therapy, researchers must collect significant laboratory data from animal trials and submit
an Investigational New Drug (IND) application to the FDA’s Center for Drug Evaluation and Research (CDER). Following a
30-day waiting period during which the FDA reviews the IND, clinical trials involving human subjects may begin. If the FDA
perceives a problem prior to or during the clinical trial, the FDA can order a “clinical hold” until any problems are addressed.

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 During clinical trials, researchers collect and analyze data on the therapy’s effectiveness and safety, including any side effects
observed. Once the therapy meets FDA standards for effectiveness and safety, the developers can submit a New Drug
Application (NDA) that details how the therapy will be manufactured, packaged, monitored, and administered.
Because new gene therapies are frequently the result of many years (even decades) of laboratory and clinical research, they
require a significant financial investment. By the time a therapy has reached the clinical trials stage, the financial stakes are
high for pharmaceutical companies and their shareholders. This creates potential conflicts of interest that can sometimes affect
the objective judgment of researchers, their funders, and even trial participants. The Jesse Gelsinger case is a classic example.
Faced with a life-threatening disease and no reasonable treatments available, it is easy to see why a patient might be eager to
participate in a clinical trial no matter the risks. It is also easy to see how a researcher might view the short-term risks for a
small group of study participants as a small price to pay for the potential benefits of a game-changing new treatment.
Gelsinger’s death led to increased scrutiny of gene therapy, and subsequent negative outcomes of gene therapy have resulted in
the temporary halting of clinical trials pending further investigation. For example, when children in France treated with gene
therapy for SCID began to develop leukemia several years after treatment, the FDA temporarily stopped clinical trials of
similar types of gene therapy occurring in the United States. 4 Cases like these highlight the need for researchers and health
professionals not only to value human well-being and patients’ rights over profitability, but also to maintain scientific
objectivity when evaluating the risks and benefits of new therapies.

Ethical Concerns
Beyond the health risks of gene therapy, the ability to genetically modify humans poses a number of ethical issues related to the
limits of such “therapy.” While current research is focused on gene therapy for genetic diseases, scientists might one day apply
these methods to manipulate other genetic traits not perceived as desirable. This raises questions such as:
1. Which genetic traits are worthy of being “corrected”?
2. Should gene therapy be used for cosmetic reasons or to enhance human abilities?
3. Should genetic manipulation be used to impart desirable traits to the unborn?
4. Is everyone entitled to gene therapy, or could the cost of gene therapy create new forms of social inequality?
5. Who should be responsible for regulating and policing inappropriate use of gene therapies?
The ability to alter reproductive cells using gene therapy could also generate new ethical dilemmas. To date, the various types of
gene therapies have been targeted to somatic cells, the non-reproductive cells within the body. Because somatic cell traits are not
inherited, any genetic changes accomplished by somatic-cell gene therapy would not be passed on to offspring. However, should
scientists successfully introduce new genes to germ cells (eggs or sperm), the resulting traits could be passed on to offspring. This
approach, called germ-line gene therapy, could potentially be used to combat heritable diseases, but it could also lead to unintended
consequences for future generations. Moreover, there is the question of informed consent, because those impacted by germ-line
gene therapy are unborn and therefore unable to choose whether they receive the therapy. For these reasons, the U.S. government
does not currently fund research projects investigating germ-line gene therapies in humans.

Exercise 10.4.2

1. Why is gene therapy research so tightly regulated?

2. What is the main ethical concern associated with germ-line gene therapy?

Clinical Focus: Resolution

Because Kayla’s symptoms were persistent and serious enough to interfere with daily activities, Kayla’s physician decided to
order some laboratory tests. The physician collected samples of Kayla’s blood, cerebrospinal fluid (CSF), and synovial fluid
(from one of her swollen knees) and requested PCR analysis on all three samples. The PCR tests on the CSF and synovial fluid
came back positive for the presence of Borrelia burgdorferi, the bacterium that causes Lyme disease.
Kayla’s physician immediately prescribed a full course of the antibiotic doxycycline. Fortunately, Kayla recovered fully within
a few weeks and did not suffer from the long-term symptoms of post-treatment Lyme disease syndrome (PTLDS), which
affects 10–20% of Lyme disease patients. To prevent future infections, Kayla’s physician advised her to use insect repellant
and wear protective clothing during her outdoor adventures. These measures can limit exposure to Lyme-bearing ticks, which

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 are common in many regions of the United States during the warmer months of the year. Kayla was also advised to make a
habit of examining herself for ticks after returning from outdoor activities, as prompt removal of a tick greatly reduces the
chances of infection.
Lyme disease is often difficult to diagnose. B. burgdorferi is not easily cultured in the laboratory, and the initial symptoms can
be very mild and resemble those of many other diseases. But left untreated, the symptoms can become quite severe and
debilitating. In addition to two antibody tests, which were inconclusive in Kayla’s case, and the PCR test, a Southern blot could
be used with B. burgdorferi-specific DNA probes to identify DNA from the pathogen. Sequencing of surface protein genes of
Borrelia species is also being used to identify strains within the species that may be more readily transmitted to humans or
cause more severe disease.

Key Concepts and Summary

While gene therapy shows great promise for the treatment of genetic diseases, there are also significant risks involved.
There is considerable federal and local regulation of the development of gene therapies by pharmaceutical companies for use in
humans.
Before gene therapy use can increase dramatically, there are many ethical issues that need to be addressed by the medical and
research communities, politicians, and society at large.

Footnotes
1. 1 Barbara Sibbald. “Death but One Unintended Consequence of Gene-Therapy Trial.” Canadian Medical Association Journal
164 no. 11 (2001): 1612–1612.
2. 2 Kerry Grens. “Report: Ease Gene Therapy Reviews.” The Scientist, December 9, 2013. http://www.the-scientist.com/?
articl...erapy-Reviews/. Accessed May 27, 2016.
3. 3 Zhen Wang and Yi Sun. “Targeting p53 for Novel Anticancer Therapy.” Translational Oncology 3, no. 1 (2010): 1–12.
4. 4 Erika Check. “Gene Therapy: A Tragic Setback.” Nature 420 no. 6912 (2002): 116–118.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 10.4: Genetic Engineering - Risks, Benefits, and Perceptions is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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Chapter 10 Exercises
Review Questions for Chapter 10
Multiple Choice

1) Which of the following is required for repairing the phosphodiester backbone of DNA during molecular cloning?
a. cDNA
b. reverse transcriptase
c. restriction enzymes
d. DNA ligase

2) All of the following are processes used to introduce DNA molecules into bacterial cells except:
a. transformation
b. transduction
c. transcription
d. conjugation

3) The enzyme that uses RNA as a template to produce a DNA copy is called:
a. a restriction enzyme
b. DNA ligase
c. reverse transcriptase
d. DNA polymerase

4) In blue-white screening, what do blue colonies represent?

a. cells that have not taken up the plasmid vector
b. cells with recombinant plasmids containing a new insert
c. cells containing empty plasmid vectors
d. cells with a non-functional lacZ gene

5) The Ti plasmid is used for introducing genes into:

a. animal cells
b. plant cells
c. bacteriophages
d. E. coli cells

6) Which technique is used to separate protein fragments based on size?

a. polyacrylamide gel electrophoresis
b. Southern blot
c. agarose gel electrophoresis
d. polymerase chain reaction

7) Which technique uses restriction enzyme digestion followed by agarose gel electrophoresis to generate a banding pattern for
comparison to another sample processed in the same way?
a. qPCR

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 b. RT-PCR
c. RFLP
d. 454 sequencing

8) All of the following techniques involve hybridization between single-stranded nucleic acid molecules except:
a. Southern blot analysis
b. RFLP analysis
c. northern blot analysis
d. microarray analysis

9) The science of studying the entire collection of mRNA molecules produced by cells, allowing scientists to monitor differences in
gene expression patterns between cells, is called:
a. genomics
b. transcriptomics
c. proteomics
d. pharmacogenomics

10) The science of studying genomic fragments from microbial communities, allowing researchers to study genes from a collection
of multiple species, is called:
a. pharmacogenomics
b. transcriptomics
c. metagenomics
d. proteomics

11) The insulin produced by recombinant DNA technology is

a. a combination of E. coli and human insulin.
b. identical to human insulin produced in the pancreas.
c. cheaper but less effective than pig insulin for treating diabetes.
d. engineered to be more effective than human insulin.

12) At what point can the FDA halt the development or use of gene therapy?
a. on submission of an IND application
b. during clinical trials
c. after manufacturing and marketing of the approved therapy
d. all of the answers are correct

Fill-in-the-Blanks

13) The process of introducing DNA molecules into eukaryotic cells is called ________.

14) The __________ blot technique is used to find an RNA fragment within a sample that is complementary to a DNA probe.

15) The PCR step during which the double-stranded template molecule becomes single-stranded is called _____________.

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16) The sequencing method involving the incorporation of ddNTPs is called __________.

17) The application of genomics to evaluate the effectiveness and safety of drugs on the basis of information from an individual’s
genomic sequence is called ____________.

18) A gene whose expression can be easily visualized and monitored is called a ________.

19) _____________ is a common viral vector used in gene therapy for introducing a new gene into a specifically targeted cell type.

Short Answers

20) Name three elements incorporated into a plasmid vector for efficient cloning.

21) When would a scientist want to generate a cDNA library instead of a genomic library?

22) What is one advantage of generating a genomic library using phages instead of plasmids?

23) Why is it important that a DNA probe be labeled with a molecular beacon?

24) When separating proteins strictly by size, why is exposure to SDS first required?

25) Why must the DNA polymerase used during PCR be heat-stable?

26) If all cellular proteins are encoded by the cell’s genes, what information does proteomics provide that genomics cannot?

27) Briefly describe the risks associated with somatic cell gene therapy.

Critical Thinking

28) Is biotechnology always associated with genetic engineering? Explain your answer.

29) Which is more efficient: blunt-end cloning or sticky-end cloning? Why?

30) Suppose you are working in a molecular biology laboratory and are having difficulty performing the PCR successfully. You
decide to double-check the PCR protocol programmed into the thermal cycler and discover that the annealing temperature was
programmed to be 65 °C instead of 50 °C, as you had intended. What effects would this mistake have on the PCR reaction?

31) What is the advantage of microarray analysis over northern blot analysis in monitoring changes in gene expression?

32) What is the difference between reverse transcriptase PCR (RT-PCR) and real-time quantitative PCR (qPCR)?

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33) What are some advantages of cloning human genes into bacteria to treat human diseases caused by specific protein
deficiencies?

34) Compare the ethical issues involved in the use of somatic cell gene therapy and germ-line gene therapy.

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 CHAPTER OVERVIEW

11: Control of Microbial Growth

11.1: Controlling Microbial Growth
11.2: Using Physical Methods to Control Microorganisms
11.3: Using Chemicals to Control Microorganisms
11.4: Discovering Antimicrobial Drugs
11.5: Drug Targets on Prokaryote Microorganisms
11.6: Drugs for Non-prokaryote Microbes
11.7: Mechanisms for Resistance
11.8: Testing the Effectiveness of Antimicrobial Chemicals and Drugs
Chapter 11 Exercises

Thumbnail: Scanning electron microscope image of Vibrio cholerae bacteria, which infect the digestive system. (Public Domain;
T.J. Kirn, M.J. Lafferty, C.M.P Sandoe and R.K. Taylor).

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1
11.1: Controlling Microbial Growth
Learning Objectives
Compare disinfectants, antiseptics, and sterilants
Describe the principles of controlling the presence of microorganisms through sterilization and disinfection
Differentiate between microorganisms of various biological safety levels and explain methods used for handling microbes
at each level

Clinical Focus: part 1

Roberta is a 46-year-old real estate agent who recently underwent a cholecystectomy (surgery to remove painful
gallstones). The surgery was performed laparoscopically with the aid of a duodenoscope, a specialized endoscope
that allows surgeons to see inside the body with the aid of a tiny camera. On returning home from the hospital,
Roberta developed abdominal pain and a high fever. She also experienced a burning sensation during urination
and noticed blood in her urine. She notified her surgeon of these symptoms, per her postoperative instructions.

Exercise 11.1.1
What are some possible causes of Roberta’s symptoms?

To prevent the spread of human disease, it is necessary to control the growth and abundance of microbes in or on various items
frequently used by humans. Inanimate items, such as doorknobs, toys, or towels, which may harbor microbes and aid in disease
transmission, are called fomites. Two factors heavily influence the level of cleanliness required for a particular fomite and, hence,
the protocol chosen to achieve this level. The first factor is the application for which the item will be used. For example, invasive
applications that require insertion into the human body require a much higher level of cleanliness than applications that do not. The
second factor is the level of resistance to antimicrobial treatment by potential pathogens. For example, foods preserved by canning
often become contaminated with the bacterium Clostridium botulinum, which produces the neurotoxin that causes botulism.
Because C. botulinum can produce endospores that can survive harsh conditions, extreme temperatures and pressures must be used
to eliminate the endospores. Other organisms may not require such extreme measures and can be controlled by a procedure such as
washing clothes in a laundry machine.
Laboratory Biological Safety Levels
For researchers or laboratory personnel working with pathogens, the risks associated with specific pathogens determine the levels
of cleanliness and control required. The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health
(NIH) have established four classification levels, called “biological safety levels” (BSLs). Various organizations around the world,
including the World Health Organization (WHO) and the European Union (EU), use a similar classification scheme. According to
the CDC, the BSL is determined by the agent’s infectivity, ease of transmission, and potential disease severity, as well as the type
of work being done with the agent.1 Each BSL requires a different level of biocontainment to prevent contamination and spread of
infectious agents to laboratory personnel and, ultimately, the community. For example, the lowest BSL, BSL-1, requires the fewest
precautions because it applies to situations with the lowest risk for microbial infection.
BSL-1 agents are those that generally do not cause infection in healthy human adults. These include noninfectious bacteria, such as
nonpathogenic strains of Escherichia coli and Bacillus subtilis, and viruses known to infect animals other than humans, such as
baculoviruses (insect viruses). Because working with BSL-1 agents poses very little risk, few precautions are necessary. Laboratory
workers use standard aseptic technique and may work with these agents at an open laboratory bench or table, wearing personal
protective equipment (PPE) such as a laboratory coat, goggles, and gloves, as needed. Other than a sink for handwashing and doors
to separate the laboratory from the rest of the building, no additional modifications are needed. Cleaning the area before and after
working with a disinfectant like bleach is also part of the protocol.
Agents classified as BSL-2 include those that pose moderate risk to laboratory workers and the community, and are typically
“indigenous,” meaning that they are commonly found in that geographical area. These include bacteria such as Staphylococcus
aureus and Salmonella spp., and viruses like hepatitis, mumps, and measles viruses. BSL-2 laboratories require additional
precautions beyond those of BSL-1, including restricted access; required PPE, including a face shield in some circumstances; and

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the use of biological safety cabinets for procedures that may disperse agents through the air (called “aerosolization”). BSL-2
laboratories are equipped with self-closing doors, an eyewash station, and an autoclave, which is a specialized device for sterilizing
materials with pressurized steam before use or disposal. BSL-1 laboratories may also have an autoclave.
BSL-3 agents have the potential to cause lethal infections by inhalation. These may be either indigenous or “exotic,” meaning that
they are derived from a foreign location, and include pathogens such as Mycobacterium tuberculosis, Bacillus anthracis, West Nile
virus, and human immunodeficiency virus (HIV). Because of the serious nature of the infections caused by BSL-3 agents,
laboratories working with them require restricted access. Laboratory workers are under medical surveillance, possibly receiving
vaccinations for the microbes with which they work. In addition to the standard PPE already mentioned, laboratory personnel in
BSL-3 laboratories must also wear a respirator and work with microbes and infectious agents in a biological safety cabinet at all
times. BSL-3 laboratories require a hands-free sink, an eyewash station near the exit, and two sets of self-closing and locking doors
at the entrance. These laboratories are equipped with directional airflow, meaning that clean air is pulled through the laboratory
from clean areas to potentially contaminated areas. This air cannot be recirculated, so a constant supply of clean air is required.
BSL-4 agents are the most dangerous and often fatal. These microbes are typically exotic, are easily transmitted by inhalation, and
cause infections for which there are no treatments or vaccinations. Examples include Ebola virus and Marburg virus, both of which
cause hemorrhagic fevers, and smallpox virus. There are only a small number of laboratories in the United States and around the
world appropriately equipped to work with these agents. In addition to BSL-3 precautions, laboratory workers in BSL-4 facilities
must also change their clothing on entering the laboratory, shower on exiting, and decontaminate all material on exiting. While
working in the laboratory, they must either wear a full-body protective suit with a designated air supply or conduct all work within
a biological safety cabinet with a high-efficiency particulate air (HEPA)-filtered air supply and a doubly HEPA-filtered exhaust. If
wearing a suit, the air pressure within the suit must be higher than that outside the suit, so that if a leak in the suit occurs, laboratory
air that may be contaminated cannot be drawn into the suit (Figure 11.1.1). The laboratory itself must be located either in a
separate building or in an isolated portion of a building and have its own air supply and exhaust system, as well as its own
decontamination system. The BSLs are summarized in Figure 11.1.2.

Figure 11.1.1 : A protective suit like this one is an additional precaution for those who work in BSL-4 laboratories. This suit has its
own air supply and maintains a positive pressure relative to the outside, so that if a leak occurs, air will flow out of the suit, not into
it from the laboratory. (credit: modification of work by Centers for Disease Control and Prevention)

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 Figure 11.1.2 : The CDC classifies infectious agents into four biosafety levels based on potential risk to laboratory personnel and
the community. Each level requires a progressively greater level of precaution. (credit “pyramid”: modification of work by Centers
for Disease Control and Prevention)
To learn more about the four BSLs, visit the CDC’s website.

Exercise 11.1.2

What are some factors used to determine the BSL necessary for working with a specific pathogen?

Sterilization
The most extreme protocols for microbial control aim to achieve sterilization: the complete removal or killing of all vegetative
cells, endospores, and viruses from the targeted item or environment. Sterilization protocols are generally reserved for laboratory,
medical, manufacturing, and food industry settings, where it may be imperative for certain items to be completely free of
potentially infectious agents. Sterilization can be accomplished through either physical means, such as exposure to high heat,
pressure, or filtration through an appropriate filter, or by chemical means. Chemicals that can be used to achieve sterilization are
called sterilants. Sterilants effectively kill all microbes and viruses, and, with appropriate exposure time, can also kill endospores.
For many clinical purposes, aseptic technique is necessary to prevent contamination of sterile surfaces. Aseptic technique involves
a combination of protocols that collectively maintain sterility, or asepsis, thus preventing contamination of the patient with
microbes and infectious agents. Failure to practice aseptic technique during many types of clinical procedures may introduce
microbes to the patient’s body and put the patient at risk for sepsis, a systemic inflammatory response to an infection that results in
high fever, increased heart and respiratory rates, shock, and, possibly, death. Medical procedures that carry risk of contamination
must be performed in a sterile field, a designated area that is kept free of all vegetative microbes, endospores, and viruses. Sterile
fields are created according to protocols requiring the use of sterilized materials, such as packaging and drapings, and strict
procedures for washing and application of sterilants. Other protocols are followed to maintain the sterile field while the medical
procedure is being performed.

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One food sterilization protocol, commercial sterilization, uses heat at a temperature low enough to preserve food quality but high
enough to destroy common pathogens responsible for food poisoning, such as C. botulinum. Because C. botulinum and its
endospores are commonly found in soil, they may easily contaminate crops during harvesting, and these endospores can later
germinate within the anaerobic environment once foods are canned. Metal cans of food contaminated with C. botulinum will bulge
due to the microbe’s production of gases; contaminated jars of food typically bulge at the metal lid. To eliminate the risk for C.
botulinum contamination, commercial food-canning protocols are designed with a large margin of error. They assume an
impossibly large population of endospores (1012 per can) and aim to reduce this population to 1 endospore per can to ensure the
safety of canned foods. For example, low- and medium-acid foods are heated to 121 °C for a minimum of 2.52 minutes, which is
the time it would take to reduce a population of 1012 endospores per can down to 1 endospore at this temperature. Even so,
commercial sterilization does not eliminate the presence of all microbes; rather, it targets those pathogens that cause spoilage and
foodborne diseases, while allowing many nonpathogenic organisms to survive. Therefore, “sterilization” is somewhat of a
misnomer in this context, and commercial sterilization may be more accurately described as “quasi-sterilization.”

Exercise 11.1.3

What is the difference between sterilization and aseptic technique?

The Association of Surgical Technologists publishes standards for aseptic technique, including creating and maintaining a sterile
field.
Disinfecting and Antiseptics
Sterilization protocols require procedures that are not practical, or necessary, in many settings. The type of protocol required to
achieve the desired level of cleanliness depends on the particular item to be cleaned. For example, those used clinically are
categorized as critical, semicritical, and noncritical. Critical items must be sterile because they will be used inside the body, often
penetrating sterile tissues or the bloodstream; examples of critical items include surgical instruments, catheters, and intravenous
fluids. Gastrointestinal endoscopes and various types of equipment for respiratory therapies are examples of semicritical items;
they may contact mucous membranes or nonintact skin but do not penetrate tissues. Semicritical items do not typically need to be
sterilized but do require a high level of disinfection. Items that may contact but not penetrate intact skin are noncritical items;
examples are bed linens, furniture, crutches, stethoscopes, and blood pressure cuffs. These articles need to be clean but not highly
disinfected. Various other methods are used in clinical and nonclinical settings to some but not all of the microbes present.
Although the terms for these methods are often used interchangeably, there are important distinctions (Figure 11.1.3).
The process of disinfection inactivates most microbes on the surface of a fomite by using antimicrobial chemicals or heat. Because
some microbes remain, the disinfected item is not considered sterile. Ideally, disinfectants should be fast acting, stable, easy to
prepare, inexpensive, and easy to use. An example of a natural disinfectant is vinegar; its acidity kills most microbes. Chemical
disinfectants, such as chlorine bleach or products containing chlorine, are used to clean nonliving surfaces such as laboratory
benches, clinical surfaces, and bathroom sinks. Typical disinfection does not lead to sterilization because endospores tend to
survive even when all vegetative cells have been killed.
Unlike disinfectants, antiseptics are antimicrobial chemicals safe for use on living skin or tissues. Examples of antiseptics include
hydrogen peroxide and isopropyl alcohol. The process of applying an antiseptic is called antisepsis. In addition to the
characteristics of a good disinfectant, antiseptics must also be selectively effective against microorganisms and able to penetrate
tissue deeply without causing tissue damage.

Sanitizing and Degerming

For the items that need to be cleaned, but are noncritical items, degerming or sanitizing are good options. These techniques reduce
the microbial load on items by removing them from the items being cleaned. Notice that these terms often do not kill microbes, and
if they do kill it is only a few of the microbes. Both of these methods rely on the fact that most humans have a functioning immune
system and can tolerate and overcome some contact with microbes.
The term sanitization refers to the cleansing of fomites (inanimate surfaces) to remove enough microbes to achieve levels deemed
safe for public health. For example, commercial dishwashers used in the food service industry typically use very hot water and air
for washing and drying; the high temperatures kill most microbes, sanitizing the dishes. Surfaces in hospital rooms are commonly
sanitized using a chemical disinfectant at lower concentrations to prevent disease transmission between patients. Figure 11.1.4
summarizes common protocols, definitions, applications, and agents used to control microbial growth.

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The act of handwashing is an example of degerming, in which microbial numbers are significantly reduced by gently scrubbing
living tissue, most commonly skin, with a mild chemical (e.g., soap) to avoid the transmission of pathogenic microbes. Wiping the
skin with an alcohol swab at an injection site is another example of degerming. These degerming methods remove most (but not
all) microbes from the skin’s surface. It makes use of soaps natural amphipathic nature to pick up and drag the microbes down the
drain. This is why its important to wash your hands for the recommended amount of time.

Handwashing the Right Way

Handwashing is critical for public health and should be emphasized in a clinical setting. For the general public, the CDC
recommends handwashing before, during, and after food handling; before eating; before and after interacting with someone
who is ill; before and after treating a wound; after using the toilet or changing diapers; after coughing, sneezing, or blowing the
nose; after handling garbage; and after interacting with an animal, its feed, or its waste. Figure 11.1.3 illustrates the five steps
of proper handwashing recommended by the CDC.
Handwashing is even more important for health-care workers, who should wash their hands thoroughly between every patient
contact, after the removal of gloves, after contact with bodily fluids and potentially infectious fomites, and before and after
assisting a surgeon with invasive procedures. Even with the use of proper surgical attire, including gloves, scrubbing for
surgery is more involved than routine handwashing. The goal of surgical scrubbing is to reduce the normal microbiota on the
skin’s surface to prevent the introduction of these microbes into a patient’s surgical wounds.
There is no single widely accepted protocol for surgical scrubbing. Protocols for length of time spent scrubbing may depend on
the antimicrobial used; health-care workers should always check the manufacturer’s recommendations. According to the
Association of Surgical Technologists (AST), surgical scrubs may be performed with or without the use of brushes (Figure
11.1.3).

Figure 11.1.3 : (a) The CDC recommends five steps as part of typical handwashing for the general public. (b) Surgical
scrubbing is more extensive, requiring scrubbing starting from the fingertips, extending to the hands and forearms, and then up
beyond the elbows, as shown here. (credit a: modification of work by World Health Organization)

To learn more about proper handwashing, visit the CDC’s website.

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 Figure 11.1.4 : Common protocols for control of microbial growth

Exercise 11.1.4
1. What is the difference between a disinfectant and an antiseptic?
2. Which is most effective at removing microbes from a product: sanitization, degerming, or sterilization? Explain.

Measuring Microbial Control

Physical and chemical methods of microbial control that kill the targeted microorganism are identified by the suffix -cide (or -
cidal). The prefix indicates the type of microbe or infectious agent killed by the treatment method: bactericides kill bacteria,
viricides kill or inactivate viruses, and fungicides kill fungi. Other methods do not kill organisms but, instead, stop their growth
(reproduction rate), making their population static; such methods are identified by the suffix -stat (or -static). For example,
bacteriostatic treatments inhibit the growth of bacteria, whereas fungistatic treatments inhibit the growth of fungi. Factors that
determine whether a particular treatment is -cidal or -static include the types of microorganisms targeted, the concentration of the
chemical used, and the nature of the treatment applied.
Although -static treatments do not actually kill infectious agents, they are often less toxic to humans and other animals, and may
also better preserve the integrity of the item treated. Such treatments are typically sufficient to keep the microbial population of an
item in check. The reduced toxicity of some of these -static chemicals also allows them to be impregnated safely into plastics to
prevent the growth of microbes on these surfaces. Such plastics are used in products such as toys for children and cutting boards for
food preparation. When used to treat an infection, -static treatments are typically sufficient in an otherwise healthy individual,
preventing the pathogen from multiplying, thus allowing the individual’s immune system to clear the infection.
The degree of microbial control can be evaluated using a microbial death curve to describe the progress and effectiveness of a
particular protocol. When exposed to a particular microbial control protocol, a fixed percentage of the microbes within the
population will die. Because the rate of killing remains constant even when the population size varies, the percentage killed is more

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useful information than the absolute number of microbes killed. Death curves are often plotted as semilog plots just like microbial
growth curves because the reduction in microorganisms is typically logarithmic (Figure 11.1.5). The amount of time it takes for a
specific protocol to produce a one order-of-magnitude decrease in the number of organisms, or the death of 90% of the population,
is called the decimal reduction time (DRT) or D-value.

Figure 11.1.5 : Microbial death is logarithmic and easily observed using a semilog plot instead of an arithmetic one. The decimal
reduction time (D-value) is the time it takes to kill 90% of the population (a 1-log decrease in the total population) when exposed to
a specific microbial control protocol, as indicated by the purple bracket.
Several factors contribute to the effectiveness of a disinfecting agent or microbial control protocol. First, as demonstrated in Figure
11.1.4, the length of time of exposure is important. Longer exposure times kill more microbes. Because microbial death of a

population exposed to a specific protocol is logarithmic, it takes longer to kill a high-population load than a low-population load
exposed to the same protocol. A shorter treatment time (measured in multiples of the D-value) is needed when starting with a
smaller number of organisms. Effectiveness also depends on the susceptibility of the agent to that disinfecting agent or protocol.
The concentration of disinfecting agent or intensity of exposure is also important. For example, higher temperatures and higher
concentrations of disinfectants kill microbes more quickly and effectively. Conditions that limit contact between the agent and the
targeted cells cells—for example, the presence of bodily fluids, tissue, organic debris (e.g., mud or feces), or biofilms on surfaces—
increase the cleaning time or intensity of the microbial control protocol required to reach the desired level of cleanliness. All these
factors must be considered when choosing the appropriate protocol to control microbial growth in a given situation.

Exercise 11.1.6

1. What are two possible reasons for choosing a bacteriostatic treatment over a bactericidal one?
2. Name at least two factors that can compromise the effectiveness of a disinfecting agent.

Key Concepts and Summary

Inanimate items that may harbor microbes and aid in their transmission are called fomites. The level of cleanliness required for
a fomite depends both on the item’s use and the infectious agent with which the item may be contaminated.
The CDC and the NIH have established four biological safety levels (BSLs) for laboratories performing research on infectious
agents. Each level is designed to protect laboratory personnel and the community. These BSLs are determined by the agent’s
infectivity, ease of transmission, and potential disease severity, as well as the type of work being performed with the agent.
Disinfection removes potential pathogens from a fomite, whereas antisepsis uses antimicrobial chemicals safe enough for
tissues; in both cases, microbial load is reduced, but microbes may remain unless the chemical used is strong enough to be a
sterilant.

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 The amount of cleanliness (sterilization versus high-level disinfection versus general cleanliness) required for items used
clinically depends on whether the item will come into contact with sterile tissues (critical item), mucous membranes
(semicritical item), or intact skin (noncritical item).
Medical procedures with a risk for contamination should be carried out in a sterile field maintained by proper aseptic
technique to prevent sepsis.
Sterilization is necessary for some medical applications as well as in the food industry, where endospores of Clostridium
botulinum are killed through commercial sterilization protocols.
Physical or chemical methods to control microbial growth that result in death of the microbe are indicated by the suffixes -cide
or -cidal (e.g., as with bactericides, viricides, and fungicides), whereas those that inhibit microbial growth are indicated by the
suffixes -stat or-static (e.g., bacteriostatic, fungistatic).
Microbial death curves display the logarithmic decline of living microbes exposed to a method of microbial control. The time
it takes for a protocol to yield a 1-log (90%) reduction in the microbial population is the decimal reduction time, or D-value.
When choosing a microbial control protocol, factors to consider include the length of exposure time, the type of microbe
targeted, its susceptibility to the protocol, the intensity of the treatment, the presence of organics that may interfere with the
protocol, and the environmental conditions that may alter the effectiveness of the protocol.

Footnotes
1. US Centers for Disease Control and Prevention. “Recognizing the Biosafety Levels.”
http://www.cdc.gov/training/quicklearns/biosafety/. Accessed June 7, 2016.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.1: Controlling Microbial Growth is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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11.2: Using Physical Methods to Control Microorganisms
Learning Objectives
Understand and compare various physical methods of controlling microbial growth, including heating, refrigeration,
freezing, high-pressure treatment, desiccation, lyophilization, irradiation, and filtration

For thousands of years, humans have used various physical methods of microbial control for food preservation. Common control
methods include the application of high temperatures, radiation, filtration, and desiccation (drying), among others. Many of these
methods nonspecifically kill cells by disrupting membranes, changing membrane permeability, or damaging proteins and nucleic
acids by denaturation, degradation, or chemical modification. Various physical methods used for microbial control are described in
this section.

Heat
Heating is one of the most common—and oldest—forms of microbial control. It is used in simple techniques like cooking and
canning. Heat can kill microbes by altering their membranes and denaturing proteins. The thermal death point (TDP) of a
microorganism is the lowest temperature at which all microbes are killed in a 10-minute exposure. Different microorganisms will
respond differently to high temperatures, with some (e.g., endospore-formers such as C. botulinum) being more heat tolerant. A
similar parameter, the thermal death time (TDT), is the length of time needed to kill all microorganisms in a sample at a given
temperature. These parameters are often used to describe sterilization procedures that use high heat, such as autoclaving. Boiling is
one of the oldest methods of moist-heat control of microbes, and it is typically quite effective at killing vegetative cells and some
viruses. However, boiling is less effective at killing endospores; some endospores are able to survive up to 20 hours of boiling.
Additionally, boiling may be less effective at higher altitudes, where the boiling point of water is lower and the boiling time needed
to kill microbes is therefore longer. For these reasons, boiling is not considered a useful sterilization technique in the laboratory or
clinical setting.
Many different heating protocols can be used for sterilization in the laboratory or clinic, and these protocols can be broken down
into two main categories: dry-heat sterilization and moist-heat sterilization. Aseptic technique in the laboratory typically involves
some dry-heat sterilization protocols using direct application of high heat, such as sterilizing inoculating loops (Figure 11.2.1).
Incineration at very high temperatures destroys all microorganisms. Dry heat can also be applied for relatively long periods of time
(at least 2 hours) at temperatures up to 170 °C by using a dry-heat sterilizer, such as an oven. However, moist-heat sterilization is
typically the more effective protocol because it penetrates cells better than dry heat does.

Figure 11.2.1 : (a) Sterilizing a loop, often referred to as “flaming a loop,” is a common component of aseptic technique in the
microbiology laboratory and is used to incinerate any microorganisms on the loop. (b) Alternatively, a bactericinerator may be used
to reduce aerosolization of microbes and remove the presence of an open flame in the laboratory. These are examples of dry-heat
sterilization by the direct application of high heat capable of incineration. (credit a: modification of work by Anh-Hue Tu; credit b:
modification of work by Brian Forster)

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Autoclaves
Autoclaves rely on moist-heat sterilization. They are used to raise temperatures above the boiling point of water to sterilize items
such as surgical equipment from vegetative cells, viruses, and especially endospores, which are known to survive boiling
temperatures, without damaging the items. Charles Chamberland (1851–1908) designed the modern autoclave in 1879 while
working in the laboratory of Louis Pasteur. The autoclave is still considered the most effective method of sterilization (Figure
11.2.2). Outside laboratory and clinical settings, large industrial autoclaves called retorts allow for moist-heat sterilization on a

large scale.
In general, the air in the chamber of an autoclave is removed and replaced with increasing amounts of steam trapped within the
enclosed chamber, resulting in increased interior pressure and temperatures above the boiling point of water. The two main types of
autoclaves differ in the way that air is removed from the chamber. In gravity displacement autoclaves, steam is introduced into the
chamber from the top or sides. Air, which is heavier than steam, sinks to the bottom of the chamber, where it is forced out through
a vent. Complete displacement of air is difficult, especially in larger loads, so longer cycles may be required for such loads. In
prevacuum sterilizers, air is removed completely using a high-speed vacuum before introducing steam into the chamber. Because
air is more completely eliminated, the steam can more easily penetrate wrapped items. Many autoclaves are capable of both gravity
and prevacuum cycles, using the former for the decontamination of waste and sterilization of media and unwrapped glassware, and
the latter for sterilization of packaged instruments.

Figure 11.2.2 : (a) An autoclave is commonly used for sterilization in the laboratory and in clinical settings. By displacing the air in
the chamber with increasing amounts of steam, pressure increases, and temperatures exceeding 100 °C can be achieved, allowing
for complete sterilization. (b) A researcher programs an autoclave to sterilize a sample. (credit a: modification of work by Courtney
Harrington; credit b: modification of work by Lackemeyer MG, Kok-Mercado Fd, Wada J, Bollinger L, Kindrachuk J, Wahl-Jensen
V, Kuhn JH, Jahrling PB)
Standard operating temperatures for autoclaves are 121 °C or, in some cases, 132 °C, typically at a pressure of 15 to 20 pounds per
square inch (psi). The length of exposure depends on the volume and nature of material being sterilized, but it is typically 20
minutes or more, with larger volumes requiring longer exposure times to ensure sufficient heat transfer to the materials being
sterilized. The steam must directly contact the liquids or dry materials being sterilized, so containers are left loosely closed and
instruments are loosely wrapped in paper or foil. The key to autoclaving is that the temperature must be high enough to kill
endospores to achieve complete sterilization.
Because sterilization is so important to safe medical and laboratory protocols, quality control is essential. Autoclaves may be
equipped with recorders to document the pressures and temperatures achieved during each run. Additionally, internal indicators of
various types should be autoclaved along with the materials to be sterilized to ensure that the proper sterilization temperature has
been reached (Figure 11.2.3). One common type of indicator is the use of heat-sensitive autoclave tape, which has white stripes
that turn black when the appropriate temperature is achieved during a successful autoclave run. This type of indicator is relatively
inexpensive and can be used during every run. However, autoclave tape provides no indication of length of exposure, so it cannot
be used as an indicator of sterility. Another type of indicator, a biological indicator spore test, uses either a strip of paper or a liquid
suspension of the endospores of Geobacillus stearothermophilus to determine whether the endospores are killed by the process.

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The endospores of the obligate thermophilic bacterium G. stearothermophilus are the gold standard used for this purpose because
of their extreme heat resistance. Biological spore indicators can also be used to test the effectiveness of other sterilization protocols,
including ethylene oxide, dry heat, formaldehyde, gamma radiation, and hydrogen peroxide plasma sterilization using either G.
stearothermophilus, Bacillus atrophaeus, B. subtilis, or B. pumilus spores. In the case of validating autoclave function, the
endospores are incubated after autoclaving to ensure no viable endospores remain. Bacterial growth subsequent to endospore
germination can be monitored by biological indicator spore tests that detect acid metabolites or fluorescence produced by enzymes
derived from viable G. stearothermophilus. A third type of autoclave indicator is the Diack tube, a glass ampule containing a
temperature-sensitive pellet that melts at the proper sterilization temperature. Spore strips or Diack tubes are used periodically to
ensure the autoclave is functioning properly.

Figure 11.2.3 : The white strips on autoclave tape (left tube) turn dark during a successful autoclave run (right tube). (credit:
modification of work by Brian Forster)

Pasteurization
Although complete sterilization is ideal for many medical applications, it is not always practical for other applications and may also
alter the quality of the product. Boiling and autoclaving are not ideal ways to control microbial growth in many foods because these
methods may ruin the consistency and other organoleptic (sensory) qualities of the food. Pasteurization is a form of microbial
control for food that uses heat but does not render the food sterile. Traditional pasteurization kills pathogens and reduces the
number of spoilage-causing microbes while maintaining food quality. The process of pasteurization was first developed by Louis
Pasteur in the 1860s as a method for preventing the spoilage of beer and wine. Today, pasteurization is most commonly used to kill
heat-sensitive pathogens in milk and other food products (e.g., apple juice and honey) (Figure 11.2.4). However, because
pasteurized food products are not sterile, they will eventually spoil.
The methods used for milk pasteurization balance the temperature and the length of time of treatment. One method, high-
temperature short-time (HTST) pasteurization, exposes milk to a temperature of 72 °C for 15 seconds, which lowers bacterial
numbers while preserving the quality of the milk. An alternative is ultra-high-temperature (UHT) pasteurization, in which the milk
is exposed to a temperature of 138 °C for 2 or more seconds. UHT pasteurized milk can be stored for a long time in sealed

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containers without being refrigerated; however, the very high temperatures alter the proteins in the milk, causing slight changes in
the taste and smell. Still, this method of pasteurization is advantageous in regions where access to refrigeration is limited.

Figure 11.2.4 : Two different methods of pasteurization, HTST and UHT, are commonly used to kill pathogens associated with milk
spoilage. (credit left: modification of work by Mark Hillary; credit right: modification of work by Kerry Ceszyk)

Exercise 11.2.1
1. In an autoclave, how are temperatures above boiling achieved?
2. How would the onset of spoilage compare between HTST-pasteurized and UHT-pasteurized milk?
3. Why is boiling not used as a sterilization method in a clinical setting?

Clinical Focus: part 2

Roberta’s physician suspected that a bacterial infection was responsible for her sudden-onset high fever,
abdominal pain, and bloody urine. Based on these symptoms, the physician diagnosed a urinary tract infection
(UTI). A wide variety of bacteria may cause UTIs, which typically occur when bacteria from the lower
gastrointestinal tract are introduced to the urinary tract. However, Roberta’s recent gallstone surgery caused the
physician to suspect that she had contracted a nosocomial (hospital-acquired) infection during her surgery. The
physician took a urine sample and ordered a urine culture to check for the presence of white blood cells, red blood
cells, and bacteria. The results of this test would help determine the cause of the infection. The physician also
prescribed a course of the antibiotic ciprofloxacin, confident that it would clear Roberta’s infection.

Exercise 11.2.5

What are some possible ways that bacteria could have been introduced to Roberta’s urinary tract during her surgery?

Refrigeration and Freezing

Just as high temperatures are effective for controlling microbial growth, exposing microbes to low temperatures can also be an easy
and effective method of microbial control, with the exception of psychrophiles, which prefer cold temperatures. Refrigerators used

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in home kitchens or in the laboratory maintain temperatures between 0 °C and 7 °C. This temperature range inhibits microbial
metabolism, slowing the growth of microorganisms significantly and helping preserve refrigerated products such as foods or
medical supplies. Certain types of laboratory cultures can be preserved by refrigeration for later use.
Freezing below −2 °C may stop microbial growth and even kill susceptible organisms. According to the US Department of
Agriculture (USDA), the only safe ways that frozen foods can be thawed are in the refrigerator, immersed in cold water changed
every 30 minutes, or in the microwave, keeping the food at temperatures not conducive for bacterial growth.1In addition, halted
bacterial growth can restart in thawed foods, so thawed foods should be treated like fresh perishables.
Bacterial cultures and medical specimens requiring long-term storage or transport are often frozen at ultra-low temperatures of −70
°C or lower. These ultra-low temperatures can be achieved by storing specimens on dry ice in an ultra-low freezer or in special
liquid nitrogen tanks, which maintain temperatures lower than −196 °C (Figure 11.2.5).

Figure 11.2.5 : Cultures and other medical specimens can be stored for long periods at ultra-low temperatures. (a) An ultra-low
freezer maintains temperatures at or below −70 °C. (b) Even lower temperatures can be achieved through freezing and storage in
liquid nitrogen. (credit a: modification of work by “Expert Infantry”/Flickr; credit b: modification of work by USDA)

Exercise 11.2.2

Does placing food in a refrigerator kill bacteria on the food?

Pressure
Exposure to high pressure kills many microbes. In the food industry, high-pressure processing (also called pascalization) is used to
kill bacteria, yeast, molds, parasites, and viruses in foods while maintaining food quality and extending shelf life. The application
of high pressure between 100 and 800 MPa (sea level atmospheric pressure is about 0.1 MPa) is sufficient to kill vegetative cells by
protein denaturation, but endospores may survive these pressures.23
In clinical settings, hyperbaric oxygen therapy is sometimes used to treat infections. In this form of therapy, a patient breathes pure
oxygen at a pressure higher than normal atmospheric pressure, typically between 1 and 3 atmospheres (atm). This is achieved by
placing the patient in a hyperbaric chamber or by supplying the pressurized oxygen through a breathing tube. Hyperbaric oxygen
therapy helps increase oxygen saturation in tissues that become hypoxic due to infection and inflammation. This increased oxygen
concentration enhances the body’s immune response by increasing the activities of neutrophils and macrophages, white blood cells
that fight infections. Increased oxygen levels also contribute to the formation of toxic free radicals that inhibit the growth of
oxygen-sensitive or anaerobic bacteria like as Clostridium perfringens, a common cause of gas gangrene. In C. perfringens
infections, hyperbaric oxygen therapy can also reduce secretion of a bacterial toxin that causes tissue destruction. Hyperbaric
oxygen therapy also seems to enhance the effectiveness of antibiotic treatments. Unfortunately, some rare risks include oxygen

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toxicity and effects on delicate tissues, such as the eyes, middle ear, and lungs, which may be damaged by the increased air
pressure.
High pressure processing is not commonly used for disinfection or sterilization of fomites. Although the application of pressure and
steam in an autoclave is effective for killing endospores, it is the high temperature achieved, and not the pressure directly, that
results in endospore death.

A Streak of Bad Potluck

One Monday in spring 2015, an Ohio woman began to experience blurred, double vision; difficulty swallowing; and drooping
eyelids. She was rushed to the emergency department of her local hospital. During the examination, she began to experience
abdominal cramping, nausea, paralysis, dry mouth, weakness of facial muscles, and difficulty speaking and breathing. Based
on these symptoms, the hospital’s incident command center was activated, and Ohio public health officials were notified of a
possible case of botulism. Meanwhile, other patients with similar symptoms began showing up at other local hospitals. Because
of the suspicion of botulism, antitoxin was shipped overnight from the CDC to these medical facilities, to be administered to
the affected patients. The first patient died of respiratory failure as a result of paralysis, and about half of the remaining victims
required additional hospitalization following antitoxin administration, with at least two requiring ventilators for breathing.
Public health officials investigated each of the cases and determined that all of the patients had attended the same church
potluck the day before. Moreover, they traced the source of the outbreak to a potato salad made with home-canned potatoes.
More than likely, the potatoes were canned using boiling water, a method that allows endospores of Clostridium botulinum to
survive. C. botulinum produces botulinum toxin, a neurotoxin that is often deadly once ingested. According to the CDC, the
Ohio case was the largest botulism outbreak in the United States in nearly 40 years. 4
Killing C. botulinum endospores requires a minimum temperature of 116 °C (240 °F), well above the boiling point of water.
This temperature can only be reached in a pressure canner, which is recommended for home canning of low-acid foods such as
meat, fish, poultry, and vegetables (Figure 11.2.6). Additionally, the CDC recommends boiling home-canned foods for about
10 minutes before consumption. Since the botulinum toxin is heat labile (meaning that it is denatured by heat), 10 minutes of
boiling will render nonfunctional any botulinum toxin that the food may contain.

Figure 11.2.6 : (a) Clostridium botulinum is the causative agent of botulism. (b) A pressure canner is recommended for home
canning because endospores of C. botulinum can survive temperatures above the boiling point of water. (credit a: modification
of work by Centers for Disease Control and Prevention; credit b: modification of work by National Center for Home Food
Preservation)

To learn more about proper home-canning techniques, visit the CDC’s website.

Desiccation
Drying, also known as desiccation or dehydration, is a method that has been used for millennia to preserve foods such as raisins,
prunes, and jerky. It works because all cells, including microbes, require water for their metabolism and survival. Although drying
controls microbial growth, it might not kill all microbes or their endospores, which may start to regrow when conditions are more
favorable and water content is restored.

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In some cases, foods are dried in the sun, relying on evaporation to achieve desiccation. Freeze-drying, or lyophilization, is another
method of dessication in which an item is rapidly frozen (“snap-frozen”) and placed under vacuum so that water is lost by
sublimation. Lyophilization combines both exposure to cold temperatures and desiccation, making it quite effective for controlling
microbial growth. In addition, lyophilization causes less damage to an item than conventional desiccation and better preserves the
item’s original qualities. Lyophilized items may be stored at room temperature if packaged appropriately to prevent moisture
acquisition. Lyophilization is used for preservation in the food industry and is also used in the laboratory for the long-term storage
and transportation of microbial cultures.
The water content of foods and materials, called the water activity, can be lowered without physical drying by the addition of
solutes such as salts or sugars. At very high concentrations of salts or sugars, the amount of available water in microbial cells is
reduced dramatically because water will be drawn from an area of low solute concentration (inside the cell) to an area of high
solute concentration (outside the cell) (Figure 11.2.7). Many microorganisms do not survive these conditions of high osmotic
pressure. Honey, for example, is 80% sucrose, an environment in which very few microorganisms are capable of growing, thereby
eliminating the need for refrigeration. Salted meats and fish, like ham and cod, respectively, were critically important foods before
the age of refrigeration. Fruits were preserved by adding sugar, making jams and jellies. However, certain microbes, such as molds
and yeasts, tend to be more tolerant of desiccation and high osmotic pressures, and, thus, may still contaminate these types of
foods.

Figure 11.2.7 : (a) The addition of a solute creates a hypertonic environment, drawing water out of cells. (b) Some foods can be
dried directly, like raisins and jerky. Other foods are dried with the addition of salt, as in the case of salted fish, or sugar, as in the
case of jam. (credit a: modification of work by “Bruce Blaus”/Wikimedia Commons; credit raisins: modification of work by
Christian Schnettelker; credit jerky: modification of work by Larry Jacobsen; credit salted fish: modification of work by “The
Photographer”/Wikimedia Commons; credit jam: modification of work by Kim Becker)

Exercise 11.2.3

How does the addition of salt or sugar to food affect its water activity?

Radiation
Radiation in various forms, from high-energy radiation to sunlight, can be used to kill microbes or inhibit their growth. Ionizing
radiation includes X-rays, gamma rays, and high-energy electron beams. Ionizing radiation is strong enough to pass into the cell,
where it alters molecular structures and damages cell components. For example, ionizing radiation introduces double-strand breaks
in DNA molecules. This may directly cause DNA mutations to occur, or mutations may be introduced when the cell attempts to
repair the DNA damage. As these mutations accumulate, they eventually lead to cell death.
Both X-rays and gamma rays easily penetrate paper and plastic and can therefore be used to sterilize many packaged materials. In
the laboratory, ionizing radiation is commonly used to sterilize materials that cannot be autoclaved, such as plastic Petri dishes and
disposable plastic inoculating loops. For clinical use, ionizing radiation is used to sterilize gloves, intravenous tubing, and other
latex and plastic items used for patient care. Ionizing radiation is also used for the sterilization of other types of delicate, heat-
sensitive materials used clinically, including tissues for transplantation, pharmaceutical drugs, and medical equipment.

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In Europe, gamma irradiation for food preservation is widely used, although it has been slow to catch on in the United States.
Packaged dried spices are also often gamma-irradiated. Because of their ability to penetrate paper, plastic, thin sheets of wood and
metal, and tissue, great care must be taken when using X-rays and gamma irradiation. These types of ionizing irradiation cannot
penetrate thick layers of iron or lead, so these metals are commonly used to protect humans who may be potentially exposed.
Another type of radiation, nonionizing radiation, is commonly used for sterilization and uses less energy than ionizing radiation. It
does not penetrate cells or packaging. Ultraviolet (UV) light is one example; it causes thymine dimers to form between adjacent
thymines within a single strand of DNA (Figure 11.2.8). When DNA polymerase encounters the thymine dimer, it does not always
incorporate the appropriate complementary nucleotides (two adenines), and this leads to formation of mutations that can ultimately
kill microorganisms.
UV light can be used effectively by both consumers and laboratory personnel to control microbial growth. UV lamps are now
commonly incorporated into water purification systems for use in homes. In addition, small portable UV lights are commonly used
by campers to purify water from natural environments before drinking. Germicidal lamps are also used in surgical suites, biological
safety cabinets, and transfer hoods, typically emitting UV light at a wavelength of 260 nm. Because UV light does not penetrate
surfaces and will not pass through plastics or glass, cells must be exposed directly to the light source.
Sunlight has a very broad spectrum that includes UV and visible light. In some cases, sunlight can be effective against certain
bacteria because of both the formation of thymine dimers by UV light and by the production of reactive oxygen products induced
in low amounts by exposure to visible light.

Figure 11.2.8 : (a) UV radiation causes the formation of thymine dimers in DNA, leading to lethal mutations in the exposed
microbes. (b) Germicidal lamps that emit UV light are commonly used in the laboratory to sterilize equipment.

Exercise 11.2.4

1. What are two advantages of ionizing radiation as a sterilization method?

2. How does the effectiveness of ionizing radiation compare with that of nonionizing radiation?

Irradiated Food: Would You Eat That?

Of all the ways to prevent food spoilage and foodborne illness, gamma irradiation may be the most unappetizing. Although
gamma irradiation is a proven method of eliminating potentially harmful microbes from food, the public has yet to buy in.
Most of their concerns, however, stem from misinformation and a poor understanding of the basic principles of radiation.
The most common method of irradiation is to expose food to cobalt-60 or cesium-137 by passing it through a radiation
chamber on a conveyor belt. The food does not directly contact the radioactive material and does not become radioactive itself.
Thus, there is no risk for exposure to radioactive material through eating gamma-irradiated foods. Additionally, irradiated
foods are not significantly altered in terms of nutritional quality, aside from the loss of certain vitamins, which is also
exacerbated by extended storage. Alterations in taste or smell may occur in irradiated foods with high fat content, such as fatty
meats and dairy products, but this effect can be minimized by using lower doses of radiation at colder temperatures.
In the United States, the CDC, Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA) have
deemed irradiation safe and effective for various types of meats, poultry, shellfish, fresh fruits and vegetables, eggs with shells,

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 and spices and seasonings. Gamma irradiation of foods has also been approved for use in many other countries, including
France, the Netherlands, Portugal, Israel, Russia, China, Thailand, Belgium, Australia, and South Africa. To help ameliorate
consumer concern and assist with education efforts, irradiated foods are now clearly labeled and marked with the international
irradiation symbol, called the “radura” (Figure 11.2.9). Consumer acceptance seems to be rising, as indicated by several recent
studies.

Figure (a) Foods are exposed to gamma radiation by passage on a conveyor

11.2.9 :
belt through a radiation chamber. (b) Gamma-irradiated foods must be clearly
labeled and display the irradiation symbol, known as the “radura.” (credit a, b:
modification of work by U.S. Department of Agriculture)

Sonication
The use of high-frequency ultrasound waves to disrupt cell structures is called sonication. Application of ultrasound waves causes
rapid changes in pressure within the intracellular liquid; this leads to cavitation, the formation of bubbles inside the cell, which can
disrupt cell structures and eventually cause the cell to lyse or collapse. Sonication is useful in the laboratory for efficiently lysing
cells to release their contents for further research; outside the laboratory, sonication is used for cleaning surgical instruments,
lenses, and a variety of other objects such as coins, tools, and musical instruments.

Filtration
Filtration is a method of physically separating microbes from samples. Air is commonly filtered through high-efficiency particulate
air (HEPA) filters (Figure 11.2.10). HEPA filters have effective pore sizes of 0.3 µm, small enough to capture bacterial cells,
endospores, and many viruses, as air passes through these filters, nearly sterilizing the air on the other side of the filter. HEPA
filters have a variety of applications and are used widely in clinical settings, in cars and airplanes, and even in the home. For
example, they may be found in vacuum cleaners, heating and air-conditioning systems, and air purifiers.

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 Figure 11.2.10: (a) HEPA filters like this one remove microbes, endospores, and viruses as air flows through them. (b) A schematic
of a HEPA filter. (credit a: modification of work by CSIRO; credit b: modification of work by “LadyofHats”/Mariana Ruiz
Villareal)

Biological Safety Cabinets

Biological safety cabinets are a good example of the use of HEPA filters. HEPA filters in biological safety cabinets (BSCs) are used
to remove particulates in the air either entering the cabinet (air intake), leaving the cabinet (air exhaust), or treating both the intake
and exhaust. Use of an air-intake HEPA filter prevents environmental contaminants from entering the BSC, creating a clean area
for handling biological materials. Use of an air-exhaust HEPA filter prevents laboratory pathogens from contaminating the
laboratory, thus maintaining a safe work area for laboratory personnel.
There are three classes of BSCs: I, II, and III. Each class is designed to provide a different level of protection for laboratory
personnel and the environment; BSC II and III are also designed to protect the materials or devices in the cabinet. Table 11.2.1
summarizes the level of safety provided by each class of BSC for each BSL.
Table 11.2.1 : Biological risks and BSCs

Biological Risk Assessed BSC Class Protection of Personnel Protection of Environment Protection of Product

BSL-1, BSL-2, BSL-3 I Yes Yes No

BSL-1, BSL-2, BSL-3 II Yes Yes Yes

III; II when used in suit

BSL-4 Yes Yes Yes
room with suit

Class I BSCs protect laboratory workers and the environment from a low to moderate risk for exposure to biological agents used in
the laboratory. Air is drawn into the cabinet and then filtered before exiting through the building’s exhaust system. Class II BSCs
use directional air flow and partial barrier systems to contain infectious agents. Class III BSCs are designed for working with
highly infectious agents like those used in BSL-4 laboratories. They are gas tight, and materials entering or exiting the cabinet must
be passed through a double-door system, allowing the intervening space to be decontaminated between uses. All air is passed
through one or two HEPA filters and an air incineration system before being exhausted directly to the outdoors (not through the
building’s exhaust system). Personnel can manipulate materials inside the Class III cabinet by using long rubber gloves sealed to
the cabinet.
This video shows how BSCs are designed and explains how they protect personnel, the environment, and the product.

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Filtration in Hospitals
HEPA filters are also commonly used in hospitals and surgical suites to prevent contamination and the spread of airborne microbes
through ventilation systems. HEPA filtration systems may be designed for entire buildings or for individual rooms. For example,
burn units, operating rooms, or isolation units may require special HEPA-filtration systems to remove opportunistic pathogens from
the environment because patients in these rooms are particularly vulnerable to infection.

Membrane Filters
Filtration can also be used to remove microbes from liquid samples using membrane filtration. Membrane filters for liquids
function similarly to HEPA filters for air. Typically, membrane filters that are used to remove bacteria have an effective pore size of
0.2 µm, smaller than the average size of a bacterium (1 µm), but filters with smaller pore sizes are available for more specific
needs. Membrane filtration is useful for removing bacteria from various types of heat-sensitive solutions used in the laboratory,
such as antibiotic solutions and vitamin solutions. Large volumes of culture media may also be filter sterilized rather than
autoclaved to protect heat-sensitive components. Often when filtering small volumes, syringe filters are used, but vacuum filters are
typically used for filtering larger volumes (Figure 11.2.11).

Figure 11.2.11: Membrane filters come in a variety of sizes, depending on the volume of solution being filtered. (a) Larger
volumes are filtered in units like these. The solution is drawn through the filter by connecting the unit to a vacuum. (b) Smaller
volumes are often filtered using syringe filters, which are units that fit on the end of a syringe. In this case, the solution is pushed
through by depressing the syringe’s plunger. (credit a, b: modification of work by Brian Forster)

Exercise 11.2.5
1. Would membrane filtration with a 0.2-µm filter likely remove viruses from a solution? Explain.
2. Name at least two common uses of HEPA filtration in clinical or laboratory settings.

Figure 11.2.12 and Figure 11.2.13 summarize the physical methods of control discussed in this section.

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 Figure 11.2.12: Physical methods of control

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 Figure 11.2.13: Physical methods of control

Key Concepts and Summary

Heat is a widely used and highly effective method for controlling microbial growth.
Dry-heat sterilization protocols are used commonly in aseptic techniques in the laboratory. However, moist-heat sterilization
is typically the more effective protocol because it penetrates cells better than dry heat does.
Pasteurization is used to kill pathogens and reduce the number of microbes that cause food spoilage. High-temperature,
short-time pasteurization is commonly used to pasteurize milk that will be refrigerated; ultra-high temperature
pasteurization can be used to pasteurize milk for long-term storage without refrigeration.
Refrigeration slows microbial growth; freezing stops growth, killing some organisms. Laboratory and medical specimens may
be frozen on dry ice or at ultra-low temperatures for storage and transport.
High-pressure processing can be used to kill microbes in food. Hyperbaric oxygen therapy to increase oxygen saturation has
also been used to treat certain infections.
Desiccation has long been used to preserve foods and is accelerated through the addition of salt or sugar, which decrease water
activity in foods.
Lyophilization combines cold exposure and desiccation for the long-term storage of foods and laboratory materials, but
microbes remain and can be rehydrated.
Ionizing radiation, including gamma irradiation, is an effective way to sterilize heat-sensitive and packaged materials.
Nonionizing radiation, like ultraviolet light, is unable to penetrate surfaces but is useful for surface sterilization.
HEPA filtration is commonly used in hospital ventilation systems and biological safety cabinets in laboratories to prevent
transmission of airborne microbes. Membrane filtration is commonly used to remove bacteria from heat-sensitive solutions.

Footnotes
1. 1 US Department of Agriculture. “Freezing and Food Safety.” 2013. http://www.fsis.usda.gov/wps/portal/...afety/CT_Index.
Accessed June 8, 2016.
2. 2 C. Ferstl. “High Pressure Processing: Insights on Technology and Regulatory Requirements.” Food for Thought/White Paper.
Series Volume 10. Livermore, CA: The National Food Lab; July 2013.
3. 3 US Food and Drug Administration. “Kinetics of Microbial Inactivation for Alternative Food Processing Technologies: High
Pressure Processing.” 2000. www.fda.gov/Food/FoodScienceR.../ucm101456.htm. Accessed July 19, 2106.
4. 4 CL McCarty et al. “Large Outbreak of Botulism Associated with a Church Potluck Meal-Ohio, 2015.” Morbidity and
Mortality Weekly Report 64, no. 29 (2015):802–803.
5. 5 AM Johnson et al. “Consumer Acceptance of Electron-Beam Irradiated Ready-to-Eat Poultry Meats.” Food Processing
Preservation, 28 no. 4 (2004):302–319.

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Contributors and Attributions
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.2: Using Physical Methods to Control Microorganisms is shared under a CC BY license and was authored, remixed, and/or
curated by OpenStax.

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11.3: Using Chemicals to Control Microorganisms
Learning Objectives
Understand and compare various chemicals used to control microbial growth, including their uses, advantages and
disadvantages, chemical structure, and mode of action

In addition to physical methods of microbial control, chemicals are also used to control microbial growth. A wide variety of
chemicals can be used as disinfectants or antiseptics. When choosing which to use, it is important to consider the type of microbe
targeted; how clean the item needs to be; the disinfectant’s effect on the item’s integrity; its safety to animals, humans, and the
environment; its expense; and its ease of use. This section describes the variety of chemicals used as disinfectants and antiseptics,
including their mechanisms of action and common uses.
Phenolics
In the 1800s, scientists began experimenting with a variety of chemicals for disinfection. In the 1860s, British surgeon Joseph
Lister (1827–1912) began using carbolic acid, known as phenol, as a disinfectant for the treatment of surgical wounds. In 1879,
Lister’s work inspired the American chemist Joseph Lawrence (1836–1909) to develop Listerine, an alcohol-based mixture of
several related compounds that is still used today as an oral antiseptic. Today, carbolic acid is no longer used as a surgical
disinfectant because it is a skin irritant, but the chemical compounds found in antiseptic mouthwashes and throat lozenges are
called phenolics.
Chemically, phenol consists of a benzene ring with an –OH group, and phenolics are compounds that have this group as part of
their chemical structure (Figure 11.3.1). Phenolics such as thymol and eucalyptol occur naturally in plants. Other phenolics can be
derived from creosote, a component of coal tar. Phenolics tend to be stable, persistent on surfaces, and less toxic than phenol. They
inhibit microbial growth by denaturing proteins and disrupting membranes.

Figure 11.3.1 : Phenol and phenolic compounds have been used to control microbial growth. (a) Chemical structure of phenol, also
known as carbolic acid. (b) o-Phenylphenol, a type of phenolic, has been used as a disinfectant as well as to control bacterial and
fungal growth on harvested citrus fruits. (c) Hexachlorophene, another phenol, known as a bisphenol (two rings), is the active
ingredient in pHisoHex.
Since Lister’s time, several phenolic compounds have been used to control microbial growth. Phenolics like cresols (methylated
phenols) and o-phenylphenol were active ingredients in various formulations of Lysol since its invention in 1889. o-Phenylphenol
was also commonly used in agriculture to control bacterial and fungal growth on harvested crops, especially citrus fruits, but its use
in the United States is now far more limited. The bisphenol hexachlorophene, a disinfectant, is the active ingredient in pHisoHex, a
topical cleansing detergent widely used for handwashing in hospital settings. pHisoHex is particularly effective against gram-
positive bacteria, including those causing staphylococcal and streptococcal skin infections. pHisoHex was formerly used for
bathing infants, but this practice has been discontinued because it has been shown that exposure to hexachlorophene can lead to
neurological problems.
Triclosan is another bisphenol compound that has seen widespread application in antibacterial products over the last several
decades. Initially used in toothpastes, triclosan is now commonly used in hand soaps and is frequently impregnated into a wide
variety of other products, including cutting boards, knives, shower curtains, clothing, and concrete, to make them antimicrobial. It
is particularly effective against gram-positive bacteria on the skin, as well as certain gram-negative bacteria and yeasts.1

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Triclosan: Antibacterial Overkill?
Hand soaps and other cleaning products are often marketed as “antibacterial,” suggesting that they provide a level of
cleanliness superior to that of conventional soaps and cleansers. But are the antibacterial ingredients in these products really
safe and effective?
About 75% of antibacterial liquid hand soaps and 30% of bar soaps contain the chemical triclosan, a phenolic, (Figure
2
11.3.2). Triclosan blocks an enzyme in the bacterial fatty acid-biosynthesis pathway that is not found in the comparable
human pathway. Although the use of triclosan in the home increased dramatically during the 1990s, more than 40 years of
research by the FDA have turned up no conclusive evidence that washing with triclosan-containing products provides
increased health benefits compared with washing with traditional soap. Although some studies indicate that fewer bacteria may
remain on a person’s hands after washing with triclosan-based soap, compared with traditional soap, no evidence points to any
reduction in the transmission of bacteria that cause respiratory and gastrointestinal illness. In short, soaps with triclosan may
remove or kill a few more germs but not enough to reduce the spread of disease.
Perhaps more disturbing, some clear risks associated with triclosan-based soaps have come to light. The widespread use of
triclosan has led to an increase in triclosan-resistant bacterial strains, including those of clinical importance, such as Salmonella
enterica; this resistance may render triclosan useless as an antibacterial in the long run. 3 4
Bacteria can easily gain
resistance to triclosan through a change to a single gene encoding the targeted enzyme in the bacterial fatty acid-synthesis
pathway. Other disinfectants with a less specific mode of action are much less prone to engendering resistance because it
would take much more than a single genetic change.
Use of triclosan over the last several decades has also led to a buildup of the chemical in the environment. Triclosan in hand
soap is directly introduced into wastewater and sewage systems as a result of the handwashing process. There, its antibacterial
properties can inhibit or kill bacteria responsible for the decomposition of sewage, causing septic systems to clog and back up.
Eventually, triclosan in wastewater finds its way into surface waters, streams, lakes, sediments, and soils, disrupting natural
populations of bacteria that carry out important environmental functions, such as inhibiting algae. Triclosan also finds its way
into the bodies of amphibians and fish, where it can act as an endocrine disruptor. Detectable levels of triclosan have also been
found in various human bodily fluids, including breast milk, plasma, and urine. 5 In fact, a study conducted by the CDC
found detectable levels of triclosan in the urine of 75% of 2,517 people tested in 2003–2004. 6 This finding is even more
troubling given the evidence that triclosan may affect immune function in humans. 7
In December 2013, the FDA gave soap manufacturers until 2016 to prove that antibacterial soaps provide a significant benefit
over traditional soaps; if unable to do so, manufacturers will be forced to remove these products from the market.

Figure 11.3.2 : Triclosan is a common ingredient in antibacterial soaps despite evidence that it poses environmental and health
risks and offers no significant health benefit compared to conventional soaps. (credit b, c: modification of work by FDA)

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 Exercise 11.3.1

Why is triclosan more like an antibiotic than a traditional disinfectant?

Heavy Metals
Some of the first chemical disinfectants and antiseptics to be used were heavy metals. Heavy metals kill microbes by binding to
proteins, thus inhibiting enzymatic activity (Figure 11.3.3). Heavy metals are oligodynamic, meaning that very small
concentrations show significant antimicrobial activity. Ions of heavy metals bind to sulfur-containing amino acids strongly and
bioaccumulate within cells, allowing these metals to reach high localized concentrations. This causes proteins to denature.
Heavy metals are not selectively toxic to microbial cells. They may bioaccumulate in human or animal cells, as well, and excessive
concentrations can have toxic effects on humans. If too much silver accumulates in the body, for example, it can result in a
condition called argyria, in which the skin turns irreversibly blue-gray. One way to reduce the potential toxicity of heavy metals is
by carefully controlling the duration of exposure and concentration of the heavy metal.

Figure 11.3.3 : Heavy metals denature proteins, impairing cell function and, thus, giving them strong antimicrobial properties. (a)
Copper in fixtures like this door handle kills microbes that otherwise might accumulate on frequently touched surfaces. (b) Eating
utensils contain small amounts of silver to inhibit microbial growth. (c) Copper commonly lines incubators to minimize
contamination of cell cultures stored inside. (d) Antiseptic mouthwashes commonly contain zinc chloride. (e) This patient is
suffering from argyria, an irreversible condition caused by bioaccumulation of silver in the body. (credit b: modification of work by
“Shoshanah”/Flickr; credit e: modification of work by Herbert L. Fred and Hendrik A. van Dijk)
Mercury
Mercury is an example of a heavy metal that has been used for many years to control microbial growth. It was used for many
centuries to treat syphilis. Mercury compounds like mercuric chloride are mainly bacteriostatic and have a very broad spectrum of
activity. Various forms of mercury bind to sulfur-containing amino acids within proteins, inhibiting their functions.
In recent decades, the use of such compounds has diminished because of mercury’s toxicity. It is toxic to the central nervous,
digestive, and renal systems at high concentrations, and has negative environmental effects, including bioaccumulation in fish.
Topical antiseptics such as mercurochrome, which contains mercury in low concentrations, and merthiolate, a tincture (a solution of
mercury dissolved in alcohol) were once commonly used. However, because of concerns about using mercury compounds, these
antiseptics are no longer sold in the United States.
Silver
Silver has long been used as an antiseptic. In ancient times, drinking water was stored in silver jugs.8 Silvadene cream is commonly
used to treat topical wounds and is particularly helpful in preventing infection in burn wounds. Silver nitrate drops were once
routinely applied to the eyes of newborns to protect against ophthalmia neonatorum, eye infections that can occur due to exposure
to pathogens in the birth canal, but antibiotic creams are more now commonly used. Silver is often combined with antibiotics,
making the antibiotics thousands of times more effective.9 Silver is also commonly incorporated into catheters and bandages,
rendering them antimicrobial; however, there is evidence that heavy metals may also enhance selection for antibiotic resistance.10

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Copper, Nickel, and Zinc
Several other heavy metals also exhibit antimicrobial activity. Copper sulfate is a common algicide used to control algal growth in
swimming pools and fish tanks. The use of metallic copper to minimize microbial growth is also becoming more widespread.
Copper linings in incubators help reduce contamination of cell cultures. The use of copper pots for water storage in underdeveloped
countries is being investigated as a way to combat diarrheal diseases. Copper coatings are also becoming popular for frequently
handled objects such as doorknobs, cabinet hardware, and other fixtures in health-care facilities in an attempt to reduce the spread
of microbes.
Nickel and zinc coatings are now being used in a similar way. Other forms of zinc, including zinc chloride and zinc oxide, are also
used commercially. Zinc chloride is quite safe for humans and is commonly found in mouthwashes, substantially increasing their
length of effectiveness. Zinc oxide is found in a variety of products, including topical antiseptic creams such as calamine lotion,
diaper ointments, baby powder, and dandruff shampoos.

Exercise 11.3.2

Why are many heavy metals both antimicrobial and toxic to humans?

Halogens
Other chemicals commonly used for disinfection are the halogens iodine, chlorine, and fluorine. Iodine works by oxidizing cellular
components, including sulfur-containing amino acids, nucleotides, and fatty acids, and destabilizing the macromolecules that
contain these molecules. It is often used as a topical tincture, but it may cause staining or skin irritation. An iodophor is a
compound of iodine complexed with an organic molecule, thereby increasing iodine’s stability and, in turn, its efficacy. One
common iodophor is povidone-iodine, which includes a wetting agent that releases iodine relatively slowly. Betadine is a brand of
povidone-iodine commonly used as a hand scrub by medical personnel before surgery and for topical antisepsis of a patient’s skin
before incision (Figure 11.3.4).

Figure 11.3.4 : (a) Betadine is a solution of the iodophor povidone-iodine. (b) It is commonly used as a topical antiseptic on a
patient’s skin before incision during surgery. (credit b: modification of work by Andrew Ratto)
Chlorine is another halogen commonly used for disinfection. When chlorine gas is mixed with water, it produces a strong oxidant
called hypochlorous acid, which is uncharged and enters cells easily. Chlorine gas is commonly used in municipal drinking water
and wastewater treatment plants, with the resulting hypochlorous acid producing the actual antimicrobial effect. Those working at
water treatment facilities need to take great care to minimize personal exposure to chlorine gas. Sodium hypochlorite is the
chemical component of common household bleach, and it is also used for a wide variety of disinfecting purposes. Hypochlorite
salts, including sodium and calcium hypochlorites, are used to disinfect swimming pools. Chlorine gas, sodium hypochlorite, and
calcium hypochlorite are also commonly used disinfectants in the food processing and restaurant industries to reduce the spread of
foodborne diseases. Workers in these industries also need to take care to use these products correctly to ensure their own safety as
well as the safety of consumers. A recent joint statement published by the Food and Agriculture Organization (FAO) of the United
Nations and WHO indicated that none of the many beneficial uses of chlorine products in food processing to reduce the spread of
foodborne illness posed risks to consumers.11

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Another class of chlorinated compounds called chloramines are widely used as disinfectants. Chloramines are relatively stable,
releasing chlorine over long periods time. Chloramines are derivatives of ammonia by substitution of one, two, or all three
hydrogen atoms with chlorine atoms (Figure 11.3.5).

Figure 11.3.5 : Monochloroamine, one of the chloramines, is derived from ammonia by the replacement of one hydrogen atom with
a chlorine atom.
Chloramines and other cholorine compounds may be used for disinfection of drinking water, and chloramine tablets are frequently
used by the military for this purpose. After a natural disaster or other event that compromises the public water supply, the CDC
recommends disinfecting tap water by adding small amounts of regular household bleach. Recent research suggests that sodium
dichloroisocyanurate (NaDCC) may also be a good alternative for drinking water disinfection. Currently, NaDCC tablets are
available for general use and for use by the military, campers, or those with emergency needs; for these uses, NaDCC is preferable
to chloramine tablets. Chlorine dioxide, a gaseous agent used for fumigation and sterilization of enclosed areas, is also commonly
used for the disinfection of water.
Although chlorinated compounds are relatively effective disinfectants, they have their disadvantages. Some may irritate the skin,
nose, or eyes of some individuals, and they may not completely eliminate certain hardy organisms from contaminated drinking
water. The fungus Cryptosporidium, for example, has a protective outer shell that makes it resistant to chlorinated disinfectants.
Thus, boiling of drinking water in emergency situations is recommended when possible.
The halogen fluorine is also known to have antimicrobial properties that contribute to the prevention of dental caries(cavities).12
Fluoride is the main active ingredient of toothpaste and is also commonly added to tap water to help communities maintain oral
health. Chemically, fluoride can become incorporated into the hydroxyapatite of tooth enamel, making it more resistant to corrosive
acids produced by the fermentation of oral microbes. Fluoride also enhances the uptake of calcium and phosphate ions in tooth
enamel, promoting remineralization. In addition to strengthening enamel, fluoride also seems to be bacteriostatic. It accumulates in
plaque-forming bacteria, interfering with their metabolism and reducing their production of the acids that contribute to tooth decay.

Exercise 11.3.3

What is a benefit of a chloramine over hypochlorite for disinfecting?

Alcohols
Alcohols make up another group of chemicals commonly used as disinfectants and antiseptics. They work by rapidly denaturing
proteins, which inhibits cell metabolism, and by disrupting membranes, which leads to cell lysis. Once denatured, the proteins may
potentially refold if enough water is present in the solution. Alcohols are typically used at concentrations of about 70% aqueous
solution and, in fact, work better in aqueous solutions than 100% alcohol solutions. This is because alcohols coagulate proteins. In
higher alcohol concentrations, rapid coagulation of surface proteins prevents effective penetration of cells. The most commonly
used alcohols for disinfection are ethyl alcohol(ethanol) and isopropyl alcohol (isopropanol, rubbing alcohol) (Figure 11.3.6).
Alcohols tend to be bactericidal and fungicidal, but may also be viricidal for enveloped viruses only. Although alcohols are not
sporicidal, they do inhibit the processes of sporulation and germination. Alcohols are volatile and dry quickly, but they may also
cause skin irritation because they dehydrate the skin at the site of application. One common clinical use of alcohols is swabbing the
skin for degerming before needle injection. Alcohols also are the active ingredients in instant hand sanitizers, which have gained
popularity in recent years. The alcohol in these hand sanitizers works both by denaturing proteins and by disrupting the microbial
cell membrane, but will not work effectively in the presence of visible dirt.
Last, alcohols are used to make tinctures with other antiseptics, such as the iodine tinctures discussed previously in this chapter. All
in all, alcohols are inexpensive and quite effective for the disinfection of a broad range of vegetative microbes. However, one
disadvantage of alcohols is their high volatility, limiting their effectiveness to immediately after application.

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 Figure 11.3.6 : (a) Ethyl alcohol, the intoxicating ingredient found in alcoholic drinks, is also used commonly as a disinfectant. (b)
Isopropyl alcohol, also called rubbing alcohol, has a related molecular structure and is another commonly used disinfectant. (credit
a photo: modification of work by D Coetzee; credit b photo: modification of work by Craig Spurrier)

Exercise 11.3.4

1. Name at least three advantages of alcohols as disinfectants.

2. Describe several specific applications of alcohols used in disinfectant products.

Surfactants
Surface-active agents, or surfactants, are a group of chemical compounds that lower the surface tension of water. Surfactants are the
major ingredients in soaps and detergents. Soaps are salts of long-chain fatty acids and have both polar and nonpolar regions,
allowing them to interact with polar and nonpolar regions in other molecules (Figure 11.3.7). They can interact with nonpolar oils
and grease to create emulsions in water, loosening and lifting away dirt and microbes from surfaces and skin. Soaps do not kill or
inhibit microbial growth and so are not considered antiseptics or disinfectants. However, proper use of soaps mechanically carries
away microorganisms, effectively degerming a surface. Some soaps contain added bacteriostatic agents such as triclocarban or
cloflucarban, compounds structurally related to triclosan, that introduce antiseptic or disinfectant properties to the soaps.

Figure 11.3.7 : Soaps are the salts (sodium salt in the illustration) of fatty acids and have the ability to emulsify lipids, fats, and oils
by interacting with water through their hydrophilic heads and with the lipid at their hydrophobic tails.
Soaps, however, often form films that are difficult to rinse away, especially in hard water, which contains high concentrations of
calcium and magnesium mineral salts. Detergents contain synthetic surfactant molecules with both polar and nonpolar regions that
have strong cleansing activity but are more soluble, even in hard water, and, therefore, leave behind no soapy deposits. Anionic
detergents, such as those used for laundry, have a negatively charged anion at one end attached to a long hydrophobic chain,
whereas cationic detergents have a positively charged cation instead. Cationic detergents include an important class of disinfectants
and antiseptics called the quaternary ammonium salts (quats), named for the characteristic quaternary nitrogen atom that confers
the positive charge (Figure 11.3.8). Overall, quats have properties similar to phospholipids, having hydrophilic and hydrophobic
ends. As such, quats have the ability to insert into the bacterial phospholipid bilayer and disrupt membrane integrity. The cationic
charge of quats appears to confer their antimicrobial properties, which are diminished when neutralized. Quats have several useful
properties. They are stable, nontoxic, inexpensive, colorless, odorless, and tasteless. They tend to be bactericidal by disrupting
membranes. They are also active against fungi, protozoans, and enveloped viruses, but endospores are unaffected. In clinical

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settings, they may be used as antiseptics or to disinfect surfaces. Mixtures of quats are also commonly found in household cleaners
and disinfectants, including many current formulations of Lysol brand products, which contain benzalkonium chlorides as the
active ingredients. Benzalkonium chlorides, along with the quat cetylpyrimidine chloride, are also found in products such as skin
antiseptics, oral rinses, and mouthwashes.

Figure 11.3.8 : (a) Two common quats are benzylalkonium chloride and cetylpyrimidine chloride. Note the hydrophobic nonpolar
carbon chain at one end and the nitrogen-containing cationic component at the other end. (b) Quats are able to infiltrate the
phospholipid plasma membranes of bacterial cells and disrupt their integrity, leading to death of the cell.

Exercise 11.3.5

Why are soaps not considered disinfectants?

Bisbiguanides
Bisbiguanides were first synthesized in the 20th century and are cationic (positively charged) molecules known for their antiseptic
properties (Figure 11.3.10). One important bisbiguanide antiseptic is chlorhexidine. It has broad-spectrum activity against yeasts,
gram-positive bacteria, and gram-negative bacteria, with the exception of Pseudomonas aeruginosa, which may develop resistance
on repeated exposure.13 Chlorhexidine disrupts cell membranes and is bacteriostatic at lower concentrations or bactericidal at
higher concentrations, in which it actually causes the cells’ cytoplasmic contents to congeal. It also has activity against enveloped
viruses. However, chlorhexidine is poorly effective against Mycobacterium tuberculosis and nonenveloped viruses, and it is not
sporicidal. Chlorhexidine is typically used in the clinical setting as a surgical scrub and for other handwashing needs for medical
personnel, as well as for topical antisepsis for patients before surgery or needle injection. It is more persistent than iodophors,
providing long-lasting antimicrobial activity. Chlorhexidine solutions may also be used as oral rinses after oral procedures or to
treat gingivitis. Another bisbiguanide, alexidine, is gaining popularity as a surgical scrub and an oral rinse because it acts faster
than chlorhexidine.

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 Figure 11.3.10: The bisbiguanides chlorhexadine and alexidine are cationic antiseptic compounds commonly used as surgical
scrubs.

Exercise 11.3.6

What two effects does chlorhexidine have on bacterial cells?

Alkylating Agents
The alkylating agents are a group of strong disinfecting chemicals that act by replacing a hydrogen atom within a molecule with an
alkyl group (CnH2n+1), thereby inactivating enzymes and nucleic acids (Figure 11.3.11). The alkylating agent formaldehyde
(CH2OH) is commonly used in solution at a concentration of 37% (known as formalin) or as a gaseous disinfectant and biocide. It
is a strong, broad-spectrum disinfectant and biocide that has the ability to kill bacteria, viruses, fungi, and endospores, leading to
sterilization at low temperatures, which is sometimes a convenient alternative to the more labor-intensive heat sterilization
methods. It also cross-links proteins and has been widely used as a chemical fixative. Because of this, it is used for the storage of
tissue specimens and as an embalming fluid. It also has been used to inactivate infectious agents in vaccine preparation.
Formaldehyde is very irritating to living tissues and is also carcinogenic; therefore, it is not used as an antiseptic.
Glutaraldehyde is structurally similar to formaldehyde but has two reactive aldehyde groups, allowing it to act more quickly than
formaldehyde. It is commonly used as a 2% solution for sterilization and is marketed under the brand name Cidex. It is used to
disinfect a variety of surfaces and surgical and medical equipment. However, similar to formaldehyde, glutaraldehyde irritates the
skin and is not used as an antiseptic.
A new type of disinfectant gaining popularity for the disinfection of medical equipment is o-phthalaldehyde (OPA), which is found
in some newer formulations of Cidex and similar products, replacing glutaraldehyde. o-Phthalaldehyde also has two reactive
aldehyde groups, but they are linked by an aromatic bridge. o-Phthalaldehyde is thought to work similarly to glutaraldehyde and
formaldehyde, but is much less irritating to skin and nasal passages, produces a minimal odor, does not require processing before
use, and is more effective against mycobacteria.
Ethylene oxide is a type of alkylating agent that is used for gaseous sterilization. It is highly penetrating and can sterilize items
within plastic bags such as catheters, disposable items in laboratories and clinical settings (like packaged Petri dishes), and other
pieces of equipment. Ethylene oxide exposure is a form of cold sterilization, making it useful for the sterilization of heat-sensitive
items. Great care needs to be taken with the use of ethylene oxide, however; it is carcinogenic, like the other alkylating agents, and
is also highly explosive. With careful use and proper aeration of the products after treatment, ethylene oxide is highly effective, and
ethylene oxide sterilizers are commonly found in medical settings for sterilizing packaged materials.
β-Propionolactone is an alkylating agent with a different chemical structure than the others already discussed. Like other alkylating
agents, β-propionolactone binds to DNA, thereby inactivating it (Figure 11.3.11). It is a clear liquid with a strong odor and has the
ability to kill endospores. As such, it has been used in either liquid form or as a vapor for the sterilization of medical instruments
and tissue grafts, and it is a common component of vaccines, used to maintain their sterility. It has also been used for the
sterilization of nutrient broth, as well as blood plasma, milk, and water. It is quickly metabolized by animals and humans to lactic
acid. It is also an irritant, however, and may lead to permanent damage of the eyes, kidneys, or liver. Additionally, it has been
shown to be carcinogenic in animals; thus, precautions are necessary to minimize human exposure to β-propionolactone.14

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 Figure 11.3.11: (a) Alkylating agents replace hydrogen atoms with alkyl groups. Here, guanine is alkylated, resulting in its
hydrogen bonding with thymine, instead of cytosine. (b) The chemical structures of several alkylating agents.

Exercise 11.3.7

1. What chemical reaction do alkylating agents participate in?

2. Why are alkylating agents not used as antiseptics?

Diehard Prions
Prions, the acellular, misfolded proteins responsible for incurable and fatal diseases such as kuru and Creutzfeldt-Jakob
disease, are notoriously difficult to destroy. Prions are extremely resistant to heat, chemicals, and radiation. They are also
extremely infectious and deadly; thus, handling and disposing of prion-infected items requires extensive training and extreme
caution.
Typical methods of disinfection can reduce but not eliminate the infectivity of prions. Autoclaving is not completely effective,
nor are chemicals such as phenol, alcohols, formalin, and β-propiolactone. Even when fixed in formalin, affected brain and
spinal cord tissues remain infectious.
Personnel who handle contaminated specimens or equipment or work with infected patients must wear a protective coat, face
protection, and cut-resistant gloves. Any contact with skin must be immediately washed with detergent and warm water
without scrubbing. The skin should then be washed with 1 N NaOH or a 1:10 dilution of bleach for 1 minute. Contaminated
waste must be incinerated or autoclaved in a strong basic solution, and instruments must be cleaned and soaked in a strong
basic solution.
For more information on the handling of animals and prion-contaminated materials, visit the guidelines published on the CDC
and WHO websites.

Peroxygens
Peroxygens are strong oxidizing agents that can be used as disinfectants or antiseptics. The most widely used peroxygen is
hydrogen peroxide (H2O2), which is often used in solution to disinfect surfaces and may also be used as a gaseous agent. Hydrogen
peroxide solutions are inexpensive skin antiseptics that break down into water and oxygen gas, both of which are environmentally
safe. This decomposition is accelerated in the presence of light, so hydrogen peroxide solutions typically are sold in brown or
opaque bottles. One disadvantage of using hydrogen peroxide as an antiseptic is that it also causes damage to skin that may delay
healing or lead to scarring. Contact lens cleaners often include hydrogen peroxide as a disinfectant.

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Hydrogen peroxide works by producing free radicals that damage cellular macromolecules. Hydrogen peroxide has broad-spectrum
activity, working against gram-positive and gram-negative bacteria (with slightly greater efficacy against gram-positive bacteria),
fungi, viruses, and endospores. However, bacteria that produce the oxygen-detoxifying enzymes catalase or peroxidase may have
inherent tolerance to low hydrogen peroxide concentrations (Figure 11.3.12). To kill endospores, the length of exposure or
concentration of solutions of hydrogen peroxide must be increased. Gaseous hydrogen peroxide has greater efficacy and can be
used as a sterilant for rooms or equipment.

Figure 11.3.12: Catalase enzymatically converts highly reactive hydrogen peroxide (H2O2) into water and oxygen. Hydrogen
peroxide can be used to clean wounds. Hydrogen peroxide is used to sterilize items such as contact lenses. (credit photos:
modification of work by Kerry Ceszyk)
Plasma, a hot, ionized gas, described as the fourth state of matter, is useful for sterilizing equipment because it penetrates surfaces
and kills vegetative cells and endospores. Hydrogen peroxide and peracetic acid, another commonly used peroxygen, each may be
introduced as a plasma. Peracetic acid can be used as a liquid or plasma sterilant insofar as it readily kills endospores, is more
effective than hydrogen peroxide even at rather low concentrations, and is immune to inactivation by catalases and peroxidases. It
also breaks down to environmentally innocuous compounds; in this case, acetic acid and oxygen.
Other examples of peroxygens include benzoyl peroxide and carbamide peroxide. Benzoyl peroxide is a peroxygen that used in
acne medication solutions. It kills the bacterium Propionibacterium acnes, which is associated with acne. Carbamide peroxide, an
ingredient used in toothpaste, is a peroxygen that combats oral biofilms that cause tooth discoloration and halitosis (bad breath).15
Last, ozone gas is a peroxygen with disinfectant qualities and is used to clean air or water supplies. Overall, peroxygens are highly
effective and commonly used, with no associated environmental hazard.

Exercise 11.3.8

How do peroxides kill cells?

Supercritical Fluids
Within the last 15 years, the use of supercritical fluids, especially supercritical carbon dioxide (scCO2), has gained popularity for
certain sterilizing applications. When carbon dioxide is brought to approximately 10 times atmospheric pressure, it reaches a
supercritical state that has physical properties between those of liquids and gases. Materials put into a chamber in which carbon
dioxide is pressurized in this way can be sterilized because of the ability of scCO2 to penetrate surfaces.
Supercritical carbon dioxide works by penetrating cells and forming carbonic acid, thereby lowering the cell pH considerably. This
technique is effective against vegetative cells and is also used in combination with peracetic acid to kill endospores. Its efficacy can
also be augmented with increased temperature or by rapid cycles of pressurization and depressurization, which more likely produce
cell lysis.
Benefits of scCO2 include the nonreactive, nontoxic, and nonflammable properties of carbon dioxide, and this protocol is effective
at low temperatures. Unlike other methods, such as heat and irradiation, that can degrade the object being sterilized, the use of
scCO2 preserves the object’s integrity and is commonly used for treating foods (including spices and juices) and medical devices

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such as endoscopes. It is also gaining popularity for disinfecting tissues such as skin, bones, tendons, and ligaments prior to
transplantation. scCO2 can also be used for pest control because it can kill insect eggs and larvae within products.

Exercise 11.3.9

Why is the use of supercritical carbon dioxide gaining popularity for commercial and medical uses?

Chemical Food Preservatives

Chemical preservatives are used to inhibit microbial growth and minimize spoilage in some foods. Commonly used chemical
preservatives include sorbic acid, benzoic acid, and propionic acid, and their more soluble salts potassium sorbate, sodium
benzoate, and calcium propionate, all of which are used to control the growth of molds in acidic foods. Each of these preservatives
is nontoxic and readily metabolized by humans. They are also flavorless, so they do not compromise the flavor of the foods they
preserve.
Sorbic and benzoic acids exhibit increased efficacy as the pH decreases. Sorbic acid is thought to work by inhibiting various
cellular enzymes, including those in the citric acid cycle, as well as catalases and peroxidases. It is added as a preservative in a
wide variety of foods, including dairy, bread, fruit, and vegetable products. Benzoic acid is found naturally in many types of fruits
and berries, spices, and fermented products. It is thought to work by decreasing intracellular pH, interfering with mechanisms such
as oxidative phosphorylation and the uptake of molecules such as amino acids into cells. Foods preserved with benzoic acid or
sodium benzoate include fruit juices, jams, ice creams, pastries, soft drinks, chewing gum, and pickles.
Propionic acid is thought to both inhibit enzymes and decrease intracellular pH, working similarly to benzoic acid. However,
propionic acid is a more effective preservative at a higher pH than either sorbic acid or benzoic acid. Propionic acid is naturally
produced by some cheeses during their ripening and is added to other types of cheese and baked goods to prevent mold
contamination. It is also added to raw dough to prevent contamination by the bacterium Bacillus mesentericus, which causes bread
to become ropy.
Other commonly used chemical preservatives include sulfur dioxide and nitrites. Sulfur dioxide prevents browning of foods and is
used for the preservation of dried fruits; it has been used in winemaking since ancient times. Sulfur dioxide gas dissolves in water
readily, forming sulfites. Although sulfites can be metabolized by the body, some people have sulfite allergies, including asthmatic
reactions. Additionally, sulfites degrade thiamine, an important nutrient in some foods. The mode of action of sulfites is not entirely
clear, but they may interfere with the disulfide bond formation in proteins, inhibiting enzymatic activity. Alternatively, they may
reduce the intracellular pH of the cell, interfering with proton motive force-driven mechanisms.
Nitrites are added to processed meats to maintain color and stop the germination of Clostridium botulinum endospores. Nitrites are
reduced to nitric oxide, which reacts with heme groups and iron-sulfur groups. When nitric oxide reacts with the heme group within
the myoglobin of meats, a red product forms, giving meat its red color. Alternatively, it is thought that when nitric acid reacts with
the iron-sulfur enzyme ferredoxin within bacteria, this electron transport-chain carrier is destroyed, preventing ATP synthesis.
Nitrosamines, however, are carcinogenic and can be produced through exposure of nitrite-preserved meats (e.g., hot dogs, lunch
meat, breakfast sausage, bacon, meat in canned soups) to heat during cooking.
Natural Chemical Food Preservatives
The discovery of natural antimicrobial substances produced by other microbes has added to the arsenal of preservatives used in
food. Nisin is an antimicrobial peptide produced by the bacterium Lactococcus lactis and is particularly effective against gram-
positive organisms. Nisin works by disrupting cell wall production, leaving cells more prone to lysis. It is used to preserve cheeses,
meats, and beverages.
Natamycin is an antifungal macrolide antibiotic produced by the bacterium Streptomyces natalensis. It was approved by the FDA in
1982 and is used to prevent fungal growth in various types of dairy products, including cottage cheese, sliced cheese, and shredded
cheese. Natamycin is also used for meat preservation in countries outside the United States.

Exercise 11.3.10

What are the advantages and drawbacks of using sulfites and nitrites as food preservatives?

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Key Concepts and Summary
Heavy metals, including mercury, silver, copper, and zinc, have long been used for disinfection and preservation, although
some have toxicity and environmental risks associated with them.
Halogens, including chlorine, fluorine, and iodine, are also commonly used for disinfection. Chlorine compounds, including
sodium hypochlorite, chloramines, and chlorine dioxide, are commonly used for water disinfection. Iodine, in both tincture
and iodophor forms, is an effective antiseptic.
Alcohols, including ethyl alcohol and isopropyl alcohol, are commonly used antiseptics that act by denaturing proteins and
disrupting membranes.
Phenolics are stable, long-acting disinfectants that denature proteins and disrupt membranes. They are commonly found in
household cleaners, mouthwashes, and hospital disinfectants, and are also used to preserve harvested crops.
The phenolic compound triclosan, found in antibacterial soaps, plastics, and textiles is technically an antibiotic because of its
specific mode of action of inhibiting bacterial fatty-acid synthesis..
Surfactants, including soaps and detergents, lower the surface tension of water to create emulsions that mechanically carry
away microbes. Soaps are long-chain fatty acids, whereas detergents are synthetic surfactants.
Quaternary ammonium compounds (quats) are cationic detergents that disrupt membranes. They are used in household
cleaners, skin disinfectants, oral rinses, and mouthwashes.
Bisbiguanides disrupt cell membranes, causing cell contents to gel. Chlorhexidine and alexidine are commonly used for
surgical scrubs, for handwashing in clinical settings, and in prescription oral rinses.
Alkylating agents effectively sterilize materials at low temperatures but are carcinogenic and may also irritate tissue.
Glutaraldehyde and o-phthalaldehyde are used as hospital disinfectants but not as antiseptics. Formaldehyde is used for the
storage of tissue specimens, as an embalming fluid, and in vaccine preparation to inactivate infectious agents. Ethylene oxide is
a gas sterilant that can permeate heat-sensitive packaged materials, but it is also explosive and carcinogenic.
Peroxygens, including hydrogen peroxide, peracetic acid, benzoyl peroxide, and ozone gas, are strong oxidizing agents that
produce free radicals in cells, damaging their macromolecules. They are environmentally safe and are highly effective
disinfectants and antiseptics.
Pressurized carbon dioxide in the form of a supercritical fluid easily permeates packaged materials and cells, forming carbonic
acid and lowering intracellular pH. Supercritical carbon dioxide is nonreactive, nontoxic, nonflammable, and effective at low
temperatures for sterilization of medical devices, implants, and transplanted tissues.
Chemical preservatives are added to a variety of foods. Sorbic acid, benzoic acid, propionic acid, and their more soluble salts
inhibit enzymes or reduce intracellular pH.
Sulfites are used in winemaking and food processing to prevent browning of foods.
Nitrites are used to preserve meats and maintain color, but cooking nitrite-preserved meats may produce carcinogenic
nitrosamines.
Nisin and natamycin are naturally produced preservatives used in cheeses and meats. Nisin is effective against gram-positive
bacteria and natamycin against fungi.

Footnotes
1. US Food and Drug Administration. “Triclosan: What Consumers Should Know.” 2015.
www.fda.gov/ForConsumers/Cons.../ucm205999.htm. Accessed June 9, 2016.
2. J. Stromberg. “Five Reasons Why You Should Probably Stop Using Antibacterial Soap.” Smithsonian.com January 3, 2014.
www.smithsonianmag.com/scienc...948078/?no-ist. Accessed June 9, 2016.
3. SP Yazdankhah et al. “Triclosan and Antimicrobial Resistance in Bacteria: An Overview.” Microbial Drug Resistance 12 no. 2
(2006):83–90.
4. L. Birošová, M. Mikulášová. “Development of Triclosan and Antibiotic Resistance in Salmonella enterica serovar
Typhimurium.” Journal of Medical Microbiology 58 no. 4 (2009):436–441.
5. AB Dann, A. Hontela. “Triclosan: Environmental Exposure, Toxicity and Mechanisms of Action.” Journal of Applied
Toxicology 31 no. 4 (2011):285–311.
6. US Centers for Disease Control and Prevention. “Triclosan Fact Sheet.” 2013.
www.cdc.gov/biomonitoring/Tri...FactSheet.html. Accessed June 9, 2016.
7. EM Clayton et al. “The Impact of Bisphenol A and Triclosan on Immune Parameters in the US Population, NHANES 2003-
2006.” Environmental Health Perspectives 119 no. 3 (2011):390.

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 8. N. Silvestry-Rodriguez et al. “Silver as a Disinfectant.” In Reviews of Environmental Contamination and Toxicology, pp. 23-45.
Edited by GW Ware and DM Whitacre. New York: Springer, 2007.
9. B. Owens. “Silver Makes Antibiotics Thousands of Times More Effective.” Nature June 19 2013.
http://www.nature.com/news/silver-ma...ective-1.13232
10. C. Seiler, TU Berendonk. “Heavy Metal Driven Co-Selection of Antibiotic Resistance in Soil and Water Bodies Impacted by
Agriculture and Aquaculture.” Frontiers in Microbiology 3 (2012):399.
11. World Health Organization. “Benefits and Risks of the Use of Chlorine-Containing Disinfectants in Food Production and Food
Processing: Report of a Joint FAO/WHO Expert Meeting.” Geneva, Switzerland: World Health Organization, 2009.
12. RE Marquis. “Antimicrobial Actions of Fluoride for Oral Bacteria.” Canadian Journal of Microbiology 41 no. 11 (1995):955–
964.
13. L. Thomas et al. “Development of Resistance to Chlorhexidine Diacetate in Pseudomonas aeruginosa and the Effect of a
‘Residual’ Concentration.” Journal of Hospital Infection 46 no. 4 (2000):297–303.
14. Institute of Medicine. “Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense).”
Washington, DC: The National Academies Press, 2007.
15. Yao, C.S. et al. “In vitro antibacterial effect of carbamide peroxide on oral biofilm.” Journal of Oral Microbiology Jun 12, 2013.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682087/. doi: 10.3402/jom.v5i0.20392.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.3: Using Chemicals to Control Microorganisms is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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11.4: Discovering Antimicrobial Drugs
Learning Objectives
Compare and contrast natural, semisynthetic, and synthetic antimicrobial drugs
Describe the chemotherapeutic approaches of ancient societies
Describe the historically important individuals and events that led to the development of antimicrobial drugs
Contrast bacteriostatic versus bactericidal antibacterial activities
Contrast broad-spectrum drugs versus narrow-spectrum drugs
Explain the significance of superinfections
Discuss the significance of dosage and the route of administration of a drug
Identify factors and variables that can influence the side effects of a drug
Describe the significance of positive and negative interactions between drugs

Most people associate the term chemotherapy with treatments for cancer. However, chemotherapy is actually a broader term that
refers to any use of chemicals or drugs to treat disease. Chemotherapy may involve drugs that target cancerous cells or tissues, or it
may involve antimicrobial drugs that target infectious microorganisms. Antimicrobial drugs typically work by destroying or
interfering with microbial structures and enzymes, either killing microbial cells or inhibiting of their growth. But before we
examine how these drugs work, we will briefly explore the history of humans’ use of antimicrobials for the purpose of
chemotherapy.

Use of Antimicrobials in Ancient Societies

Although the discovery of antimicrobials and their subsequent widespread use is commonly associated with modern medicine,
there is evidence that humans have been exposed to antimicrobial compounds for millennia. Chemical analyses of the skeletal
remains of people from Nubia1 (now found in present-day Sudan) dating from between 350 and 550 AD have shown residue of the
antimicrobial agent tetracycline in high enough quantities to suggest the purposeful fermentation of tetracycline-producing
Streptomyces during the beer-making process. The resulting beer, which was thick and gruel-like, was used to treat a variety of
ailments in both adults and children, including gum disease and wounds. The antimicrobial properties of certain plants may also
have been recognized by various cultures around the world, including Indian and Chinese herbalists (Figure 11.4.1) who have long
used plants for a wide variety of medical purposes. Healers of many cultures understood the antimicrobial properties of fungi and
their use of moldy bread or other mold-containing products to treat wounds has been well documented for centuries.2 Today, while
about 80% of the world’s population still relies on plant-derived medicines,3 scientists are now discovering the active compounds
conferring the medicinal benefits contained in many of these traditionally used plants.

Figure 11.4.1 : For millennia, Chinese herbalists have used many different species of plants for the treatment of a wide variety of
human ailments.

Exercise 11.4.2

Give examples of how antimicrobials were used in ancient societies

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The First Antimicrobial Drugs
Societies relied on traditional medicine for thousands of years; however, the first half of the 20th century brought an era of strategic
drug discovery. In the early 1900s, the German physician and scientist Paul Ehrlich (1854–1915) set out to discover or synthesize
chemical compounds capable of killing infectious microbes without harming the patient. In 1909, after screening more than 600
arsenic-containing compounds, Ehrlich’s assistant Sahachiro Hata (1873–1938) found one such “magic bullet.” Compound 606
targeted the bacterium Treponema pallidum, the causative agent of syphilis. Compound 606 was found to successfully cure syphilis
in rabbits and soon after was marketed under the name Salvarsan as a remedy for the disease in humans (Figure 11.4.2). Ehrlich’s
innovative approach of systematically screening a wide variety of compounds remains a common strategy for the discovery of new
antimicrobial agents even today.

Figure 11.4.2 : Paul Ehrlich was influential in the discovery of Compound 606, an antimicrobial agent that proved to be an effective
treatment for syphilis.
A few decades later, German scientists Josef Klarer, Fritz Mietzsch, and Gerhard Domagk discovered the antibacterial activity of a
synthetic dye, prontosil, that could treat streptococcal and staphylococcal infections in mice. Domagk’s own daughter was one of
the first human recipients of the drug, which completely cured her of a severe streptococcal infection that had resulted from a poke
with an embroidery needle. Gerhard Domagk (1895–1964) was awarded the Nobel Prize in Medicine in 1939 for his work with
prontosil and sulfanilamide, the active breakdown product of prontosil in the body. Sulfanilamide, the first synthetic antimicrobial
created, served as the foundation for the chemical development of a family of sulfa drugs. A synthetic antimicrobial is a drug that is
developed from a chemical not found in nature. The success of the sulfa drugs led to the discovery and production of additional
important classes of synthetic antimicrobials, including the quinolines and oxazolidinones.
A few years before the discovery of prontosil, scientist Alexander Fleming (1881–1955) made his own accidental discovery that
turned out to be monumental. In 1928, Fleming returned from holiday and examined some old plates of staphylococci in his
research laboratory at St. Mary’s Hospital in London. He observed that contaminating mold growth (subsequently identified as a
strain of Penicillium notatum) inhibited staphylococcal growth on one plate. Fleming, therefore, is credited with the discovery of
penicillin, the first natural antibiotic, (Figure 11.4.3). Further experimentation showed that penicillin from the mold was
antibacterial against streptococci, meningococci, and Corynebacterium diphtheriae, the causative agent of diphtheria.

Figure 11.4.3 : (a) Alexander Fleming was the first to discover a naturally produced antimicrobial, penicillin, in 1928. (b) Howard
Florey and Ernst Chain discovered how to scale up penicillin production. Then they figured out how to purify it and showed its
efficacy as an antimicrobial in animal and human trials in the early 1940s.
Fleming and his colleagues were credited with discovering and identifying penicillin, but its isolation and mass production were
accomplished by a team of researchers at Oxford University under the direction of Howard Florey(1898–1968) and Ernst Chain
(1906–1979) (Figure 11.4.3). In 1940, the research team purified penicillin and reported its success as an antimicrobial agent

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against streptococcal infections in mice. Their subsequent work with human subjects also showed penicillin to be very effective.
Because of their important work, Fleming, Florey, and Chain were awarded the Nobel Prize in Physiology and Medicine in 1945.
In the early 1940s, scientist Dorothy Hodgkin (1910–1994), who studied crystallography at Oxford University, used X-rays to
analyze the structure of a variety of natural products. In 1946, she determined the structure of penicillin, for which she was awarded
the Nobel Prize in Chemistry in 1964. Once the structure was understood, scientists could modify it to produce a variety of
semisynthetic penicillins. A semisynthetic antimicrobial is a chemically modified derivative of a natural antibiotic. The chemical
modifications are generally designed to increase the range of bacteria targeted, increase stability, decrease toxicity, or confer other
properties beneficial for treating infections.
Penicillin is only one example of a natural antibiotic. Also in the 1940s, Selman Waksman (1888–1973) (Figure 11.4.4), a
prominent soil microbiologist at Rutgers University, led a research team that discovered several antimicrobials, including
actinomycin, streptomycin, and neomycin. The discoveries of these antimicrobials stemmed from Waksman’s study of fungi and
the Actinobacteria, including soil bacteria in the genus Streptomyces, known for their natural production of a wide variety of
antimicrobials. His work earned him the Nobel Prize in Physiology and Medicine in 1952. The actinomycetes are the source of
more than half of all natural antibiotics4 and continue to serve as an excellent reservoir for the discovery of novel antimicrobial
agents. Some researchers argue that we have not yet come close to tapping the full antimicrobial potential of this group.5

Figure 11.4.4 : Selman Waksman was the first to show the vast antimicrobial production capabilities of a group of soil bacteria, the
actinomycetes.

Exercise 11.4.3

Why is the soil a reservoir for antimicrobial resistance genes?

Choosing Antimicrobial Drugs

Several factors are important in choosing the most appropriate antimicrobial drug therapy, including bacteriostatic versus
bactericidal mechanisms, spectrum of activity, dosage and route of administration, the potential for side effects, and the potential
interactions between drugs. The following discussion will focus primarily on antibacterial drugs, but the concepts translate to other
antimicrobial classes.

Bacteriostatic Versus Bactericidal

Antibacterial drugs can be either bacteriostatic or bactericidal in their interactions with target bacteria. Bacteriostatic drugs cause a
reversible inhibition of growth, with bacterial growth restarting after elimination of the drug. By contrast, bactericidal drugs kill
their target bacteria. The decision of whether to use a bacteriostatic or bactericidal drugs depends on the type of infection and the
immune status of the patient. In a patient with strong immune defenses, bacteriostatic and bactericidal drugs can be effective in
achieving clinical cure. However, when a patient is immunocompromised, a bactericidal drug is essential for the successful
treatment of infections. Regardless of the immune status of the patient, life-threatening infections such as acute endocarditis require
the use of a bactericidal drug.

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Spectrum of Activity
The spectrum of activity of an antibacterial drug relates to diversity of targeted bacteria. A narrow-spectrum antimicrobial targets
only specific subsets of bacterial pathogens. For example, some narrow-spectrum drugs only target gram-positive bacteria, whereas
others target only gram-negative bacteria. If the pathogen causing an infection has been identified, it is best to use a narrow-
spectrum antimicrobial and minimize collateral damage to the normal microbiota. A broad-spectrum antimicrobial targets a wide
variety of bacterial pathogens, including both gram-positive and gram-negative species, and is frequently used as empiric therapy
to cover a wide range of potential pathogens while waiting on the laboratory identification of the infecting pathogen. Broad-
spectrum antimicrobials are also used for polymicrobic infections (mixed infection with multiple bacterial species), or as
prophylactic prevention of infections with surgery/invasive procedures. Finally, broad-spectrum antimicrobials may be selected to
treat an infection when a narrow-spectrum drug fails because of development of drug resistance by the target pathogen.
The risk associated with using broad-spectrum antimicrobials is that they will also target a broad spectrum of the normal
microbiota, increasing the risk of a superinfection, a secondary infection in a patient having a preexisting infection. A
superinfection develops when the antibacterial intended for the preexisting infection kills the protective microbiota, allowing
another pathogen resistant to the antibacterial to proliferate and cause a secondary infection (Figure 11.4.5). Common examples of
superinfections that develop as a result of antimicrobial usage include yeast infections (candidiasis) and pseudomembranous colitis
caused by Clostridium difficile, which can be fatal.

Figure 11.4.5 : Broad-spectrum antimicrobial use may lead to the development of a superinfection. (credit: modification of work by
Centers for Disease Control and Prevention)

Exercise 11.4.4

What is a superinfection and how does one arise?

Dosage and Route of Administration

The amount of medication given during a certain time interval is the dosage, and it must be determined carefully to ensure that
optimum therapeutic drug levels are achieved at the site of infection without causing significant toxicity (side effects) to the patient.
Each drug class is associated with a variety of potential side effects, and some of these are described for specific drugs later in this
chapter. Despite best efforts to optimize dosing, allergic reactions and other potentially serious side effects do occur. Therefore, the
goal is to select the optimum dosage that will minimize the risk of side effects while still achieving clinical cure, and there are
important factors to consider when selecting the best dose and dosage interval. For example, in children, dose is based upon the
patient’s mass. However, the same is not true for adults and children 12 years of age and older, for which there is typically a single
standard dose regardless of the patient’s mass. With the great variability in adult body mass, some experts have argued that mass
should be considered for all patients when determining appropriate dosage.6 An additional consideration is how drugs are
metabolized and eliminated from the body. In general, patients with a history of liver or kidney dysfunction may experience
reduced drug metabolism or clearance from the body, resulting in increased drug levels that may lead to toxicity and make them
more prone to side effects.
There are also some factors specific to the drugs themselves that influence appropriate dose and time interval between doses. For
example, the half-life, or rate at which 50% of a drug is eliminated from the plasma, can vary significantly between drugs. Some
drugs have a short half-life of only 1 hour and must be given multiple times a day, whereas other drugs have half-lives exceeding
12 hours and can be given as a single dose every 24 hours. Although a longer half-life can be considered an advantage for an

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antibacterial when it comes to convenient dosing intervals, the longer half-life can also be a concern for a drug that has serious side
effects because drug levels may remain toxic for a longer time. Last, some drugs are dose dependent, meaning they are more
effective when administered in large doses to provide high levels for a short time at the site of infection. Others are time dependent,
meaning they are more effective when lower optimum levels are maintained over a longer period of time.
The route of administration, the method used to introduce a drug into the body, is also an important consideration for drug therapy.
Drugs that can be administered orally are generally preferred because patients can more conveniently take these drugs at home.
However, some drugs are not absorbed easily from the gastrointestinal (GI) tract into the bloodstream. These drugs are often useful
for treating diseases of the intestinal tract, such as tapeworms treated with niclosamide, or for decontaminating the bowel, as with
colistin. Some drugs that are not absorbed easily, such as bacitracin, polymyxin, and several antifungals, are available as topical
preparations for treatment of superficial skin infections. Sometimes, patients may not initially be able to take oral medications
because of their illness (e.g., vomiting, intubation for respirator). When this occurs, and when a chosen drug is not absorbed in the
GI tract, administration of the drug by a parenteral route (intravenous or intramuscular injection) is preferred and typically is
performed in health-care settings. For most drugs, the plasma levels achieved by intravenous administration is substantially higher
than levels achieved by oral or intramuscular administration, and this can also be an important consideration when choosing the
route of administration for treating an infection (Figure 11.4.6).

Figure 11.4.6 : On this graph, t0 represents the time at which a drug dose is administered. The curves illustrate how plasma
concentration of the drug changes over specific intervals of time (t1 through t4). As the graph shows, when a drug is administered
intravenously, the concentration peaks very quickly and then gradually decreases. When drugs are administered orally or
intramuscularly, it takes longer for the concentration to reach its peak.

Exercise 11.4.5

1. List five factors to consider when determining the dosage of a drug.

2. Name some typical side effects associated with drugs and identify some factors that might contribute to these side effects.

Drug Interactions
For the optimum treatment of some infections, two antibacterial drugs may be administered together to provide a synergistic
interaction that is better than the efficacy of either drug alone. A classic example of synergistic combinations is trimethoprim and
sulfamethoxazole (Bactrim). Individually, these two drugs provide only bacteriostatic inhibition of bacterial growth, but combined,
the drugs are bactericidal.
Whereas synergistic drug interactions provide a benefit to the patient, antagonistic interactions produce harmful effects.
Antagonism can occur between two antimicrobials or between antimicrobials and nonantimicrobials being used to treat other
conditions. The effects vary depending on the drugs involved, but antagonistic interactions may cause loss of drug activity,

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decreased therapeutic levels due to increased metabolism and elimination, or increased potential for toxicity due to decreased
metabolism and elimination. As an example, some antibacterials are absorbed most effectively from the acidic environment of the
stomach. If a patient takes antacids, however, this increases the pH of the stomach and negatively impacts the absorption of these
antimicrobials, decreasing their effectiveness in treating an infection. Studies have also shown an association between use of some
antimicrobials and failure of oral contraceptives.7

Exercise 11.4.6

Explain the difference between synergistic and antagonistic drug interactions.

Resistance Police

In the United States and many other countries, most antimicrobial drugs are self-administered by patients at home.
Unfortunately, many patients stop taking antimicrobials once their symptoms dissipate and they feel better. If a 10-day course
of treatment is prescribed, many patients only take the drug for 5 or 6 days, unaware of the negative consequences of not
completing the full course of treatment. A shorter course of treatment not only fails to kill the target organisms to expected
levels, it also selects for drug-resistant variants within the target population and within the patient’s microbiota.
Patients’ nonadherence especially amplifies drug resistance when the recommended course of treatment is long. Treatment for
tuberculosis (TB) is a case in point, with the recommended treatment lasting from 6 months to a year. The CDC estimates that
about one-third of the world’s population is infected with TB, most living in underdeveloped or underserved regions where
antimicrobial drugs are available over the counter. In such countries, there may be even lower rates of adherence than in
developed areas. Nonadherence leads to antibiotic resistance and more difficulty in controlling pathogens. As a direct result,
the emergence of multidrug-resistant and extensively drug-resistant strains of TB is becoming a huge problem.
Overprescription of antimicrobials also contributes to antibiotic resistance. Patients often demand antibiotics for diseases that
do not require them, like viral colds and ear infections. Pharmaceutical companies aggressively market drugs to physicians and
clinics, making it easy for them to give free samples to patients, and some pharmacies even offer certain antibiotics free to low-
income patients with a prescription.
In recent years, various initiatives have aimed to educate parents and clinicians about the judicious use of antibiotics. However,
a recent study showed that, between 2000 and 2013, the parental expectation for antimicrobial prescriptions for children
actually increased (Figure 11.4.7).
One possible solution is a regimen called directly observed therapy (DOT), which involves the supervised administration of
medications to patients. Patients are either required to visit a health-care facility to receive their medications, or health-care
providers must administer medication in patients’ homes or another designated location. DOT has been implemented in many
cases for the treatment of TB and has been shown to be effective; indeed, DOT is an integral part of WHO’s global strategy for
eradicating TB.8,9 But is this a practical strategy for all antibiotics? Would patients taking penicillin, for example, be more or
less likely to adhere to the full course of treatment if they had to travel to a health-care facility for each dose? And who would
pay for the increased cost associated with DOT? When it comes to overprescription, should someone be policing physicians or
drug companies to enforce best practices? What group should assume this responsibility, and what penalties would be effective
in discouraging overprescription?

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 Figure 11.4.7 : This graph indicates trends in parental expectations related to prescription of antibiotics based on a recent
study.10 Among parents of Medicaid-insured children, there was a clear upward trend in parental expectations for prescription
antibiotics. Expectations were relatively stable (and lesser) among parents whose children were commercially insured,
suggesting that these parents were somewhat better informed than those with Medicaid-insured children.

Key Concepts and Summary

Antimicrobial drugs produced by purposeful fermentation and/or contained in plants have been used as traditional medicines
in many cultures for millennia.
The purposeful and systematic search for a chemical “magic bullet” that specifically target infectious microbes was initiated by
Paul Ehrlich in the early 20th century.
The discovery of the natural antibiotic, penicillin, by Alexander Fleming in 1928 started the modern age of antimicrobial
discovery and research.
Sulfanilamide, the first synthetic antimicrobial, was discovered by Gerhard Domagk and colleagues and is a breakdown
product of the synthetic dye, prontosil.
Antimicrobial drugs can be bacteriostatic or bactericidal, and these characteristics are important considerations when
selecting the most appropriate drug.
The use of narrow-spectrum antimicrobial drugs is preferred in many cases to avoid superinfection and the development of
antimicrobial resistance.
Broad-spectrum antimicrobial use is warranted for serious systemic infections when there is no time to determine the causative
agent, when narrow-spectrum antimicrobials fail, or for the treatment or prevention of infections with multiple types of
microbes.
The dosage and route of administration are important considerations when selecting an antimicrobial to treat and infection.
Other considerations include the patient’s age, mass, ability to take oral medications, liver and kidney function, and possible
interactions with other drugs the patient may be taking.

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Footnotes
1. M.L. Nelson et al. “Brief Communication: Mass Spectroscopic Characterization of Tetracycline in the Skeletal Remains of an
Ancient Population from Sudanese Nubia 350–550 CE.” American Journal of Physical Anthropology 143 no. 1 (2010):151–
154.
2. M. Wainwright. “Moulds in Ancient and More Recent Medicine.” Mycologist 3 no. 1 (1989):21–23.
3. S. Verma, S.P. Singh. “Current and Future Status of Herbal Medicines.” Veterinary World 1 no. 11 (2008):347–350.
4. J. Berdy. “Bioactive Microbial Metabolites.” The Journal of Antibiotics 58 no. 1 (2005):1–26.
5. M. Baltz. “Antimicrobials from Actinomycetes: Back to the Future.” Microbe 2 no. 3 (2007):125–131.
6. M.E. Falagas, D.E. Karageorgopoulos. “Adjustment of Dosing of Antimicrobial Agents for Bodyweight in Adults.” The Lancet
375 no. 9710 (2010):248–251.
7. B.D. Dickinson et al. “Drug Interactions between Oral Contraceptives and Antibiotics.” Obstetrics & Gynecology 98, no. 5
(2001):853–860.
8. Centers for Disease Control and Prevention. “Tuberculosis (TB).” www.cdc.gov/tb/education/ssmo...s9reading2.htm. Accessed
June 2, 2016.
9. World Health Organization. “Tuberculosis (TB): The Five Elements of DOTS.” http://www.who.int/tb/dots/whatisdots/en/.
Accessed June 2, 2016.
10. Naz, L.E., et al. “Prevalence of Parental Misconceptions About Antibiotic Use.” Pediatrics 136 no.2 (August 2015). DOI:
10.1542/peds.2015-0883.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.4: Discovering Antimicrobial Drugs is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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11.5: Drug Targets on Prokaryote Microorganisms
Learning Objectives
Describe the mechanisms of action associated with drugs that inhibit cell wall biosynthesis, protein synthesis, membrane
function, nucleic acid synthesis, and metabolic pathway.

An important quality for an antimicrobial drug is selective toxicity, meaning that it selectively kills or inhibits the growth of
microbial targets while causing minimal or no harm to the host. Most antimicrobial drugs currently in clinical use are antibacterial
because the prokaryotic cell provides a greater variety of unique targets for selective toxicity, in comparison to fungi, parasites, and
viruses. Each class of antibacterial drugs has a unique mode of action (the way in which a drug affects microbes at the cellular
level), and these are summarized in Figure 11.5.1 and Table 11.5.1.

Figure 11.5.1 : There are several classes of antibacterial compounds that are typically classified based on their bacterial target.
Table 11.5.1 : Common Antibacterial Drugs by Mode of Action
Mode of Action Target Drug Class

β-lactams: penicillins, cephalosporins,

Penicillin-binding proteins
monobactams, carbapenems
Inhibit cell wall biosynthesis
Peptidoglycan subunits Glycopeptides

Peptidoglycan subunit transport Bacitracin

30S ribosomal subunit Aminoglycosides, tetracyclines

Inhibit biosynthesis of proteins Macrolides, lincosamides, chloramphenicol,
50S ribosomal subunit
oxazolidinones

Lipopolysaccharide, inner and outer

Disrupt membranes Polymyxin B, colistin, daptomycin
membranes

RNA Rifamycin
Inhibit nucleic acid synthesis
DNA Fluoroquinolones

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 Mode of Action Target Drug Class

Folic acid synthesis enzyme Sulfonamides, trimethoprim

Antimetabolites
Mycolic acid synthesis enzyme Isonicotinic acid hydrazide

Mycobacterial adenosine triphosphate (ATP)

Mycobacterial ATP synthase Diarylquinoline
synthase inhibitor

Inhibitors of Cell Wall Biosynthesis

Several different classes of antibacterials block steps in the biosynthesis of peptidoglycan, making cells more susceptible to
osmotic lysis (Table 11.5.2). Therefore, antibacterials that target cell wall biosynthesis are bactericidal in their action. Because
human cells do not make peptidoglycan, this mode of action is an excellent example of selective toxicity.
Penicillin, the first antibiotic discovered, is one of several antibacterials within a class called β-lactams. This group of compounds
includes the penicillins, cephalosporins, monobactams, and carbapenems, and is characterized by the presence of a β-lactam ring
found within the central structure of the drug molecule (Figure 11.5.2). The β-lactam antibacterials block the crosslinking of
peptide chains during the biosynthesis of new peptidoglycan in the bacterial cell wall. They are able to block this process because
the β-lactam structure is similar to the structure of the peptidoglycan subunit component that is recognized by the crosslinking
transpeptidase enzyme, also known as a penicillin-binding protein (PBP). Although the β-lactam ring must remain unchanged for
these drugs to retain their antibacterial activity, strategic chemical changes to the R groups have allowed for development of a wide
variety of semisynthetic β-lactam drugs with increased potency, expanded spectrum of activity, and longer half-lives for better
dosing, among other characteristics.
Penicillin G and penicillin V are natural antibiotics from fungi and are primarily active against gram-positive bacterial pathogens,
and a few gram-negative bacterial pathogens such as Pasteurella multocida. Figure 11.5.2 summarizes the semisynthetic
development of some of the penicillins. Adding an amino group (-NH2) to penicillin G created the aminopenicillins (i.e., ampicillin
and amoxicillin) that have increased spectrum of activity against more gram-negative pathogens. Furthermore, the addition of a
hydroxyl group (-OH) to amoxicillin increased acid stability, which allows for improved oral absorption. Methicillin is a
semisynthetic penicillin that was developed to address the spread of enzymes (penicillinases) that were inactivating the other
penicillins. Changing the R group of penicillin G to the more bulky dimethoxyphenyl group provided protection of the β-lactam
ring from enzymatic destruction by penicillinases, giving us the first penicillinase-resistant penicillin.
Similar to the penicillins, cephalosporins contain a β-lactam ring (Figure 11.5.2) and block the transpeptidase activity of penicillin-
binding proteins. However, the β-lactam ring of cephalosporins is fused to a six-member ring, rather than the five-member ring
found in penicillins. This chemical difference provides cephalosporins with an increased resistance to enzymatic inactivation by β-
lactamases. The drug cephalosporin C was originally isolated from the fungus Cephalosporium acremonium in the 1950s and has a
similar spectrum of activity to that of penicillin against gram-positive bacteria but is active against more gram-negative bacteria
than penicillin. Another important structural difference is that cephalosporin C possesses two R groups, compared with just one R
group for penicillin, and this provides for greater diversity in chemical alterations and development of semisynthetic
cephalosporins. The family of semisynthetic cephalosporins is much larger than the penicillins, and these drugs have been
classified into generations based primarily on their spectrum of activity, increasing in spectrum from the narrow-spectrum, first-
generation cephalosporins to the broad-spectrum, fourth-generation cephalosporins. A new fifth-generation cephalosporin has been
developed that is active against methicillin-resistant Staphylococcus aureus (MRSA).
The carbapenems and monobactams also have a β-lactam ring as part of their core structure, and they inhibit the transpeptidase
activity of penicillin-binding proteins. The only monobactam used clinically is aztreonam. It is a narrow-spectrum antibacterial
with activity only against gram-negative bacteria. In contrast, the carbapenem family includes a variety of semisynthetic drugs
(imipenem, meropenem, and doripenem) that provide very broad-spectrum activity against gram-positive and gram-negative
bacterial pathogens.
The drug vancomycin, a member of a class of compounds called the glycopeptides, was discovered in the 1950s as a natural
antibiotic from the actinomycete Amycolatopsis orientalis. Similar to the β-lactams, vancomycin inhibits cell wall biosynthesis and
is bactericidal. However, in contrast to the β-lactams, the structure of vancomycin is not similar to that of cell-wall peptidoglycan
subunits and does not directly inactivate penicillin-binding proteins. Rather, vancomycin is a very large, complex molecule that
binds to the end of the peptide chain of cell wall precursors, creating a structural blockage that prevents the cell wall subunits from
being incorporated into the growing N-acetylglucosamine and N-acetylmuramic acid (NAM-NAG) backbone of the peptidoglycan

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structure (transglycosylation). Vancomycin also structurally blocks transpeptidation. Vancomycin is bactericidal against gram-
positive bacterial pathogens, but it is not active against gram-negative bacteria because of its inability to penetrate the protective
outer membrane.
The drug bacitracin consists of a group of structurally similar peptide antibiotics originally isolated from Bacillus subtilis.
Bacitracin blocks the activity of a specific cell-membrane molecule that is responsible for the movement of peptidoglycan
precursors from the cytoplasm to the exterior of the cell, ultimately preventing their incorporation into the cell wall. Bacitracin is
effective against a wide range of bacteria, including gram-positive organisms found on the skin, such as Staphylococcus and
Streptococcus. Although it may be administered orally or intramuscularly in some circumstances, bacitracin has been shown to be
nephrotoxic (damaging to the kidneys). Therefore, it is more commonly combined with neomycin and polymyxin in topical
ointments such as Neosporin.

Figure 11.5.2 : Penicillins, cephalosporins, monobactams, and carbapenems all contain a β-lactam ring, the site of attack by
inactivating β-lactamase enzymes. Although they all share the same nucleus, various penicillins differ from each other in the
structure of their R groups. Chemical changes to the R groups provided increased spectrum of activity, acid stability, and resistance
to β-lactamase degradation.
Table 11.5.2 : Drugs that Inhibit Bacterial Cell Wall Synthesis
Mechanism of Action Drug Class Specific Drugs Natural or Semisynthetic Spectrum of Activity

Interact directly with PBPs Penicillins Narrow-spectrum against

and inhibit transpeptidase Penicillin G, penicillin V Natural gram-positive and a few
activity gram-negative bacteria
Narrow-spectrum against
gram-positive bacteria but
Ampicillin, amoxicillin Semisynthetic
with increased gram-
negative spectrum

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 Mechanism of Action Drug Class Specific Drugs Natural or Semisynthetic Spectrum of Activity

Narrow-spectrum against
gram-positive bacteria
Methicillin Semisynthetic
only, including strains
producing penicillinase
Narrow-spectrum similar to
penicillin but with
Cephalosporin C Natural
increased gram-negative
spectrum

First-generation Narrow-spectrum similar to

Semisynthetic
cephalosporins cephalosporin C

Narrow-spectrum but with

Second-generation increased gram-negative
Semisynthetic
cephalosporins spectrum compared with
Cephalosporins first generation
Broad-spectrum against
gram-positive and gram-
Third- and fourth-
Semisynthetic negative bacteria, including
generation cephalosporins
some β-lactamase
producers
Broad-spectrum against
Fifth-generation gram-positive and gram-
Semisynthetic
cephalosporins negative bacteria, including
MRSA
Narrow-spectrum against
gram-negative bacteria,
Monobactams Aztreonam Semisynthetic
including some β-lactamase
producers
Broadest spectrum of the β-
lactams against gram-
Imipenem, meropenem,
Carbapenems Semisynthetic positive and gram-negative
doripenem
bacteria, including many β-
lactamase producers

Large molecules that bind

to the peptide chain of Narrow spectrum against
peptidoglycan subunits, gram-positive bacteria
Glycopeptides Vancomycin Natural
blocking only, including multidrug-
transglycosylation and resistant strains
transpeptidation

Block transport of
Broad-spectrum against
peptidoglycan subunits
Bacitracin Bacitracin Natural gram-positive and gram-
across cytoplasmic
negative bacteria
membrane

Exercise 11.5.1

Describe the mode of action of β-lactams.

Inhibitors of Protein Biosynthesis

The cytoplasmic ribosomes found in animal cells (80S) are structurally distinct from those found in bacterial cells (70S), making
protein biosynthesis a good selective target for antibacterial drugs. Several types of protein biosynthesis inhibitors are discussed in

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this section and are summarized in Figure 11.5.3.

Protein Synthesis Inhibitors That Bind the 30S Subunit

Aminoglycosides are large, highly polar antibacterial drugs that bind to the 30S subunit of bacterial ribosomes, impairing the
proofreading ability of the ribosomal complex. This impairment causes mismatches between codons and anticodons, resulting in
the production of proteins with incorrect amino acids and shortened proteins that insert into the cytoplasmic membrane. Disruption
of the cytoplasmic membrane by the faulty proteins kills the bacterial cells. The aminoglycosides, which include drugs such as
streptomycin, gentamicin, neomycin, and kanamycin, are potent broad-spectrum antibacterials. However, aminoglycosides have
been shown to be nephrotoxic (damaging to kidney), neurotoxic (damaging to the nervous system), and ototoxic (damaging to the
ear).
Another class of antibacterial compounds that bind to the 30S subunit is the tetracyclines. In contrast to aminoglycosides, these
drugs are bacteriostatic and inhibit protein synthesis by blocking the association of tRNAs with the ribosome during translation.
Naturally occurring tetracyclines produced by various strains of Streptomyces were first discovered in the 1940s, and several
semisynthetic tetracyclines, including doxycycline and tigecycline have also been produced. Although the tetracyclines are broad
spectrum in their coverage of bacterial pathogens, side effects that can limit their use include phototoxicity, permanent
discoloration of developing teeth, and liver toxicity with high doses or in patients with kidney impairment.

Protein Synthesis Inhibitors That Bind the 50S Subunit

There are several classes of antibacterial drugs that work through binding to the 50S subunit of bacterial ribosomes. The macrolide
antibacterial drugs have a large, complex ring structure and are part of a larger class of naturally produced secondary metabolites
called polyketides, complex compounds produced in a stepwise fashion through the repeated addition of two-carbon units by a
mechanism similar to that used for fatty acid synthesis. Macrolides are broad-spectrum, bacteriostatic drugs that block elongation
of proteins by inhibiting peptide bond formation between specific combinations of amino acids. The first macrolide was
erythromycin. It was isolated in 1952 from Streptomyces erythreus and prevents translocation. Semisynthetic macrolides include
azithromycin and telithromycin. Compared with erythromycin, azithromycin has a broader spectrum of activity, fewer side effects,
and a significantly longer half-life (1.5 hours for erythromycin versus 68 hours for azithromycin) that allows for once-daily dosing
and a short 3-day course of therapy (i.e., Zpac formulation) for most infections. Telithromycin is the first semisynthetic within the
class known as ketolides. Although telithromycin shows increased potency and activity against macrolide-resistant pathogens, the
US Food and Drug Administration (FDA) has limited its use to treatment of community-acquired pneumonia and requires the
strongest “black box warning” label for the drug because of serious hepatotoxicity.
The lincosamides include the naturally produced lincomycin and semisynthetic clindamycin. Although structurally distinct from
macrolides, lincosamides are similar in their mode of action to the macrolides through binding to the 50S ribosomal subunit and
preventing peptide bond formation. Lincosamides are particularly active against streptococcal and staphylococcal infections.
The drug chloramphenicol represents yet another structurally distinct class of antibacterials that also bind to the 50S ribosome,
inhibiting peptide bond formation. Chloramphenicol, produced by Streptomyces venezuelae, was discovered in 1947; in 1949, it
became the first broad-spectrum antibiotic that was approved by the FDA. Although it is a natural antibiotic, it is also easily
synthesized and was the first antibacterial drug synthetically mass produced. As a result of its mass production, broad-spectrum
coverage, and ability to penetrate into tissues efficiently, chloramphenicol was historically used to treat a wide range of infections,
from meningitis to typhoid fever to conjunctivitis. Unfortunately, serious side effects, such as lethal gray baby syndrome, and
suppression of bone marrow production, have limited its clinical role. Chloramphenicol also causes anemia in two different ways.
One mechanism involves the targeting of mitochondrial ribosomes within hematopoietic stem cells, causing a reversible, dose-
dependent suppression of blood cell production. Once chloramphenicol dosing is discontinued, blood cell production returns to
normal. This mechanism highlights the similarity between 70S ribosomes of bacteria and the 70S ribosomes within our
mitochondria. The second mechanism of anemia is idiosyncratic (i.e., the mechanism is not understood), and involves an
irreversible lethal loss of blood cell production known as aplastic anemia. This mechanism of aplastic anemia is not dose dependent
and can develop after therapy has stopped. Because of toxicity concerns, chloramphenicol usage in humans is now rare in the
United States and is limited to severe infections unable to be treated by less toxic antibiotics. Because its side effects are much less
severe in animals, it is used in veterinary medicine.
The oxazolidinones, including linezolid, are a new broad-spectrum class of synthetic protein synthesis inhibitors that bind to the
50S ribosomal subunit of both gram-positive and gram-negative bacteria. However, their mechanism of action seems somewhat
different from that of the other 50S subunit-binding protein synthesis inhibitors already discussed. Instead, they seem to interfere

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with formation of the initiation complex (association of the 50S subunit, 30S subunit, and other factors) for translation, and they
prevent translocation of the growing protein from the ribosomal A site to the P site. Table 11.5.3 summarizes the protein synthesis
inhibitors.

Figure 11.5.3 : The major classes of protein synthesis inhibitors target the 30S or 50S subunits of cytoplasmic ribosomes.
Table 11.5.3 : Drugs That Inhibit Bacterial Protein Synthesis
Mechanism of Bacteriostatic or
Molecular Target Drug Class Specific Drugs Spectrum of Activity
Action Bactericidal

Causes mismatches
between codons and
anticodons, leading to Streptomycin,
faulty proteins that Aminoglycosides gentamicin, Bactericidal Broad spectrum
insert into and disrupt neomycin, kanamycin
30S subunit cytoplasmic
membrane

Tetracycline,
Blocks association of
Tetracyclines doxycycline, Bacteriostatic Broad spectrum
tRNAs with ribosome
tigecycline

Erythromycin,
Macrolides azithromycin, Bacteriostatic Broad spectrum
Blocks peptide bond telithromycin
formation between
Lincomycin, Narrow spectrum
amino acids Lincosamides Bacteriostatic
clindamycin Broad spectrum

50S subunit Not applicable Chloramphenicol Bacteriostatic Broad spectrum

Interferes with the

formation of the
initiation complex
Oxazolidinones Linezolid Bacteriostatic Broad spectrum
between 50S and 30S
subunits and other
factors.

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 Exercise 11.5.2

Compare and contrast the different types of protein synthesis inhibitors.

Inhibitors of Membrane Function

A small group of antibacterials target the bacterial membrane as their mode of action (Table 11.5.4). The polymyxins are natural
polypeptide antibiotics that were first discovered in 1947 as products of Bacillus polymyxa; only polymyxin B and polymyxin E
(colistin) have been used clinically. They are lipophilic with detergent-like properties and interact with the lipopolysaccharide
component of the outer membrane of gram-negative bacteria, ultimately disrupting both their outer and inner membranes and
killing the bacterial cells. Unfortunately, the membrane-targeting mechanism is not a selective toxicity, and these drugs also target
and damage the membrane of cells in the kidney and nervous system when administered systemically. Because of these serious side
effects and their poor absorption from the digestive tract, polymyxin B is used in over-the-counter topical antibiotic ointments (e.g.,
Neosporin), and oral colistin was historically used only for bowel decontamination to prevent infections originating from bowel
microbes in immunocompromised patients or for those undergoing certain abdominal surgeries. However, the emergence and
spread of multidrug-resistant pathogens has led to increased use of intravenous colistin in hospitals, often as a drug of last resort to
treat serious infections. The antibacterial daptomycin is a cyclic lipopeptide produced by Streptomyces roseosporus that seems to
work like the polymyxins, inserting in the bacterial cell membrane and disrupting it. However, in contrast to polymyxin B and
colistin, which target only gram-negative bacteria, daptomycin specifically targets gram-positive bacteria. It is typically
administered intravenously and seems to be well tolerated, showing reversible toxicity in skeletal muscles.
Table 11.5.4 : Drugs That Inhibit Bacterial Membrane Function
Mechanism of Action Drug Class Specific Drugs Spectrum of Activity Clinical Use

Narrow spectrum against

Topical preparations to
gram-negative bacteria,
Polymyxin B prevent infections in
including multidrug-
wounds
resistant strains
Interacts with
Oral dosing to
lipopolysaccharide in the
decontaminate bowels to
outer membrane of gram-
prevent infections in
negative bacteria, killing
Polymyxins immunocompromised
the cell through the Narrow spectrum against patients or patients
eventual disruption of the gram-negative bacteria,
Polymyxin E (colistin) undergoing invasive
outer membrane and including multidrug- surgery/procedures.
cytoplasmic membrane resistant strains
Intravenous dosing to treat
serious systemic infections
caused by multidrug-
resistant pathogens

Inserts into the Complicated skin and skin-

Narrow spectrum against
cytoplasmic membrane of structure infections and
gram-positive bacteria,
gram-positive bacteria, Lipopeptide Daptomycin bacteremia caused by
including multidrug-
disrupting the membrane gram-positive pathogens,
resistant strains
and killing the cell including MRSA

Exercise 11.5.3

How do polymyxins inhibit membrane function?

Inhibitors of Nucleic Acid Synthesis

Some antibacterial drugs work by inhibiting nucleic acid synthesis (Table 11.5.5). For example, metronidazole is a semisynthetic
member of the nitroimidazole family that is also an antiprotozoan. It interferes with DNA replication in target cells. The drug
rifampin is a semisynthetic member of the rifamycin family and functions by blocking RNA polymerase activity in bacteria. The
RNA polymerase enzymes in bacteria are structurally different from those in eukaryotes, providing for selective toxicity against
bacterial cells. It is used for the treatment of a variety of infections, but its primary use, often in a cocktail with other antibacterial

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drugs, is against mycobacteria that cause tuberculosis. Despite the selectivity of its mechanism, rifampin can induce liver enzymes
to increase metabolism of other drugs being administered (antagonism), leading to hepatotoxicity (liver toxicity) and negatively
influencing the bioavailability and therapeutic effect of the companion drugs.
One member of the quinolone family, a group of synthetic antimicrobials, is nalidixic acid. It was discovered in 1962 as a
byproduct during the synthesis of chloroquine, an antimalarial drug. Nalidixic acid selectively inhibits the activity of bacterial
DNA gyrase, blocking DNA replication. Chemical modifications to the original quinolone backbone have resulted in the
production of fluoroquinolones, like ciprofloxacin and levofloxacin, which also inhibit the activity of DNA gyrase. Ciprofloxacin
and levofloxacin are effective against a broad spectrum of gram-positive or gram-negative bacteria, and are among the most
commonly prescribed antibiotics used to treat a wide range of infections, including urinary tract infections, respiratory infections,
abdominal infections, and skin infections. However, despite their selective toxicity against DNA gyrase, side effects associated
with different fluoroquinolones include phototoxicity, neurotoxicity, cardiotoxicity, glucose metabolism dysfunction, and increased
risk for tendon rupture.
Table 11.5.5 : Drugs That Inhibit Bacterial Nucleic Acid Synthesis
Mechanisms of Action Drug Class Specific Drugs Spectrum of activity Clinical Use

Narrow spectrum with

activity against gram-
Inhibits bacterial RNA
positive and limited
polymerase activity and Combination therapy for
Rifamycin Rifampin numbers of gram-negative
blocks transcription, treatment of tuberculosis
bacteria. Also active
killing the cell
against Mycobacterium
tuberculosis.

Inhibits the activity of

Broad spectrum against
DNA gyrase and blocks Ciprofloxacin, ofloxacin, Wide variety of skin and
Fluoroquinolones gram-positive and gram-
DNA replication, killing moxifloxacin systemic infections
negative bacteria
the cell

Exercise 11.5.4

Why do inhibitors of bacterial nucleic acid synthesis not target host cells?

Inhibitors of Metabolic Pathways

Some synthetic drugs control bacterial infections by functioning as antimetabolites, competitive inhibitors for bacterial metabolic
enzymes (Table 11.5.6). The sulfonamides (sulfa drugs) are the oldest synthetic antibacterial agents and are structural analogues of
para-aminobenzoic acid (PABA), an early intermediate in folic acid synthesis (Figure 11.5.4). By inhibiting the enzyme involved
in the production of dihydrofolic acid, sulfonamides block bacterial biosynthesis of folic acid and, subsequently, pyrimidines and
purines required for nucleic acid synthesis. This mechanism of action provides bacteriostatic inhibition of growth against a wide
spectrum of gram-positive and gram-negative pathogens. Because humans obtain folic acid from food instead of synthesizing it
intracellularly, sulfonamides are selectively toxic for bacteria. However, allergic reactions to sulfa drugs are common. The sulfones
are structurally similar to sulfonamides but are not commonly used today except for the treatment of Hansen’s disease (leprosy).
Trimethoprim is a synthetic antimicrobial compound that serves as an antimetabolite within the same folic acid synthesis pathway
as sulfonamides. However, trimethoprim is a structural analogue of dihydrofolic acid and inhibits a later step in the metabolic
pathway (Figure 11.5.4). Trimethoprim is used in combination with the sulfa drug sulfamethoxazole to treat urinary tract
infections, ear infections, and bronchitis. As discussed, the combination of trimethoprim and sulfamethoxazole is an example of
antibacterial synergy. When used alone, each antimetabolite only decreases production of folic acid to a level where bacteriostatic
inhibition of growth occurs. However, when used in combination, inhibition of both steps in the metabolic pathway decreases folic
acid synthesis to a level that is lethal to the bacterial cell. Because of the importance of folic acid during fetal development, sulfa
drugs and trimethoprim use should be carefully considered during early pregnancy.
The drug isoniazid is an antimetabolite with specific toxicity for mycobacteria and has long been used in combination with
rifampin or streptomycin in the treatment of tuberculosis. It is administered as a prodrug, requiring activation through the action of
an intracellular bacterial peroxidase enzyme, forming isoniazid-nicotinamide adenine dinucleotide (NAD) and isoniazid-
nicotinamide adenine dinucleotide phosphate (NADP), ultimately preventing the synthesis of mycolic acid, which is essential for

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mycobacterial cell walls. Possible side effects of isoniazid use include hepatotoxicity, neurotoxicity, and hematologic toxicity
(anemia).

Figure 11.5.4 : Sulfonamides and trimethoprim are examples of antimetabolites that interfere in the bacterial synthesis of folic acid
by blocking purine and pyrimidine biosynthesis, thus inhibiting bacterial growth.
Table 11.5.6 : Antimetabolite Drugs
Metabolic Pathway
Mechanism of Action Drug Class Specific Drugs Spectrum of Activity
Target

Inhibits the enzyme Sulfonamides Sulfamethoxazole Broad spectrum against

involved in production of gram-positive and gram-
dihydrofolic acid Sulfones Dapsone negative bacteria
Folic acid synthesis
Inhibits the enzyme Broad spectrum against
involved in the production Not applicable Trimethoprim gram-positive and gram-
of tetrahydrofolic acid negative bacteria

Narrow spectrum against

Interferes with the
Mycolic acid synthesis Not applicable Isoniazid Mycobacterium spp.,
synthesis of mycolic acid
including M. tuberculosis

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 Exercise 11.5.5

How do sulfonamides and trimethoprim selectively target bacteria?

Inhibitor of ATP Synthase

Bedaquiline, representing the synthetic antibacterial class of compounds called the diarylquinolones, uses a novel mode of action
that specifically inhibits mycobacterial growth. Although the specific mechanism has yet to be elucidated, this compound appears
to interfere with the function of ATP synthases, perhaps by interfering with the use of the hydrogen ion gradient for ATP synthesis
by oxidative phosphorylation, leading to reduced ATP production. Due to its side effects, including hepatotoxicity and potentially
lethal heart arrhythmia, its use is reserved for serious, otherwise untreatable cases of tuberculosis.
To learn more about the general principles of antimicrobial therapy and bacterial modes of action, visit Michigan State University’s
Antimicrobial Resistance Learning Site, particularly pages 6 through 9.

Key Concepts and Summary

Antibacterial compounds exhibit selective toxicity, largely due to differences between prokaryotic and eukaryotic cell structure.
Cell wall synthesis inhibitors, including the β-lactams, the glycopeptides, and bacitracin, interfere with peptidoglycan
synthesis, making bacterial cells more prone to osmotic lysis.
There are a variety of broad-spectrum, bacterial protein synthesis inhibitors that selectively target the prokaryotic 70S ribosome,
including those that bind to the 30S subunit (aminoglycosides and tetracyclines) and others that bind to the 50S subunit
(macrolides, lincosamides, chloramphenicol, and oxazolidinones).
Polymyxins are lipophilic polypeptide antibiotics that target the lipopolysaccharide component of gram-negative bacteria and
ultimately disrupt the integrity of the outer and inner membranes of these bacteria.
The nucleic acid synthesis inhibitors rifamycins and fluoroquinolones target bacterial RNA transcription and DNA replication,
respectively.
Some antibacterial drugs are antimetabolites, acting as competitive inhibitors for bacterial metabolic enzymes. Sulfonamides
and trimethoprim are antimetabolites that interfere with bacterial folic acid synthesis. Isoniazid is an antimetabolite that
interferes with mycolic acid synthesis in mycobacteria.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.5: Drug Targets on Prokaryote Microorganisms is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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11.6: Drugs for Non-prokaryote Microbes
Learning Objectives
Explain the differences between modes of action of drugs that target fungi, protozoa, helminths, and viruses

Because fungi, protozoa, and helminths are eukaryotic, their cells are very similar to human cells, making it more difficult to
develop drugs with selective toxicity. Additionally, viruses replicate within human host cells, making it difficult to develop drugs
that are selectively toxic to viruses or virus-infected cells. Despite these challenges, there are antimicrobial drugs that target fungi,
protozoa, helminths, and viruses, and some even target more than one type of microbe. Table 11.6.1, Table 11.6.2, Table 11.6.3,
and Table 11.6.4 provide examples for antimicrobial drugs in these various classes.

Antifungal Drugs
The most common mode of action for antifungal drugs is the disruption of the cell membrane. Antifungals take advantage of small
differences between fungi and humans in the biochemical pathways that synthesize sterols. The sterols are important in maintaining
proper membrane fluidity and, hence, proper function of the cell membrane. For most fungi, the predominant membrane sterol is
ergosterol. Because human cell membranes use cholesterol, instead of ergosterol, antifungal drugs that target ergosterol synthesis
are selectively toxic (Figure 11.6.1).

Figure 11.6.1 : The predominant sterol found in human cells is cholesterol, whereas the predominant sterol found in fungi is
ergosterol, making ergosterol a good target for antifungal drug development.
The imidazoles are synthetic fungicides that disrupt ergosterol biosynthesis; they are commonly used in medical applications and
also in agriculture to keep seeds and harvested crops from molding. Examples include miconazole, ketoconazole, and clotrimazole,
which are used to treat fungal skin infections such as ringworm, specifically tinea pedis (athlete’s foot), tinea cruris (jock itch), and
tinea corporis. These infections are commonly caused by dermatophytes of the genera Trichophyton, Epidermophyton, and
Microsporum. Miconazole is also used predominantly for the treatment of vaginal yeast infections caused by the fungus Candida,
and ketoconazole is used for the treatment of tinea versicolor and dandruff, which both can be caused by the fungus Malassezia.
The triazole drugs, including fluconazole, also inhibit ergosterol biosynthesis. However, they can be administered orally or
intravenously for the treatment of several types of systemic yeast infections, including oral thrush and cryptococcal meningitis,
both of which are prevalent in patients with AIDS. The triazoles also exhibit more selective toxicity, compared with the imidazoles,
and are associated with fewer side effects.
The allylamines, a structurally different class of synthetic antifungal drugs, inhibit an earlier step in ergosterol biosynthesis. The
most commonly used allylamine is terbinafine (marketed under the brand name Lamisil), which is used topically for the treatment
of dermatophytic skin infections like athlete’s foot, ringworm, and jock itch. Oral treatment with terbinafine is also used for the
treatment of fingernail and toenail fungus, but it can be associated with the rare side effect of hepatotoxicity.
The polyenes are a class of antifungal agents naturally produced by certain actinomycete soil bacteria and are structurally related to
macrolides. These large, lipophilic molecules bind to ergosterol in fungal cytoplasmic membranes, thus creating pores. Common
examples include nystatin and amphotericin B. Nystatin is typically used as a topical treatment for yeast infections of the skin,
mouth, and vagina, but may also be used for intestinal fungal infections. The drug amphotericin B is used for systemic fungal
infections like aspergillosis, cryptococcal meningitis, histoplasmosis, blastomycosis, and candidiasis. Amphotericin B was the only

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antifungal drug available for several decades, but its use is associated with some serious side effects, including nephrotoxicity
(kidney toxicity).
Amphotericin B is often used in combination with flucytosine, a fluorinated pyrimidine analog that is converted by a fungal-
specific enzyme into a toxic product that interferes with both DNA replication and protein synthesis in fungi. Flucytosine is also
associated with hepatotoxicity (liver toxicity) and bone marrow depression.
Beyond targeting ergosterol in fungal cell membranes, there are a few antifungal drugs that target other fungal structures (Figure
11.6.2). The echinocandins, including caspofungin, are a group of naturally produced antifungal compounds that block the

synthesis of β(1→3) glucan found in fungal cell walls but not found in human cells. This drug class has the nickname “penicillin
for fungi.” Caspofungin is used for the treatment of aspergillosis as well as systemic yeast infections.
Although chitin is only a minor constituent of fungal cell walls, it is also absent in human cells, making it a selective target. The
polyoxins and nikkomycins are naturally produced antifungals that target chitin synthesis. Polyoxins are used to control fungi for
agricultural purposes, and nikkomycin Z is currently under development for use in humans to treat yeast infections and Valley fever
(coccidioidomycosis), a fungal disease prevalent in the southwestern US.1
The naturally produced antifungal griseofulvin is thought to specifically disrupt fungal cell division by interfering with
microtubules involved in spindle formation during mitosis. It was one of the first antifungals, but its use is associated with
hepatotoxicity. It is typically administered orally to treat various types of dermatophytic skin infections when other topical
antifungal treatments are ineffective.
There are a few drugs that act as antimetabolites against fungal processes. For example, atovaquone, a representative of the
naphthoquinone drug class, is a semisynthetic antimetabolite for fungal and protozoal versions of a mitochondrial cytochrome
important in electron transport. Structurally, it is an analog of coenzyme Q, with which it competes for electron binding. It is
particularly useful for the treatment of Pneumocystis pneumonia caused by Pneumocystis jirovecii. The antibacterial
sulfamethoxazole-trimethoprim combination also acts as an antimetabolite against P. jirovecii.
Table 11.6.1 shows the various therapeutic classes of antifungal drugs, categorized by mode of action, with examples of each.

Figure 11.6.2 : Antifungal drugs target several different cell structures. (credit right: modification of work by “Maya and
Rike”/Wikimedia Commons)
Table 11.6.1 : Common Antifungal Drugs
Mechanism of Action Drug Class Specific Drugs Clinical Uses

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 Mechanism of Action Drug Class Specific Drugs Clinical Uses

Miconazole, ketoconazole, Fungal skin infections and vaginal

Imidazoles
clotrimazole yeast infections

Systemic yeast infections, oral

Triazoles Fluconazole thrush, and cryptococcal
Inhibit ergosterol synthesis meningitis
Dermatophytic skin infections
(athlete’s foot, ring worm, jock
Allylamines Terbinafine
itch), and infections of fingernails
and toenails
Used topically for yeast infections
Bind ergosterol in the cell of skin, mouth, and vagina; also
Nystatin
membrane and create pores that Polyenes used for fungal infections of the
disrupt the membrane intestine

Amphotericin B Variety systemic fungal infections

Aspergillosis and systemic yeast

Echinocandins Caspofungin
infections
Inhibit cell wall synthesis
Coccidioidomycosis (Valley fever)
Not applicable Nikkomycin Z
and yeast infections

Inhibit microtubules and cell

Not applicable Griseofulvin Dermatophytic skin infections
division

Exercise 11.6.1

How is disruption of ergosterol biosynthesis an effective mode of action for antifungals?

Treating a Fungal Infection of the Lungs

Jack, a 48-year-old engineer, is HIV positive but generally healthy thanks to antiretroviral therapy (ART). However, after a
particularly intense week at work, he developed a fever and a dry cough. He assumed that he just had a cold or mild flu due to
overexertion and didn’t think much of it. However, after about a week, he began to experience fatigue, weight loss, and
shortness of breath. He decided to visit his physician, who found that Jack had a low level of blood oxygenation. The physician
ordered blood testing, a chest X-ray, and the collection of an induced sputum sample for analysis. His X-ray showed a fine
cloudiness and several pneumatoceles (thin-walled pockets of air), which indicated Pneumocystis pneumonia (PCP), a type of
pneumonia caused by the fungus Pneumocystis jirovecii. Jack’s physician admitted him to the hospital and prescribed Bactrim,
a combination of sulfamethoxazole and trimethoprim, to be administered intravenously.
P. jirovecii is a yeast-like fungus with a life cycle similar to that of protozoans. As such, it was classified as a protozoan until
the 1980s. It lives only in the lung tissue of infected persons and is transmitted from person to person, with many people
exposed as children. Typically, P. jirovecii only causes pneumonia in immunocompromised individuals. Healthy people may
carry the fungus in their lungs with no symptoms of disease. PCP is particularly problematic among HIV patients with
compromised immune systems.
PCP is usually treated with oral or intravenous Bactrim, but atovaquone or pentamidine (another antiparasitic drug) are
alternatives. If not treated, PCP can progress, leading to a collapsed lung and nearly 100% mortality. Even with antimicrobial
drug therapy, PCP still is responsible for 10% of HIV-related deaths.
The cytological examination, using direct immunofluorescence assay (DFA), of a smear from Jack’s sputum sample confirmed
the presence of P. jirovecii (Figure 11.6.3). Additionally, the results of Jack’s blood tests revealed that his white blood cell
count had dipped, making him more susceptible to the fungus. His physician reviewed his ART regimen and made
adjustments. After a few days of hospitalization, Jack was released to continue his antimicrobial therapy at home. With the
adjustments to his ART therapy, Jack’s CD4 counts began to increase and he was able to go back to work.

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 Figure 11.6.3 : Microscopic examination of an induced sputum sample or bronchoaveolar lavage sample typically reveals the
organism, as shown here. (credit: modification of work by the Centers for Disease Control and Prevention)

Antiprotozoan Drugs
There are a few mechanisms by which antiprotozoan drugs target infectious protozoans (Table 11.6.3). Some are antimetabolites,
such as atovaquone, proguanil, and artemisinins. Atovaquone, in addition to being antifungal, blocks electron transport in
protozoans and is used for the treatment of protozoan infections including malaria, babesiosis, and toxoplasmosis. Proguanil is
another synthetic antimetabolite that is processed in parasitic cells into its active form, which inhibits protozoan folic acid
synthesis. It is often used in combination with atovaquone, and the combination is marketed as Malarone for both malaria treatment
and prevention.
Artemisinin, a plant-derived antifungal first discovered by Chinese scientists in the 1970s, is quite effective against malaria.
Semisynthetic derivatives of artemisinin are more water soluble than the natural version, which makes them more bioavailable.
Although the exact mechanism of action is unclear, artemisinins appear to act as prodrugs that are metabolized by target cells to
produce reactive oxygen species (ROS) that damage target cells. Due to the rise in resistance to antimalarial drugs, artemisinins are
also commonly used in combination with other antimalarial compounds in artemisinin-based combination therapy (ACT).
Several antimetabolites are used for the treatment of toxoplasmosis caused by the parasite Toxoplasma gondii. The synthetic sulfa
drug sulfadiazine competitively inhibits an enzyme in folic acid production in parasites and can be used to treat malaria and
toxoplasmosis. Pyrimethamine is a synthetic drug that inhibits a different enzyme in the folic acid production pathway and is often
used in combination with sulfadoxine (another sulfa drug) for the treatment of malariaor in combination with sulfadiazine for the
treatment of toxoplasmosis. Side effects of pyrimethamine include decreased bone marrow activity that may cause increased
bruising and low red blood cell counts. When toxicity is a concern, spiramycin, a macrolide protein synthesis inhibitor, is typically
administered for the treatment of toxoplasmosis.
Two classes of antiprotozoan drugs interfere with nucleic acid synthesis: nitroimidazoles and quinolines. Nitroimidazoles,
including semisynthetic metronidazole, which was discussed previously as an antibacterial drug, and synthetic tinidazole, are useful
in combating a wide variety of protozoan pathogens, such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis.
Upon introduction into these cells in low-oxygen environments, nitroimidazoles become activated and introduce DNA strand
breakage, interfering with DNA replication in target cells. Unfortunately, metronidazole is associated with carcinogenesis (the
development of cancer) in humans.
Another type of synthetic antiprotozoan drug that has long been thought to specifically interfere with DNA replication in certain
pathogens is pentamidine. It has historically been used for the treatment of African sleeping sickness (caused by the protozoan
Trypanosoma brucei) and leishmaniasis (caused by protozoa of the genus Leishmania), but it is also an alternative treatment for the
fungus Pneumocystis. Some studies indicate that it specifically binds to the DNA found within kinetoplasts (kDNA; long
mitochondrion-like structures unique to trypanosomes), leading to the cleavage of kDNA. However, nuclear DNA of both the

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parasite and host remain unaffected. It also appears to bind to tRNA, inhibiting the addition of amino acids to tRNA, thus
preventing protein synthesis. Possible side effects of pentamidine use include pancreatic dysfunction and liver damage.
The quinolines are a class of synthetic compounds related to quinine, which has a long history of use against malaria. Quinolines
are thought to interfere with heme detoxification, which is necessary for the parasite’s effective breakdown of hemoglobin into
amino acids inside red blood cells. The synthetic derivatives chloroquine, quinacrine (also called mepacrine), and mefloquine are
commonly used as antimalarials, and chloroquine is also used to treat amebiasis typically caused by Entamoeba histolytica. Long-
term prophylactic use of chloroquine or mefloquine may result in serious side effects, including hallucinations or cardiac issues.
Patients with glucose-6-phosphate dehydrogenase deficiency experience severe anemia when treated with chloroquine.
Table 11.6.2 : Common Antiprotozoan Drugs
Mechanism of Action Drug Class Specific Drugs Clinical Uses

Inhibit electron transport in Malaria, babesiosis, and

Naphthoquinone Atovaquone
mitochondria toxoplasmosis

Combination therapy with

Not applicable Proquanil atovaquone for malaria treatment
and prevention

Inhibit folic acid synthesis Sulfonamide Sulfadiazine Malaria and toxoplasmosis

Combination therapy with

Not applicable Pyrimethamine sulfadoxine (sulfa drug) for
malaria

Produces damaging reactive Combination therapy to treat

Not applicable Artemisinin
oxygen species malaria

Infections caused by Giardia

Nitroimidazoles Metronidazole, tinidazole lamblia, Entamoeba histolytica,
Inhibit DNA synthesis and Trichomonas vaginalis
African sleeping sickness and
Not applicable Pentamidine
leishmaniasis
Malaria and infections with E.
Chloroquine
Inhibit heme detoxification Quinolines histolytica

Mepacrine, mefloquine Malaria

Exercise 11.6.2

List two modes of action for antiprotozoan drugs.

Antihelminthic Drugs
Because helminths are multicellular animal eukaryotes like humans, developing drugs with selective toxicity against them is
extremely challenging. Despite this, several effective classes have been developed (Table 11.6.3). Synthetic benzimidazoles, like
mebendazole and albendazole, bind to helminthic β-tubulin, preventing microtubule formation. Microtubules in the intestinal cells
of the worms seem to be particularly affected, leading to a reduction in glucose uptake. Besides their activity against a broad range
of helminths, benzimidazoles are also active against many protozoans, fungi, and viruses, and their use for inhibiting mitosis and
cell cycle progression in cancer cells is under study.2 Possible side effects of their use include liver damage and bone marrow
suppression.
The avermectins are members of the macrolide family that were first discovered from a Japanese soil isolate, Streptomyces
avermectinius. A more potent semisynthetic derivative of avermectin is ivermectin, which binds to glutamate-gated chloride
channels specific to invertebrates including helminths, blocking neuronal transmission and causing starvation, paralysis, and death
of the worms. Ivermectin is used to treat roundworm diseases, including onchocerciasis (also called river blindness, caused by the
worm Onchocerca volvulus) and strongyloidiasis (caused by the worm Strongyloides stercoralis or S. fuelleborni). Ivermectin also
can also treat parasitic insects like mites, lice, and bed bugs, and is nontoxic to humans.

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Niclosamide is a synthetic drug that has been used for over 50 years to treat tapeworm infections. Although its mode of action is
not entirely clear, niclosamide appears to inhibit ATP formation under anaerobic conditions and inhibit oxidative phosphorylation
in the mitochondria of its target pathogens. Niclosamide is not absorbed from the gastrointestinal tract, thus it can achieve high
localized intestinal concentrations in patients. Recently, it has been shown to also have antibacterial, antiviral, and antitumor
activities.345
Another synthetic antihelminthic drug is praziquantel, which used for the treatment of parasitic tapeworms and liver flukes, and is
particularly useful for the treatment of schistosomiasis (caused by blood flukes from three genera of Schistosoma). Its mode of
action remains unclear, but it appears to cause the influx of calcium into the worm, resulting in intense spasm and paralysis of the
worm. It is often used as a preferred alternative to niclosamide in the treatment of tapeworms when gastrointestinal discomfort
limits niclosamide use.
The thioxanthenones, another class of synthetic drugs structurally related to quinine, exhibit antischistosomal activity by inhibiting
RNA synthesis. The thioxanthenone lucanthone and its metabolite hycanthone were the first used clinically, but serious
neurological, gastrointestinal, cardiovascular, and hepatic side effects led to their discontinuation. Oxamniquine, a less toxic
derivative of hycanthone, is only effective against S. mansoni, one of the three species known to cause schistosomiasis in humans.
Praziquantel was developed to target the other two schistosome species, but concerns about increasing resistance have renewed
interest in developing additional derivatives of oxamniquine to target all three clinically important schistosome species.
Table 11.6.3 : Common Antihelminthic Drugs
Mechanism of Action Drug Class Specific Drugs Clinical Uses

Inhibit microtubule formation,

Benzimidazoles Mebendazole, albendazole Variety of helminth infections
reducing glucose uptake

Roundworm diseases, including

Block neuronal transmission, river blindness and
Avermectins Ivermectin
causing paralysis and starvation strongyloidiasis, and treatment of
parasitic insects

Inhibit ATP production Not applicable Niclosamide Intestinal tapeworm infections

Induce calcium influx Not applicable Praziquantel Schistosomiasis (blood flukes)

Lucanthone, hycanthone,
Inhibit RNA synthesis Thioxanthenones Schistosomiasis (blood flukes)
oxamniquine

Exercise 11.6.3

Why are antihelminthic drugs difficult to develop?

Antiviral Drugs
Unlike the complex structure of fungi, protozoa, and helminths, viral structure is simple, consisting of nucleic acid, a protein coat,
viral enzymes, and, sometimes, a lipid envelope. Furthermore, viruses are obligate intracellular pathogens that use the host’s
cellular machinery to replicate. These characteristics make it difficult to develop drugs with selective toxicity against viruses.
Many antiviral drugs are nucleoside analogs and function by inhibiting nucleic acid biosynthesis. For example, acyclovir(marketed
as Zovirax) is a synthetic analog of the nucleoside guanosine (Figure 11.6.4). It is activated by the herpes simplex viral enzyme
thymidine kinase and, when added to a growing DNA strand during replication, causes chain termination. Its specificity for virus-
infected cells comes from both the need for a viral enzyme to activate it and the increased affinity of the activated form for viral
DNA polymerase compared to host cell DNA polymerase. Acyclovir and its derivatives are frequently used for the treatment of
herpes virus infections, including genital herpes, chickenpox, shingles, Epstein-Barr virus infections, and cytomegalovirus
infections. Acyclovir can be administered either topically or systemically, depending on the infection. One possible side effect of its
use includes nephrotoxicity. The drug adenine-arabinoside, marketed as vidarabine, is a synthetic analog to deoxyadenosine that
has a mechanism of action similar to that of acyclovir. It is also effective for the treatment of various human herpes viruses.
However, because of possible side effects involving low white blood cell counts and neurotoxicity, treatment with acyclovir is now
preferred.

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Ribavirin, another synthetic guanosine analog, works by a mechanism of action that is not entirely clear. It appears to interfere with
both DNA and RNA synthesis, perhaps by reducing intracellular pools of guanosine triphosphate (GTP). Ribavarin also appears to
inhibit the RNA polymerase of hepatitis C virus. It is primarily used for the treatment of the RNA viruses like hepatitis C (in
combination therapy with interferon) and respiratory syncytial virus. Possible side effects of ribavirin use include anemia and
developmental effects on unborn children in pregnant patients. In recent years, another nucleotide analog, sofosbuvir (Solvaldi),
has also been developed for the treatment of hepatitis C. Sofosbuvir is a uridine analog that interferes with viral polymerase
activity. It is commonly coadministered with ribavirin, with and without interferon.
Inhibition of nucleic acid synthesis is not the only target of synthetic antivirals. Although the mode of action of amantadine and its
relative rimantadine are not entirely clear, these drugs appear to bind to a transmembrane protein that is involved in the escape of
the influenza virus from endosomes. Blocking escape of the virus also prevents viral RNA release into host cells and subsequent
viral replication. Increasing resistance has limited the use of amantadine and rimantadine in the treatment of influenza A. Use of
amantadine can result in neurological side effects, but the side effects of rimantadine seem less severe. Interestingly, because of
their effects on brain chemicals such as dopamine and NMDA (N-methyl D-aspartate), amantadine and rimantadine are also used
for the treatment of Parkinson’s disease.
Neuraminidase inhibitors, including olsetamivir (Tamiflu), zanamivir (Relenza), and peramivir (Rapivab), specifically target
influenza viruses by blocking the activity of influenza virus neuraminidase, preventing the release of the virus from infected cells.
These three antivirals can decrease flu symptoms and shorten the duration of illness, but they differ in their modes of
administration: olsetamivir is administered orally, zanamivir is inhaled, and peramivir is administered intravenously. Resistance to
these neuraminidase inhibitors still seems to be minimal.
Pleconaril is a synthetic antiviral under development that showed promise for the treatment of picornaviruses. Use of pleconaril for
the treatment of the common cold caused by rhinoviruses was not approved by the FDA in 2002 because of lack of proven
effectiveness, lack of stability, and association with irregular menstruation. Its further development for this purpose was halted in
2007. However, pleconaril is still being investigated for use in the treatment of life-threatening complications of enteroviruses, such
as meningitis and sepsis. It is also being investigated for use in the global eradication of a specific enterovirus, polio.6 Pleconaril
seems to work by binding to the viral capsid and preventing the uncoating of viral particles inside host cells during viral infection.
Viruses with complex life cycles, such as HIV, can be more difficult to treat. First, HIV targets CD4-positive white blood cells,
which are necessary for a normal immune response to infection. Second, HIV is a retrovirus, meaning that it converts its RNA
genome into a DNA copy that integrates into the host cell’s genome, thus hiding within host cell DNA. Third, the HIV reverse
transcriptase lacks proofreading activity and introduces mutations that allow for rapid development of antiviral drug resistance. To
help prevent the emergence of resistance, a combination of specific synthetic antiviral drugs is typically used in ART for HIV
(Figure).
The reverse transcriptase inhibitors block the early step of converting viral RNA genome into DNA, and can include competitive
nucleoside analog inhibitors (e.g., azidothymidine/zidovudine, or AZT) and non-nucleoside noncompetitive inhibitors (e.g.,
etravirine) that bind reverse transcriptase and cause an inactivating conformational change. Drugs called protease inhibitors (e.g.,
ritonavir) block the processing of viral proteins and prevent viral maturation. Protease inhibitors are also being developed for the
treatment of other viral types.7 For example, simeprevir (Olysio) has been approved for the treatment of hepatitis C and is
administered with ribavirin and interferon in combination therapy. The integrase inhibitors (e.g., raltegravir), block the activity of
the HIV integrase responsible for the recombination of a DNA copy of the viral genome into the host cell chromosome. Additional
drug classes for HIV treatment include the CCR5 antagonists and the fusion inhibitors (e.g., enfuviritide), which prevent the
binding of HIV to the host cell coreceptor (chemokine receptor type 5 [CCR5]) and the merging of the viral envelope with the host
cell membrane, respectively. Table 11.6.4 shows the various therapeutic classes of antiviral drugs, categorized by mode of action,
with examples of each.

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Figure 11.6.4 : Acyclovir is a structural analog of guanosine. It is specifically activated by the viral enzyme thymidine kinase and
then preferentially binds to viral DNA polymerase, leading to chain termination during DNA replication.

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 Figure 11.6.5 : Antiretroviral therapy (ART) is typically used for the treatment of HIV. The targets of drug classes currently in use
are shown here. (credit: modification of work by Thomas Splettstoesser)
Table 11.6.4 : Common Antiviral Drugs
Mechanism of Action Drug Clinical Uses

Acyclovir Herpes virus infections

Azidothymidine/zidovudine (AZT) HIV infections

Nucleoside analog inhibition of nucleic acid Hepatitis C virus and respiratory syncytial
Ribavirin
synthesis virus infections

Vidarabine Herpes virus infections

Sofosbuvir Hepatitis C virus infections

Non-nucleoside noncompetitive inhibition Etravirine HIV infections

Inhibit escape of virus from endosomes Amantadine, rimantadine Infections with influenza virus

Inhibit neuraminadase Olsetamivir, zanamivir, peramivir Infections with influenza virus

Inhibit viral uncoating Pleconaril Serious enterovirus infections

Ritonavir HIV infections

Inhibition of protease
Simeprevir Hepatitis C virus infections

Inhibition of integrase Raltegravir HIV infections

Inhibition of membrane fusion Enfuviritide HIV infections

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 Exercise 11.6.4

Why is HIV difficult to treat with antivirals?

To learn more about the various classes of antiretroviral drugs used in the ART of HIV infection, explore each of the drugs in the
HIV drug classes provided by US Department of Health and Human Services at this website.

Key Concepts and Summary

Because fungi, protozoans, and helminths are eukaryotic organisms like human cells, it is more challenging to develop
antimicrobial drugs that specifically target them. Similarly, it is hard to target viruses because human viruses replicate inside of
human cells.
Antifungal drugs interfere with ergosterol synthesis, bind to ergosterol to disrupt fungal cell membrane integrity, or target cell
wall-specific components or other cellular proteins.
Antiprotozoan drugs increase cellular levels of reactive oxygen species, interfere with protozoal DNA replication (nuclear
versus kDNA, respectively), and disrupt heme detoxification.
Antihelminthic drugs disrupt helminthic and protozoan microtubule formation; block neuronal transmissions; inhibit
anaerobic ATP formation and/or oxidative phosphorylation; induce a calcium influx in tapeworms, leading to spasms and
paralysis; and interfere with RNA synthesis in schistosomes.
Antiviral drugs inhibit viral entry, inhibit viral uncoating, inhibit nucleic acid biosynthesis, prevent viral escape from
endosomes in host cells, and prevent viral release from infected cells.
Because it can easily mutate to become drug resistant, HIV is typically treated with a combination of several antiretroviral
drugs, which may include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and drugs that interfere
with viral binding and fusion to initiate infection.

Footnotes
1. Centers for Disease Control and Prevention. “Valley Fever: Awareness Is Key.” www.cdc.gov/features/valleyfever/. Accessed
June 1, 2016.
2. B. Chu et al. “A Benzimidazole Derivative Exhibiting Antitumor Activity Blocks EGFR and HER2 Activity and Upregulates
DR5 in Breast Cancer Cells.” Cell Death and Disease 6 (2015):e1686
3. J.-X. Pan et al. “Niclosamide, An Old Antihelminthic Agent, Demonstrates Antitumor Activity by Blocking Multiple Signaling
Pathways of Cancer Stem Cells.” Chinese Journal of Cancer 31 no. 4 (2012):178–184.
4. F. Imperi et al. “New Life for an Old Drug: The Anthelmintic Drug Niclosamide Inhibits Pseudomonas aeruginosa Quorum
Sensing.” Antimicrobial Agents and Chemotherapy 57 no. 2 (2013):996-1005.
5. A. Jurgeit et al. “Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects.” PLoS
Pathogens 8 no. 10 (2012):e1002976.
6. M.J. Abzug. “The Enteroviruses: Problems in Need of Treatments.” Journal of Infection 68 no. S1 (2014):108–14.
7. B.L. Pearlman. “Protease Inhibitors for the Treatment of Chronic Hepatitis C Genotype-1 Infection: The New Standard of
Care.” Lancet Infectious Diseases 12 no. 9 (2012):717–728.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.6: Drugs for Non-prokaryote Microbes is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

Access for free at OpenStax 11.6.10 https://bio.libretexts.org/@go/page/31841

11.7: Mechanisms for Resistance
Learning Objectives
Explain the concept of drug resistance
Describe how microorganisms develop or acquire drug resistance
Describe the different mechanisms of antimicrobial drug resistance

Antimicrobial resistance is not a new phenomenon. In nature, microbes are constantly evolving in order to overcome the
antimicrobial compounds produced by other microorganisms. Human development of antimicrobial drugs and their widespread
clinical use has simply provided another selective pressure that promotes further evolution. Several important factors can accelerate
the evolution of drug resistance. These include the overuse and misuse of antimicrobials, inappropriate use of antimicrobials,
subtherapeutic dosing, and patient noncompliance with the recommended course of treatment.
Exposure of a pathogen to an antimicrobial compound can select for chromosomal mutations conferring resistance, which can be
transferred vertically to subsequent microbial generations and eventually become predominant in a microbial population that is
repeatedly exposed to the antimicrobial. Alternatively, many genes responsible for drug resistance are found on plasmids or in
transposons that can be transferred easily between microbes through horizontal gene transfer. Transposons also have the ability to
move resistance genes between plasmids and chromosomes to further promote the spread of resistance.

Mechanisms for Drug Resistance

There are several common mechanisms for drug resistance, which are summarized in Figure 11.7.1. These mechanisms include
enzymatic modification of the drug, modification of the antimicrobial target, and prevention of drug penetration or accumulation.

Figure 11.7.1 : There are multiple strategies that microbes use to develop resistance to antimicrobial drugs. (Not shown: target
overproduction, target mimicry, and enzymatic bypass). (credit: modification of work by Gerard D Wright)

Drug Modification or Inactivation

Resistance genes may code for enzymes that chemically modify an antimicrobial, thereby inactivating it, or destroy an
antimicrobial through hydrolysis. Resistance to many types of antimicrobials occurs through this mechanism. For example,

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aminoglycoside resistance can occur through enzymatic transfer of chemical groups to the drug molecule, impairing the binding of
the drug to its bacterial target. For β-lactams, bacterial resistance can involve the enzymatic hydrolysis of the β-lactam bond within
the β-lactam ring of the drug molecule. Once the β-lactam bond is broken, the drug loses its antibacterial activity. This mechanism
of resistance is mediated by β-lactamases, which are the most common mechanism of β-lactam resistance. Inactivation of rifampin
commonly occurs through glycosylation, phosphorylation, or adenosine diphosphate (ADP) ribosylation, and resistance to
macrolides and lincosamides can also occur due to enzymatic inactivation of the drug or modification.

Prevention of Cellular Uptake or Efflux

Microbes may develop resistance mechanisms that involve inhibiting the accumulation of an antimicrobial drug, which then
prevents the drug from reaching its cellular target. This strategy is common among gram-negative pathogens and can involve
changes in outer membrane lipid composition, porin channel selectivity, and/or porin channel concentrations. For example, a
common mechanism of carbapenem resistance among Pseudomonas aeruginosa is to decrease the amount of its OprD porin, which
is the primary portal of entry for carbapenems through the outer membrane of this pathogen. Additionally, many gram-positive and
gram-negative pathogenic bacteria produce efflux pumps that actively transport an antimicrobial drug out of the cell and prevent
the accumulation of drug to a level that would be antibacterial. For example, resistance to β-lactams, tetracyclines, and
fluoroquinolones commonly occurs through active efflux out of the cell, and it is rather common for a single efflux pump to have
the ability to translocate multiple types of antimicrobials.

Target Modification
Because antimicrobial drugs have very specific targets, structural changes to those targets can prevent drug binding, rendering the
drug ineffective. Through spontaneous mutations in the genes encoding antibacterial drug targets, bacteria have an evolutionary
advantage that allows them to develop resistance to drugs. This mechanism of resistance development is quite common. Genetic
changes impacting the active site of penicillin-binding proteins (PBPs) can inhibit the binding of β-lactam drugs and provide
resistance to multiple drugs within this class. This mechanism is very common among strains of Streptococcus pneumoniae, which
alter their own PBPs through genetic mechanisms. In contrast, strains of Staphylococcus aureus develop resistance to methicillin
(MRSA) through the acquisition of a new low-affinity PBP, rather than structurally alter their existing PBPs. Not only does this
new low-affinity PBP provide resistance to methicillin but it provides resistance to virtually all β-lactam drugs, with the exception
of the newer fifth-generation cephalosporins designed specifically to kill MRSA. Other examples of this resistance strategy include
alterations in
ribosome subunits, providing resistance to macrolides, tetracyclines, and aminoglycosides;
lipopolysaccharide (LPS) structure, providing resistance to polymyxins;
RNA polymerase, providing resistance to rifampin;
DNA gyrase, providing resistance to fluoroquinolones;
metabolic enzymes, providing resistance to sulfa drugs, sulfones, and trimethoprim; and
peptidoglycan subunit peptide chains, providing resistance to glycopeptides.

Target Overproduction or Enzymatic Bypass

When an antimicrobial drug functions as an antimetabolite, targeting a specific enzyme to inhibit its activity, there are additional
ways that microbial resistance may occur. First, the microbe may overproduce the target enzyme such that there is a sufficient
amount of antimicrobial-free enzyme to carry out the proper enzymatic reaction. Second, the bacterial cell may develop a bypass
that circumvents the need for the functional target enzyme. Both of these strategies have been found as mechanisms of sulfonamide
resistance. Vancomycin resistance among S. aureus has been shown to involve the decreased cross-linkage of peptide chains in the
bacterial cell wall, which provides an increase in targets for vancomycin to bind to in the outer cell wall. Increased binding of
vancomycin in the outer cell wall provides a blockage that prevents free drug molecules from penetrating to where they can block
new cell wall synthesis.

Target Mimicry
A recently discovered mechanism of resistance called target mimicry involves the production of proteins that bind and sequester
drugs, preventing the drugs from binding to their target. For example, Mycobacterium tuberculosis produces a protein with regular
pentapeptide repeats that appears to mimic the structure of DNA. This protein binds fluoroquinolones, sequestering them and
keeping them from binding to DNA, providing M. tuberculosis resistance to fluoroquinolones. Proteins that mimic the A-site of the
bacterial ribosome have been found to contribute to aminoglycoside resistance as well.1

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 Exercise 11.7.1

List several mechanisms for drug resistance.

Multidrug-Resistant Microbes and Cross Resistance

From a clinical perspective, our greatest concerns are multidrug-resistant microbes (MDRs) and cross resistance. MDRs are
colloquially known as “superbugs” and carry one or more resistance mechanism(s), making them resistant to multiple
antimicrobials. In cross-resistance, a single resistance mechanism confers resistance to multiple antimicrobial drugs. For example,
having an efflux pump that can export multiple antimicrobial drugs is a common way for microbes to be resistant to multiple drugs
by using a single resistance mechanism. In recent years, several clinically important superbugs have emerged, and the CDC reports
that superbugs are responsible for more than 2 million infections in the US annually, resulting in at least 23,000 fatalities.2 Several
of the superbugs discussed in the following sections have been dubbed the ESKAPE pathogens. This acronym refers to the names
of the pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa and Enterobacter spp.) but it is also fitting in that these pathogens are able to “escape” many conventional forms of
antimicrobial therapy. As such, infections by ESKAPE pathogens can be difficult to treat and they cause a large number of
nosocomial infections.

Methicillin-Resistant Staphylococcus aureus (MRSA)

Methicillin, a semisynthetic penicillin, was designed to resist inactivation by β-lactamases. Unfortunately, soon after the
introduction of methicillin to clinical practice, methicillin-resistant strains of S. aureus appeared and started to spread. The
mechanism of resistance, acquisition of a new low-affinity PBP, provided S. aureus with resistance to all available β-lactams.
Strains of methicillin-resistant S. aureus (MRSA) are widespread opportunistic pathogens and a particular concern for skin and
other wound infections, but may also cause pneumonia and septicemia. Although originally a problem in health-care settings
(hospital-acquired MRSA [HA-MRSA]), MRSA infections are now also acquired through contact with contaminated members of
the general public, called community-associated MRSA (CA-MRSA). Approximately one-third of the population carries S. aureus
as a member of their normal nasal microbiota without illness, and about 6% of these strains are methicillin resistant.3,4

Clavulanic Acid: Penicillin's Little Helper

With the introduction of penicillin in the early 1940s, and its subsequent mass production, society began to think of antibiotics
as miracle cures for a wide range of infectious diseases. Unfortunately, as early as 1945, penicillin resistance was first
documented and started to spread. Greater than 90% of current S. aureus clinical isolates are resistant to penicillin.5
Although developing new antimicrobial drugs is one solution to this problem, scientists have explored new approaches,
including the development of compounds that inactivate resistance mechanisms. The development of clavulanic acid represents
an early example of this strategy. Clavulanic acid is a molecule produced by the bacterium Streptococcus clavuligerus. It
contains a β-lactam ring, making it structurally similar to penicillin and other β-lactams, but shows no clinical effectiveness
when administered on its own. Instead, clavulanic acid binds irreversibly within the active site of β-lactamases and prevents
them from inactivating a coadministered penicillin.
Clavulanic acid was first developed in the 1970s and was mass marketed in combination with amoxicillin beginning in the
1980s under the brand name Augmentin. As is typically the case, resistance to the amoxicillin-clavulanic acid combination
soon appeared. Resistance most commonly results from bacteria increasing production of their β-lactamase and overwhelming
the inhibitory effects of clavulanic acid, mutating their β-lactamase so it is no longer inhibited by clavulanic acid, or from
acquiring a new β-lactamase that is not inhibited by clavulanic acid. Despite increasing resistance concerns, clavulanic acid
and related β-lactamase inhibitors (sulbactam and tazobactam) represent an important new strategy: the development of
compounds that directly inhibit antimicrobial resistance-conferring enzymes.

Vancomycin-Resistant Enterococci and Staphylococcus aureus

Vancomycin is only effective against gram-positive organisms, and it is used to treat wound infections, septic infections,
endocarditis, and meningitis that are caused by pathogens resistant to other antibiotics. It is considered one of the last lines of
defense against such resistant infections, including MRSA. With the rise of antibiotic resistance in the 1970s and 1980s,
vancomycin use increased, and it is not surprising that we saw the emergence and spread of vancomycin-resistant enterococci
(VRE), vancomycin-resistant S. aureus (VRSA), and vancomycin-intermediate S. aureus (VISA). The mechanism of vancomycin

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resistance among enterococci is target modification involving a structural change to the peptide component of the peptidoglycan
subunits, preventing vancomycin from binding. These strains are typically spread among patients in clinical settings by contact
with health-care workers and contaminated surfaces and medical equipment.
VISA and VRSA strains differ from each other in the mechanism of resistance and the degree of resistance each mechanism
confers. VISA strains exhibit intermediate resistance, with a minimum inhibitory concentration (MIC) of 4–8 μg/mL, and the
mechanism involves an increase in vancomycin targets. VISA strains decrease the crosslinking of peptide chains in the cell wall,
providing an increase in vancomycin targets that trap vancomycin in the outer cell wall. In contrast, VRSA strains acquire
vancomycin resistance through horizontal transfer of resistance genes from VRE, an opportunity provided in individuals coinfected
with both VRE and MRSA. VRSA exhibit a higher level of resistance, with MICs of 16 μg/mL or higher.6 In the case of all three
types of vancomycin-resistant bacteria, rapid clinical identification is necessary so proper procedures to limit spread can be
implemented. The oxazolidinones like linezolid are useful for the treatment of these vancomycin-resistant, opportunistic pathogens,
as well as MRSA.

Extended-Spectrum β-Lactamase–Producing Gram-Negative Pathogens

Gram-negative pathogens that produce extended-spectrum β-lactamases (ESBLs) show resistance well beyond just penicillins. The
spectrum of β-lactams inactivated by ESBLs provides for resistance to all penicillins, cephalosporins, monobactams, and the β-
lactamase-inhibitor combinations, but not the carbapenems. An even greater concern is that the genes encoding for ESBLs are
usually found on mobile plasmids that also contain genes for resistance to other drug classes (e.g., fluoroquinolones,
aminoglycosides, tetracyclines), and may be readily spread to other bacteria by horizontal gene transfer. These multidrug-resistant
bacteria are members of the intestinal microbiota of some individuals, but they are also important causes of opportunistic infections
in hospitalized patients, from whom they can be spread to other people.

Carbapenem-Resistant Gram-Negative Bacteria

The occurrence of carbapenem-resistant Enterobacteriaceae (CRE) and carbapenem resistance among other gram-negative bacteria
(e.g., P. aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophila) is a growing health-care concern. These pathogens
develop resistance to carbapenems through a variety of mechanisms, including production of carbapenemases (broad-spectrum β-
lactamases that inactivate all β-lactams, including carbapenems), active efflux of carbapenems out of the cell, and/or prevention of
carbapenem entry through porin channels. Similar to concerns with ESBLs, carbapenem-resistant, gram-negative pathogens are
usually resistant to multiple classes of antibacterials, and some have even developed pan-resistance (resistance to all available
antibacterials). Infections with carbapenem-resistant, gram-negative pathogens commonly occur in health-care settings through
interaction with contaminated individuals or medical devices, or as a result of surgery.

Multidrug-Resistant Mycobacterium tuberculosis

The emergence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant Mycobacterium
tuberculosis (XDR-TB) is also of significant global concern. MDR-TB strains are resistant to both rifampin and isoniazid, the drug
combination typically prescribed for treatment of tuberculosis. XDR-TB strains are additionally resistant to any fluoroquinolone
and at least one of three other drugs (amikacin, kanamycin, or capreomycin) used as a second line of treatment, leaving these
patients very few treatment options. Both types of pathogens are particularly problematic in immunocompromised persons,
including those suffering from HIV infection. The development of resistance in these strains often results from the incorrect use of
antimicrobials for tuberculosistreatment, selecting for resistance.

Exercise 11.7.2

How does drug resistance lead to superbugs?

To learn more about the top 18 drug-resistant threats to the US, visit the CDC’s website.

Factory Farming and Drug Resistance

Although animal husbandry has long been a major part of agriculture in America, the rise of concentrated animal feeding
operations (CAFOs) since the 1950s has brought about some new environmental issues, including the contamination of water
and air with biological waste, and ethical issues regarding animal rights also are associated with growing animals in this way.
Additionally, the increase in CAFOs involves the extensive use of antimicrobial drugs in raising livestock. Antimicrobials are

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 used to prevent the development of infectious disease in the close quarters of CAFOs; however, the majority of antimicrobials
used in factory farming are for the promotion of growth—in other words, to grow larger animals.
The mechanism underlying this enhanced growth remains unclear. These antibiotics may not necessarily be the same as those
used clinically for humans, but they are structurally related to drugs used for humans. As a result, use of antimicrobial drugs in
animals can select for antimicrobial resistance, with these resistant bacteria becoming cross-resistant to drugs typically used in
humans. For example, tylosin use in animals appears to select for bacteria also cross-resistant to other macrolides, including
erythromycin, commonly used in humans.
Concentrations of the drug-resistant bacterial strains generated by CAFOs become increased in water and soil surrounding
these farms. If not directly pathogenic in humans, these resistant bacteria may serve as a reservoir of mobile genetic elements
that can then pass resistance genes to human pathogens. Fortunately, the cooking process typically inactivates any
antimicrobials remaining in meat, so humans typically are not directly ingesting these drugs. Nevertheless, many people are
calling for more judicious use of these drugs, perhaps charging farmers user fees to reduce indiscriminate use. In fact, in 2012,
the FDA published guidelines for farmers who voluntarily phase out the use of antimicrobial drugs except under veterinary
supervision and when necessary to ensure animal health. Although following the guidelines is voluntary at this time, the FDA
does recommend what it calls “judicious” use of antimicrobial drugs in food-producing animals in an effort to decrease
antimicrobial resistance.

Key Concepts and Summary

Antimicrobial resistance is on the rise and is the result of selection of drug-resistant strains in clinical environments, the
overuse and misuse of antibacterials, the use of subtherapeutic doses of antibacterial drugs, and poor patient compliance with
antibacterial drug therapies.
Drug resistance genes are often carried on plasmids or in transposons that can undergo vertical transfer easily and between
microbes through horizontal gene transfer.
Common modes of antimicrobial drug resistance include drug modification or inactivation, prevention of cellular uptake or
efflux, target modification, target overproduction or enzymatic bypass, and target mimicry.
Problematic microbial strains showing extensive antimicrobial resistance are emerging; many of these strains can reside as
members of the normal microbiota in individuals but also can cause opportunistic infection. The transmission of many of these
highly resistant microbial strains often occurs in clinical settings, but can also be community-acquired.

Footnotes
1. D.H. Fong, A.M. Berghuis. “Substrate Promiscuity of an Aminoglycoside Antibiotic Resistance Enzyme Via Target Mimicry.”
EMBO Journal 21 no. 10 (2002):2323–2331.
2. Centers for Disease Control and Prevention. “Antibiotic/Antimicrobial Resistance.”
http://www.cdc.gov/drugresistance/index.html. Accessed June 2, 2016.
3. A.S. Kalokhe et al. “Multidrug-Resistant Tuberculosis Drug Susceptibility and Molecular Diagnostic Testing: A Review of the
Literature. American Journal of the Medical Sciences 345 no. 2 (2013):143–148.
4. Centers for Disease Control and Prevention. “Methicillin-Resistant Staphylococcus aureus (MRSA): General Information
About MRSA in the Community.” http://www.cdc.gov/mrsa/community/index.html. Accessed June 2, 2016
5. F.D. Lowy. “Antimicrobial Resistance: The Example of Staphylococcus aureus.” Journal of Clinical Investigation 111 no. 9
(2003):1265–1273.
6. Centers for Disease Control and Prevention. “Healthcare-Associated Infections (HIA): General Information about
VISA/VRSA.” www.cdc.gov/HAI/organisms/vis...visa_vrsa.html. Accessed June 2, 2016.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.7: Mechanisms for Resistance is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

Access for free at OpenStax 11.7.5 https://bio.libretexts.org/@go/page/31842

11.8: Testing the Effectiveness of Antimicrobial Chemicals and Drugs
Learning Objectives
Describe why the phenol coefficient is used
Describe how the Kirby-Bauer disk diffusion test determines the susceptibility of a microbe to an antibacterial drug.
Explain the significance of the minimal inhibitory concentration and the minimal bactericidal concentration relative to the
effectiveness of an antimicrobial drug.
Compare and contrast the disk-diffusion, use-dilution, and in-use methods for testing the effectiveness of antiseptics,
disinfectants, and sterilants

The effectiveness of various chemical disinfectants is reflected in the terms used to describe them. Chemical disinfectants are
grouped by the power of their activity, with each category reflecting the types of microbes and viruses its component disinfectants
are effective against. High-level germicides have the ability to kill vegetative cells, fungi, viruses, and endospores, leading to
sterilization, with extended use. Intermediate-level germicides, as their name suggests, are less effective against endospores and
certain viruses, and low-level germicides kill only vegetative cells and certain enveloped viruses, and are ineffective against
endospores.
However, several environmental conditions influence the potency of an antimicrobial agent and its effectiveness. For example,
length of exposure is particularly important, with longer exposure increasing efficacy. Similarly, the concentration of the chemical
agent is also important, with higher concentrations being more effective than lower ones. Temperature, pH, and other factors can
also affect the potency of a disinfecting agent.
One method to determine the effectiveness of a chemical agent includes swabbing surfaces before and after use to confirm whether
a sterile field was maintained during use. Additional tests are described in the sections that follow. These tests allow for the
maintenance of appropriate disinfection protocols in clinical settings, controlling microbial growth to protect patients, health-care
workers, and the community.

Phenol Coefficient
The effectiveness of a disinfectant or antiseptic can be determined in a number of ways. Historically, a chemical agent’s
effectiveness was often compared with that of phenol, the first chemical agent used by Joseph Lister. In 1903, British chemists
Samuel Rideal (1863–1929) and J. T. Ainslie Walker (1868–1930) established a protocol to compare the effectiveness of a variety
of chemicals with that of phenol, using as their test organisms Staphylococcus aureus (a gram-positive bacterium) and Salmonella
enterica serovar Typhi (a gram-negative bacterium). They exposed the test bacteria to the antimicrobial chemical solutions diluted
in water for 7.5 minutes. They then calculated a phenol coefficient for each chemical for each of the two bacteria tested. A phenol
coefficient of 1.0 means that the chemical agent has about the same level of effectiveness as phenol. A chemical agent with a
phenol coefficient of less than 1.0 is less effective than phenol. An example is formalin, with phenol coefficients of 0.3 (S. aureus)
and 0.7 (S. enterica serovar Typhi). A chemical agent with a phenol coefficient greater than 1.0 is more effective than phenol, such
as chloramine, with phenol coefficients of 133 and 100, respectively. Although the phenol coefficient was once a useful measure of
effectiveness, it is no longer commonly used because the conditions and organisms used were arbitrarily chosen.

Exercise 11.8.1

What are the differences between the three levels of disinfectant effectiveness?

Disk-Diffusion (Kirby-Bauer) Method

The Kirby-Bauer disk diffusion test has long been used as a starting point for determining the susceptibility of specific microbes to
various antimicrobial drugs or chemicals. The Kirby-Bauer assay starts with a Mueller-Hinton agar plate on which a confluent lawn
(bacteria is spread across the entire surface of the plate) is inoculated with a sample or patient’s isolated bacterial pathogen. Filter
paper disks impregnated with known amounts of antibacterial drugs or chemicals to be tested are then placed on the agar plate. As
the bacterial inoculum grows, drug or chemical diffuses from the circular disk into the agar and interacts with the growing bacteria.
Antimicrobial activity is observed as a clear circular zone of inhibition around the drug/chemical-impregnated disk. The diameter
of the zone of inhibition, measured in millimeters and compared to a standardized chart, determines the susceptibility or resistance
of the bacterial pathogen to the drug or chemical.(Figure 11.8.1)

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There are multiple factors that determine the size of a zone of inhibition in this assay, including drug solubility (whether the agent
is water soluble and able to diffuse in the agar), rate of drug diffusion through agar, the thickness of the agar medium, and the drug
concentration impregnated into the disk. Due to a lack of standardization of these factors, interpretation of the Kirby-Bauer disk
diffusion assay provides only limited information on susceptibility and resistance to the drugs tested. The assay cannot distinguish
between bacteriostatic and bactericidal activities, and differences in zone sizes cannot be used to compare drug potencies or
efficacies. Comparison of zone sizes to a standardized chart will only provide information on the antibacterials to which a bacterial
pathogen is susceptible or resistant. However it is common to correlate larger zones to increased inhibition effectiveness of the
chemical agent.

Figure 11.8.1 : A disk-diffusion assay is used to determine the effectiveness of chemical agents against a particular microbe. (a) A
plate is inoculated with various antimicrobial discs. The zone of inhibition around each disc indicates how effective that
antimicrobial is against the particular species being tested. (b) On these plates, four antimicrobial agents are tested for efficacy in
killing Pseudomonas aeruginosa (left) and Staphylococcus aureus (right). These antimicrobials are much more effective at killing
S. aureus, as indicated by the size of the zones of inhibition. (credit b: modification of work by American Society for
Microbiology)

Exercise 11.8.2

When comparing the activities of two disinfectants against the same microbe, using the disk-diffusion assay, and assuming
both are water soluble and can easily diffuse in the agar, would a more effective disinfectant have a larger zone of inhibition or
a smaller one?
How does one use the information from a Kirby-Bauer assay to predict the therapeutic effectiveness of an antimicrobial drug in
a patient?

Antibiograms: Taking Some of the Guesswork Out of Prescriptions

Unfortunately, infectious diseases don’t take a time-out for lab work. As a result, physicians rarely have the luxury of
conducting susceptibility testing before they write a prescription. Instead, they rely primarily on the empirical evidence (i.e.,
the signs and symptoms of disease) and their professional experience to make an educated guess as to the diagnosis, causative
agent(s), and drug most likely to be effective. This approach allows treatment to begin sooner so the patient does not have to
wait for lab test results. In many cases, the prescription is effective; however, in an age of increased antimicrobial resistance, it
is becoming increasingly more difficult to select the most appropriate empiric therapy. Selecting an inappropriate empiric
therapy not only puts the patient at risk but may promote greater resistance to the drug prescribed.
Recently, studies have shown that antibiograms are useful tools in the decision-making process of selecting appropriate empiric
therapy. An antibiogram is a compilation of local antibiotic susceptibility data broken down by bacterial pathogen. In a
November 2014 study published in the journal Infection Control and Hospital Epidemiology, researchers determined that 85%
of the prescriptions ordered in skilled nursing facilities were decided upon empirically, but only 35% of those prescriptions
were deemed appropriate when compared with the eventual pathogen identification and susceptibility profile obtained from the

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 clinical laboratory. However, in one nursing facility where use of antibiograms was implemented to direct selection of empiric
therapy, appropriateness of empiric therapy increased from 32% before antibiogram implementation to 45% after
implementation of antibiograms.1 Although these data are preliminary, they do suggest that health-care facilities can reduce the
number of inappropriate prescriptions by using antibiograms to select empiric therapy, thus benefiting patients and minimizing
opportunities for antimicrobial resistance to develop.

Dilution Tests
As discussed, the limitations of the Kirby-Bauer disk diffusion test do not allow for a direct comparison of antibacterial potencies
to guide selection of the best therapeutic choice. However, antibacterial dilution tests can be used to determine a particular drug’s
minimal inhibitory concentration (MIC), the lowest concentration of drug that inhibits visible bacterial growth, and minimal
bactericidal concentration (MBC), the lowest drug concentration that kills ≥99.9% of the starting inoculum. Determining these
concentrations helps identify the correct drug for a particular pathogen. For the macrobroth dilution assay, a dilution series of the
drug in broth is made in test tubes and the same number of cells of a test bacterial strain is added to each tube (Figure 11.8.2). The
MIC is determined by examining the tubes to find the lowest drug concentration that inhibits visible growth; this is observed as
turbidity (cloudiness) in the broth. Tubes with no visible growth are then inoculated onto agar media without antibiotic to
determine the MBC. Generally, serum levels of an antibacterial should be at least three to five times above the MIC for treatment of
an infection.

Figure 11.8.2 : In a dilution test, the lowest dilution that inhibits turbidity (cloudiness) is the MIC. In this example, the MIC is 8
μg/mL. Broth from samples without turbidity can be inoculated onto plates lacking the antimicrobial drug. The lowest dilution that
kills ≥99.9% of the starting inoculum is observed on the plates is the MBC. (credit: modification of work by Suzanne Wakim)
The MIC assay can also be performed using 96-well microdilution trays, which allow for the use of small volumes and automated
dispensing devices, as well as the testing of multiple antimicrobials and/or microorganisms in one tray (Figure 11.8.3). MICs are
interpreted as the lowest concentration that inhibits visible growth, the same as for the macrobroth dilution in test tubes. Growth
may also be interpreted visually or by using a spectrophotometer or similar device to detect turbidity or a color change if an
appropriate biochemical substrate that changes color in the presence of bacterial growth is also included in each well.

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 Figure 11.8.3 : A microdilution tray can also be used to determine MICs of multiple antimicrobial drugs in a single assay. In this
example, the drug concentrations increase from left to right and the rows with clindamycin, penicillin, and erythromycin have been
indicated to the left of the plate. For penicillin and erythromycin, the lowest concentrations that inhibited visible growth are
indicated by red circles and were 0.06 μg/mL for penicillin and 8 μg/mL for erythromycin. For clindamycin, visible bacterial
growth was observed at every concentration up to 32 μg/mL and the MIC is interpreted as >32 μg/mL. (credit: modification of
work by Centers for Disease Control and Prevention)
The Etest is an alternative method used to determine MIC, and is a combination of the Kirby-Bauer disk diffusion test and dilution
methods. Similar to the Kirby-Bauer assay, a confluent lawn of a bacterial isolate is inoculated onto the surface of an agar plate.
Rather than using circular disks impregnated with one concentration of drug, however, commercially available plastic strips that
contain a gradient of an antibacterial are placed on the surface of the inoculated agar plate (Figure 11.8.4). As the bacterial
inoculum grows, antibiotic diffuses from the plastic strips into the agar and interacts with the bacterial cells. Because the rate of
drug diffusion is directly related to concentration, an elliptical zone of inhibition is observed with the Etest drug gradient, rather
than a circular zone of inhibition observed with the Kirby-Bauer assay. To interpret the results, the intersection of the elliptical zone
with the gradient on the drug-containing strip indicates the MIC. Because multiple strips containing different antimicrobials can be
placed on the same plate, the MIC of multiple antimicrobials can be determined concurrently and directly compared. However,
unlike the macrobroth and microbroth dilution methods, the MBC cannot be determined with the Etest.

Figure 11.8.4 : The Etest can be used to determine the MIC of an antibiotic. In this Etest, vancomycin is shown to have a MIC of
1.5 μg/mL against Staphylococcus aureus.

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 Exercise 11.8.3

Compare and contrast MIC and MBC.

Use-Dilution Test
Other methods are also used for measuring the effectiveness of a chemical agent in clinical settings. The use-dilution test is
commonly used to determine a chemical’s disinfection effectiveness on an inanimate surface. For this test, a cylinder of stainless
steel is dipped in a culture of the targeted microorganism and then dried. The cylinder is then dipped in solutions of disinfectant at
various concentrations for a specified amount of time. Finally, the cylinder is transferred to a new test tube containing fresh sterile
medium that does not contain disinfectant, and this test tube is incubated. Bacterial survival is demonstrated by the presence of
turbidity in the medium, whereas killing of the target organism on the cylinder by the disinfectant will produce no turbidity.
The Association of Official Agricultural Chemists International (AOAC), a nonprofit group that establishes many protocol
standards, has determined that a minimum of 59 of 60 replicates must show no growth in such a test to achieve a passing result, and
the results must be repeatable from different batches of disinfectant and when performed on different days. Disinfectant
manufacturers perform use-dilution tests to validate the efficacy claims for their products, as designated by the EPA.

Exercise 11.8.4

Is the use-dilution test performed in a clinical setting? Why?

In-Use Test
An in-use test can determine whether an actively used solution of disinfectant in a clinical setting is microbially contaminated
(Figure 11.8.5). A 1-mL sample of the used disinfectant is diluted into 9 mL of sterile broth medium that also contains a compound
to inactivate the disinfectant. Ten drops, totaling approximately 0.2 mL of this mixture, are then inoculated onto each of two agar
plates. One plate is incubated at 37 °C for 3 days and the other is incubated at room temperature for 7 days. The plates are
monitored for growth of microbial colonies. Growth of five or more colonies on either plate suggests that viable microbial cells
existed in the disinfectant solution and that it is contaminated. Such in-use tests monitor the effectiveness of disinfectants in the
clinical setting.

Figure 11.8.5 : Used disinfectant solutions in a clinical setting can be checked with the in-use test for contamination with microbes.

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Exercise 11.8.5

What does a positive in-use test indicate?

Clinical Focus- Resolution

Despite antibiotic treatment, Roberta’s symptoms worsened. She developed pyelonephritis, a severe kidney infection, and was
rehospitalized in the intensive care unit (ICU). Her condition continued to deteriorate, and she developed symptoms of septic
shock. At this point, her physician ordered a culture from her urine to determine the exact cause of her infection, as well as a
drug sensitivity test to determine what antibiotics would be effective against the causative bacterium. The results of this test
indicated resistance to a wide range of antibiotics, including the carbapenems, a class of antibiotics that are used as the last
resort for many types of bacterial infections. This was an alarming outcome, suggesting that Roberta’s infection was caused by
a so-called superbug: a bacterial strain that has developed resistance to the majority of commonly used antibiotics. In this case,
the causative agent belonged to the carbapenem-resistant Enterobacteriaceae (CRE), a drug-resistant family of bacteria
normally found in the digestive system . When CRE is introduced to other body systems, as might occur through improperly
cleaned surgical instruments, catheters, or endoscopes, aggressive infections can occur.
CRE infections are notoriously difficult to treat, with a 40%–50% fatality rate. To treat her kidney infection and septic shock,
Roberta was treated with dialysis, intravenous fluids, and medications to maintain blood pressure and prevent blood clotting.
She was also started on aggressive treatment with intravenous administration of a new drug called tigecycline, which has been
successful in treating infections caused by drug-resistant bacteria.
After several weeks in the ICU, Roberta recovered from her CRE infection. However, public health officials soon noticed that
Roberta’s case was not isolated. Several patients who underwent similar procedures at the same hospital also developed CRE
infections, some dying as a result. Ultimately, the source of the infection was traced to the duodenoscopes used in the
procedures. Despite the hospital staff meticulously following manufacturer protocols for disinfection, bacteria, including CRE,
remained within the instruments and were introduced to patients during procedures.

Figure 11.8.6 : CRE is an extremely drug-resistant strain of bacteria that is typically associated with nosocomial infections.
(credit: Centers for Disease Control and Prevention)

Who is Responsible?
Carbapenem-resistant Enterobacteriaceae infections due to contaminated endoscopes have become a high-profile problem in
recent years. Several CRE outbreaks have been traced to endoscopes, including a case at Ronald Reagan UCLA Medical
Center in early 2015 in which 179 patients may have been exposed to a contaminated endoscope. Seven of the patients
developed infections, and two later died. Several lawsuits have been filed against Olympus, the manufacturer of the
endoscopes. Some claim that Olympus did not obtain FDA approval for design changes that may have led to contamination,
and others claim that the manufacturer knowingly withheld information from hospitals concerning defects in the endoscopes.
Lawsuits like these raise difficult-to-answer questions about liability. Invasive procedures are inherently risky, but negative
outcomes can be minimized by strict adherence to established protocols. Who is responsible, however, when negative
outcomes occur due to flawed protocols or faulty equipment? Can hospitals or health-care workers be held liable if they have
strictly followed a flawed procedure? Should manufacturers be held liable—and perhaps be driven out of business—if their

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lifesaving equipment fails or is found defective? What is the government’s role in ensuring that use and maintenance of
medical equipment and protocols are fail-safe?
Protocols for cleaning or sterilizing medical equipment are often developed by government agencies like the FDA, and other
groups, like the AOAC, a nonprofit scientific organization that establishes many protocols for standard use globally. These
procedures and protocols are then adopted by medical device and equipment manufacturers. Ultimately, the end-users
(hospitals and their staff) are responsible for following these procedures and can be held liable if a breach occurs and patients
become ill from improperly cleaned equipment.
Unfortunately, protocols are not infallible, and sometimes it takes negative outcomes to reveal their flaws. In 2008, the FDA
had approved a disinfection protocol for endoscopes, using glutaraldehyde (at a lower concentration when mixed with phenol),
o-phthalaldehyde, hydrogen peroxide, peracetic acid, and a mix of hydrogen peroxide with peracetic acid. However,
subsequent CRE outbreaks from endoscope use showed that this protocol alone was inadequate.
As a result of CRE outbreaks, hospitals, manufacturers, and the FDA are investigating solutions. Many hospitals are instituting
more rigorous cleaning procedures than those mandated by the FDA. Manufacturers are looking for ways to redesign
duodenoscopes to minimize hard-to-reach crevices where bacteria can escape disinfectants, and the FDA is updating its
protocols. In February 2015, the FDA added new recommendations for careful hand cleaning of the duodenoscope elevator
mechanism (the location where microbes are most likely to escape disinfection), and issued more careful documentation about
quality control of disinfection protocols (Figure 11.8.7).
There is no guarantee that new procedures, protocols, or equipment will completely eliminate the risk for infection associated
with endoscopes. Yet these devices are used successfully in 500,000–650,000 procedures annually in the United States, many
of them lifesaving. At what point do the risks outweigh the benefits of these devices, and who should be held responsible when
negative outcomes occur?

Figure 11.8.7 : The elevator mechanism in a duodenoscope contains crevices that are difficult to disinfect. Pathogens that
survive disinfection protocols can be passed from one patient to another, causing serious infections. (credit “photos”:
modification of work by Centers for Disease Control and Prevention)

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Key Concepts and Summary
Chemical disinfectants are grouped by the types of microbes and infectious agents they are effective against. High-level
germicides kill vegetative cells, fungi, viruses, and endospores, and can ultimately lead to sterilization. Intermediate-level
germicides cannot kill all viruses and are less effective against endospores. Low-level germicides kill vegetative cells and
some enveloped viruses, but are ineffective against endospores.
The effectiveness of a disinfectant is influenced by several factors, including length of exposure, concentration of disinfectant,
temperature, and pH.
Historically, the effectiveness of a chemical disinfectant was compared with that of phenol at killing Staphylococcus aureus and
Salmonella enterica serovar Typhi, and a phenol coefficient was calculated.
The disk-diffusion method is used to test the effectiveness of a chemical disinfectant against a particular microbe.
The Kirby-Bauer disk diffusion test helps determine the susceptibility of a microorganism to various antimicrobial drugs.
However, the zones of inhibition measured must be correlated to known standards to determine susceptibility and resistance,
and do not provide information on bactericidal versus bacteriostatic activity, or allow for direct comparison of drug potencies.
Antibiograms are useful for monitoring local trends in antimicrobial resistance/susceptibility and for directing appropriate
selection of empiric antibacterial therapy.
There are several laboratory methods available for determining the minimum inhibitory concentration (MIC) of an
antimicrobial drug against a specific microbe. The minimal bactericidal concentration (MBC) can also be determined,
typically as a follow-up experiment to MIC determination using the tube dilution method.
The use-dilution test determines the effectiveness of a disinfectant on a surface. In-use tests can determine whether
disinfectant solutions are being used correctly in clinical settings.

Footnotes
1. J.P. Furuno et al. “Using Antibiograms to Improve Antibiotic Prescribing in Skilled Nursing Facilities.” Infection Control and
Hospital Epidemiology 35 no. Suppl S3 (2014):S56–61.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 11.8: Testing the Effectiveness of Antimicrobial Chemicals and Drugs is shared under a CC BY license and was authored,
remixed, and/or curated by OpenStax.

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Chapter 11 Exercises
Review Questions for Chapter 10
Multiple Choice
1) Which of the following types of medical items requires sterilization?
A. needles
B. bed linens
C. respiratory masks
D. blood pressure cuffs

2) Which of the following is suitable for use on tissues for microbial control to prevent infection?
A. disinfectant
B. antiseptic
C. sterilant
D. water

3) Which biosafety level is appropriate for research with microbes or infectious agents that pose moderate risk to laboratory
workers and the community, and are typically indigenous?
A. BSL-1
B. BSL-2
C. BSL-3
D. BSL-4

4) Which of the following best describes a microbial control protocol that inhibits the growth of molds and yeast?
A. bacteriostatic
B. fungicidal
C. bactericidal
D. fungistatic

5) The decimal reduction time refers to the amount of time it takes to which of the following?
A. reduce a microbial population by 10%
B. reduce a microbial population by 0.1%
C. reduce a microbial population by 90%
D. completely eliminate a microbial population

6) Which of the following methods brings about cell lysis due to cavitation induced by rapid localized pressure changes?
A. microwaving
B. gamma irradiation
C. ultraviolet radiation
D. sonication

7) Which of the following terms is used to describe the time required to kill all of the microbes within a sample at a given
temperature?
A. D-value

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B. thermal death point
C. thermal death time
D. decimal reduction time

8) Which of the following microbial control methods does not actually kill microbes or inhibit their growth but instead removes
them physically from samples?
A. filtration
B. desiccation
C. lyophilization
D. nonionizing radiation

9) Which of the following refers to a disinfecting chemical dissolved in alcohol?

A. iodophor
B. tincture
C. phenolic
D. peroxygen

10) Which of the following peroxygens is widely used as a household disinfectant, is inexpensive, and breaks down into water and
oxygen gas?
A. hydrogen peroxide
B. peracetic acid
C. benzoyl peroxide
D. ozone

11) Which of the following chemical food preservatives is used in the wine industry but may cause asthmatic reactions in some
individuals?
A. nitrites
B. sulfites
C. propionic acid
D. benzoic acid

12) Bleach is an example of which group of chemicals used for disinfection?

A. heavy metals
B. halogens
C. quats
D. bisbiguanides

13) Which chemical disinfectant works by methylating enzymes and nucleic acids and is known for being toxic and carcinogenic?
A. sorbic acid
B. triclosan
C. formaldehyde
D. hexaclorophene

14) Which type of test is used to determine whether disinfectant solutions actively used in a clinical setting are being used
correctly?

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A. disk-diffusion assay
B. phenol coefficient test
C. in-use test
D. use-dilution test

15) The effectiveness of chemical disinfectants has historically been compared to that of which of the following?
A. phenol
B. ethyl alcohol
C. bleach
D. formaldehyde

16) Which of the following refers to a germicide that can kill vegetative cells and certain enveloped viruses but not endospores?
A. high-level germicide
B. intermediate-level germicide
C. low-level germicide
D. sterilant

17) A scientist discovers that a soil bacterium he has been studying produces an antimicrobial that kills gram-negative bacteria. She
isolates and purifies the antimicrobial compound, then chemically converts a chemical side chain to a hydroxyl group. When she
tests the antimicrobial properties of this new version, she finds that this antimicrobial drug can now also kill gram-positive bacteria.
The new antimicrobial drug with broad-spectrum activity is considered to be which of the following?
A. resistant
B. semisynthetic
C. synthetic
D. natural

18) Which of the following antimicrobial drugs is synthetic?

A. sulfanilamide
B. penicillin
C. actinomycin
D. neomycin

19) Which of the following combinations would most likely contribute to the development of a superinfection?
A. long-term use of narrow-spectrum antimicrobials
B. long-term use of broad-spectrum antimicrobials
C. short-term use of narrow-spectrum antimicrobials
D. short-term use of broad-spectrum antimicrobials

20) Which of the following routes of administration would be appropriate and convenient for home administration of an
antimicrobial to treat a systemic infection?
A. oral
B. intravenous
C. topical
D. parenteral

21) Which clinical situation would be appropriate for treatment with a narrow-spectrum antimicrobial drug?

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A. treatment of a polymicrobic mixed infection in the intestine
B. prophylaxis against infection after a surgical procedure
C. treatment of strep throat caused by culture identified Streptococcus pyogenes
D. empiric therapy of pneumonia while waiting for culture results

22) Which of the following terms refers to the ability of an antimicrobial drug to harm the target microbe without harming the host?
A. mode of action
B. therapeutic level
C. spectrum of activity
D. selective toxicity

23) Which of the following is not a type of β-lactam antimicrobial?

A. penicillins
B. glycopeptides
C. cephalosporins
D. monobactams

24) Which of the following does not bind to the 50S ribosomal subunit?
A. tetracyclines
B. lincosamides
C. macrolides
D. chloramphenicol

25) Which of the following antimicrobials inhibits the activity of DNA gyrase?
A. polymyxin B
B. clindamycin
C. nalidixic acid
D. rifampin

26) Which of the following is not an appropriate target for antifungal drugs?
A. ergosterol
B. chitin
C. cholesterol
D. β(1→3) glucan

27) Which of the following drug classes specifically inhibits neuronal transmission in helminths?
A. quinolines
B. avermectins
C. amantadines
D. imidazoles

28) Which of the following is a nucleoside analog commonly used as a reverse transcriptase inhibitor in the treatment of HIV?
A. acyclovir
B. ribavirin
C. adenine-arabinoside

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D. azidothymidine

29) Which of the following is an antimalarial drug that is thought to increase ROS levels in target cells?
A. artemisinin
B. amphotericin b
C. praziquantel
D. pleconaril

30) Which of the following resistance mechanisms describes the function of β-lactamase?
A. efflux pump
B. target mimicry
C. drug inactivation
D. target overproduction

31) Which of the following resistance mechanisms is commonly effective against a wide range of antimicrobials in multiple
classes?
A. efflux pump
B. target mimicry
C. target modification
D. target overproduction

32) Which of the following resistance mechanisms is the most nonspecific to a particular class of antimicrobials?
A. drug modification
B. target mimicry
C. target modification
D. efflux pump

33) Which of the following types of drug-resistant bacteria do not typically persist in individuals as a member of their intestinal
microbiota?
A. MRSA
B. VRE
C. CRE
D. ESBL-producing bacteria

34) In the Kirby-Bauer disk diffusion test, the _______ of the zone of inhibition is measured and used for interpretation.
A. diameter
B. microbial population
C. circumference
D. depth

35) Which of the following techniques cannot be used to determine the minimum inhibitory concentration of an antimicrobial drug
against a particular microbe?
A. Etest
B. microbroth dilution test
C. Kirby-Bauer disk diffusion test

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D. macrobroth dilution test

36) The utility of an antibiogram is that it shows antimicrobial susceptibility trends

A. over a large geographic area.
B. for an individual patient.
C. in research laboratory strains.
D. in a localized population.

37) Which of the following has yielded compounds with the most antimicrobial activity?
A. water
B. air
C. volcanoes
D. soil

Fill-in-the-Blanks

38) A medical item that comes into contact with intact skin and does not penetrate sterile tissues or come into contact with mucous
membranes is called a(n) ________ item.

39) The goal of ________ ________ protocols is to rid canned produce of Clostridium botulinum endospores.

40) In an autoclave, the application of pressure to ________ is increased to allow the steam to achieve temperatures above the
boiling point of water.

41) Doorknobs and other surfaces in clinical settings are often coated with ________, ________, or ________ to prevent the
transmission of microbes.

42) If a chemical disinfectant is more effective than phenol, then its phenol coefficient would be ________ than 1.0.

43) If used for extended periods of time, ________ germicides may lead to sterility.

44) In the disk-diffusion assay, a large zone of inhibition around a disk to which a chemical disinfectant has been applied indicates
________ of the test microbe to the chemical disinfectant.

45) The group of soil bacteria known for their ability to produce a wide variety of antimicrobials is called the ________.

46) The bacterium known for causing pseudomembranous colitis, a potentially deadly superinfection, is ________.

47) Selective toxicity antimicrobials are easier to develop against bacteria because they are ________ cells, whereas human cells
are eukaryotic.

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48) Antiviral drugs, like Tamiflu and Relenza, that are effective against the influenza virus by preventing viral escape from host
cells are called ________.

49) Staphylococcus aureus, including MRSA strains, may commonly be carried as a normal member of the ________ microbiota in
some people.

50) The method that can determine the MICs of multiple antimicrobial drugs against a microbial strain using a single agar plate is
called the ________.

Short Answers

51) What are some characteristics of microbes and infectious agents that would require handling in a BSL-3 laboratory?

52) What is the purpose of degerming? Does it completely eliminate microbes?

53) What are some factors that alter the effectiveness of a disinfectant?

54) What is the advantage of HTST pasteurization compared with sterilization? What is an advantage of UHT treatment?

55) How does the addition of salt or sugar help preserve food?

56) Which is more effective at killing microbes: autoclaving or freezing? Explain.

57) Which solution of ethyl alcohol is more effective at inhibiting microbial growth: a 70% solution or a 100% solution? Why?

58) When might a gas treatment be used to control microbial growth instead of autoclaving? What are some examples?

59) What is the advantage of using an iodophor rather than iodine or an iodine tincture?

60) Why were chemical disinfectants once commonly compared with phenol?

61) Why is length of exposure to a chemical disinfectant important for its activity?

62) Where do antimicrobials come from naturally? Why?

63) Why was Salvarsan considered to be a “magic bullet” for the treatment of syphilis?

64) When prescribing antibiotics, what aspects of the patient’s health history should the clinician ask about and why?

65) When is using a broad-spectrum antimicrobial drug warranted?

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66) If human cells and bacterial cells perform transcription, how are the rifamycins specific for bacterial infections?

67) What bacterial structural target would make an antibacterial drug selective for gram-negative bacteria? Provide one example of
an antimicrobial compound that targets this structure.

68) How does the biology of HIV necessitate the need to treat HIV infections with multiple drugs?

69) Niclosamide is insoluble and thus is not readily absorbed from the stomach into the bloodstream. How does the insolubility of
niclosamide aid its effectiveness as a treatment for tapeworm infection?

70) Why does the length of time of antimicrobial treatment for tuberculosis contribute to the rise of resistant strains?

71) What is the difference between multidrug resistance and cross-resistance?

72) How is the information from a Kirby-Bauer disk diffusion test used for the recommendation of the clinical use of an
antimicrobial drug?

73) What is the difference between MIC and MBC?

Critical Thinking

74) When plotting microbial death curves, how might they look different for bactericidal versus bacteriostatic treatments?

75) What are the benefits of cleaning something to a level of cleanliness beyond what is required? What are some possible
disadvantages of doing so?

76) In 2001, endospores of Bacillus anthracis, the causative agent of anthrax, were sent to government officials and news agencies
via the mail. In response, the US Postal Service began to irradiate mail with UV light. Was this an effective strategy? Why or why
not?

77) Do you think naturally produced antimicrobial products like nisin and natamycin should replace sorbic acid for food
preservation? Why or why not?

78) Why is the use of skin disinfecting compounds required for surgical scrubbing and not for everyday handwashing?

79) What are some advantages of use-dilution and in-use tests compared with the disk-diffusion assay?

80) In nature, why do antimicrobial-producing microbes commonly also have antimicrobial resistance genes?

81) Why are yeast infections a common type of superinfection that results from long-term use of broad-spectrum antimicrobials?

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82) Too often patients will stop taking antimicrobial drugs before the prescription is finished. What are factors that cause a patient
to stop too soon, and what negative impacts could this have?

83) In considering the cell structure of prokaryotes compared with that of eukaryotes, propose one possible reason for side effects
in humans due to treatment of bacterial infections with protein synthesis inhibitors.

84) Which of the following molecules is an example of a nucleoside analog?

85) Why can’t drugs used to treat influenza, like amantadines and neuraminidase inhibitors, be used to treat a wider variety of viral
infections?

86) Can an Etest be used to find the MBC of a drug? Explain.

87) Who should be responsible for discovering and developing new antibiotics? Support your answer with reasoning.

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 CHAPTER OVERVIEW

12: Microbial Interactions Flora, Pathogenicity and Epidemiology

12.1: Normal Microbiota of the Body
12.2: Characteristics and Steps of Infectious Diseases
12.3: Virulence Factors in Infection
12.4: How Diseases Spread
12.5: The Language of Epidemiologists
12.6: Tracking Infectious Diseases
Chapter 12 Exercises

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12.1: Normal Microbiota of the Body
Learning Objectives
Compare and contrast the microbiomes of various body sites
Identify features of the body sites that might affect with microbes reside in each space

PART 1
Michael, a 10-year-old boy in generally good health, went to a birthday party on Sunday with his family. He ate many different
foods but was the only one in the family to eat the undercooked hot dogs served by the hosts. Monday morning, he woke up
feeling achy and nauseous, and he was running a fever of 38 °C (100.4 °F). His parents, assuming Michael had caught the flu,
made him stay home from school and limited his activities. But after 4 days, Michael began to experience severe headaches,
and his fever spiked to 40 °C (104 °F). Growing worried, his parents finally decide to take Michael to a nearby clinic.

Exercise 12.1.1
1. What signs and symptoms is Michael experiencing?
2. What do these signs and symptoms tell us about the stage of Michael’s disease?

Normal Flora
Microbes are ubiquitous is a phrase that has been repeated often, but many people do not realize how close to home it is. Microbes
not only live all around us, but also on our bodies- skin, nose, and gut. Many are living with us side-by-side in a mutualistic
relationship that benefits everyone. In this section we explore those that live in harmony with us, at least temporarily.

Normal Microbiota of the Skin

The skin is home to a wide variety of normal microbiota, consisting of commensal organisms that derive nutrition from skin cells
and secretions such as sweat and sebum. The normal microbiota of skin tends to inhibit transient-microbe colonization by
producing antimicrobial substances and outcompeting other microbes that land on the surface of the skin. This helps to protect the
skin from pathogenic infection.
The skin’s properties differ from one region of the body to another, as does the composition of the skin’s microbiota. The
availability of nutrients and moisture partly dictates which microorganisms will thrive in a particular region of the skin. Relatively
moist skin, such as that of the nares (nostrils) and underarms, has a much different microbiota than the dryer skin on the arms, legs,
hands, and top of the feet. Some areas of the skin have higher densities of sebaceous glands. These sebum-rich areas, which include
the back, the folds at the side of the nose, and the back of the neck, harbor distinct microbial communities that are less diverse than
those found on other parts of the body.
Different types of bacteria dominate the dry, moist, and sebum-rich regions of the skin. The most abundant microbes typically
found in the dry and sebaceous regions are Betaproteobacteria and Propionibacteria, respectively. In the moist regions,
Corynebacterium and Staphylococcus are most commonly found (Figure 12.1.1). Viruses and fungi are also found on the skin, with
Malassezia being the most common type of fungus found as part of the normal microbiota. The role and populations of viruses in
the microbiota, known as viromes, are still not well understood, and there are limitations to the techniques used to identify them.
However, Circoviridae, Papillomaviridae, and Polyomaviridaeappear to be the most common residents in the healthy skin
virome.123

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 Figure 12.1.1 : The normal microbiota varies on different regions of the skin, especially in dry versus moist areas. The figure shows
the major organisms commonly found in different locations of a healthy individual’s skin and external mucosa. Note that there is
significant variation among individuals. (credit: modification of work by National Human Genome Research Institute)

Exercise 12.1.2

What are the four most common bacteria that are part of the normal skin microbiota?

Microbiota of the Eye

The surfaces of the eyeball and inner eyelid are mucous membranes called conjunctiva. The normal conjunctival microbiota has not
been well characterized, but does exist. One small study (part of the Ocular Microbiome project) found twelve genera that were
consistently present in the conjunctiva.4 These microbes are thought to help defend the membranes against pathogens. However, it
is still unclear which microbes may be transient and which may form a stable microbiota.5
Use of contact lenses can cause changes in the normal microbiota of the conjunctiva by introducing another surface into the natural
anatomy of the eye. Research is currently underway to better understand how contact lenses may impact the normal microbiota and
contribute to eye disease. The watery material inside of the eyeball is called the vitreous humor. Unlike the conjunctiva, it is
protected from contact with the environment and is almost always sterile, with no normal microbiota (Figure 12.1.2).

Figure 12.1.2 : Some microbes live on the conjunctiva of the human eye, but the vitreous humor is sterile.

Mouth and GI tract

Microbes such as bacteria and archaea are abundant in the mouth and coat all of the surfaces of the oral cavity. The most well
known genus is Streptococcus, but there are many others that have not been well characterized. Even within the mouth, different

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structures, such as the teeth or cheeks, host unique communities of both aerobic and anaerobic microbes. The number of microbes
goes down and is essentially zero by the stomach. But after the stomach, microorganisms present in the small intestine can include
lactobacilli, diptherioids and the fungus Candida. On the other hand, the large intestine (colon) contains a diverse and abundant
microbiota that is important for normal function. These microbes include Bacteriodetes (especially the genera Bacteroides and
Prevotella) and Firmicutes (especially members of the genus Clostridium). Methanogenic archaea and some fungi are also present,
among many other species of bacteria. These microbes all aid in digestion and contribute to the production of feces, the waste
excreted from the digestive tract, and flatus, the gas produced from microbial fermentation of undigested food. They can also
produce valuable nutrients. For example, lactic acid bacteria such as bifidobacteria can synthesize vitamins, such as vitamin B12,
folate, and riboflavin, that humans cannot synthesize themselves. E. coli found in the intestine can also break down food and help
the body produce vitamin K, which is important for blood coagulation.
Diagram of the digestive system. The system begins with the mouth and tongue. There are salivary glands in this region: the sublingual gland is under the tongue, the submandibular gland is below the jaw and the
parotid gland is in the very back of the mouth. The mouth leads to the pharynx (a tube) that leads to the esophagus, that leads to the stomach, that leads to the small intestines. The small intestine is divided into 3
regions: first is the duodenum, next is the jejunim and finally the ileum. This leads to the large intestines which is divided into regions: first the cecum, then the ascending colon, then the transverse colon, then the
descending colon, then the sigmoid colon, and finally the rectum, anal canal and anus. The appendix is a small projection off the cecum. Also part of the digestive system is the large liver (above and to the right of the
stomach), the gallbladder (a small sac under the liver), the pancrease (a structure below and behind the stomach) and the spleen (a structure below and to the left of the stomach).

Figure 12.1.3: Microbes abound in the mouth down to the end of the esophagus. The stomach is generally not colonized, but the
intestines have many microbes.

Normal Microbiota of the Respiratory System

The upper respiratory tract contains an abundant and diverse microbiota. The nasal passages and sinuses are primarily colonized by
members of the Firmicutes, Actinobacteria, and Proteobacteria. The most common bacteria identified include Staphylococcus
epidermidis, viridans group streptococci (VGS), Corynebacterium spp. (diphtheroids), Propionibacterium spp., and Haemophilus
spp. The oropharynx includes many of the same isolates as the nose and sinuses, with the addition of variable numbers of bacteria
like species of Prevotella, Fusobacterium, Moraxella, and Eikenella, as well as some Candida fungal isolates. In addition, many
healthy humans asymptomatically carry potential pathogens in the upper respiratory tract. As much as 20% of the population carry
Staphylococcus aureus in their nostrils.6 The pharynx, too, can be colonized with pathogenic strains of Streptococcus,
Haemophilus, and Neisseria.
The lower respiratory tract, by contrast, is scantily populated with microbes. Of the organisms identified in the lower respiratory
tract, species of Pseudomonas, Streptococcus, Prevotella, Fusobacterium, and Veillonella are the most common. It is not clear at
this time if these small populations of bacteria constitute a normal microbiota or if they are transients.
A drawA drawing of the lower respiratory system. The epiglottis is a flap that can allow material into the larynx. The larynx is a tube that leads to the trachea. The trachea branches to become the primary bronchi.
These branch to become the secondary bronchi, these branch to become the tertiary bronchi. These branch to become the bronchioles. Terminal bronchioles end in clusters of balloon shapes called alveolar sacs. Each
balloon shape is an alveolus. Thin, webbed capillaries cover the outside of the alveolus and are connected to pulmonary veins and pulmonary arteries. Oxygen from the alveolus travels into the capillary and carbon
dioxide from the capillary travels into the alveolus. ing of the lower respiratory system. The epiglottis is a flap that can allow material into the larynx. The larynx is a tube that leads to the trachea. The trachea branches to
become the primary bronchi. These branch to become the secondary bronchi, these branch to become the tertiary bronchi. These branch to become the bronchioles. Terminal bronchioles end in clusters of balloon shapes
called alveolar sacs. Each balloon shape is an alveolus. Thin, webbed capillaries cover the outside of the alveolus and are connected to pulmonary veins and pulmonary arteries. Oxygen from the alveolus travels into the
capillary and carbon dioxide from the capillary travels into the alveolus.

Figure 12.1.4: Microbes generally have a lot of mixing with the GI tract until the trachea. Microbes then decrease until the lungs,
which should be sterile.

Normal Microbiota of the Urogenital System

Below the bladder, the normal microbiota of the male urogenital system is found primarily within the distal urethra and includes
bacterial species that are commonly associated with the skin microbiota- think Staphylococcus. In women, the normal microbiota is
found within the distal one third of the urethra and the vagina. The normal microbiota of the vagina becomes established shortly
after birth and is a complex and dynamic population of bacteria that fluctuates in response to environmental changes. Members of
the vaginal microbiota play an important role in the nonspecific defense against vaginal infections and sexually transmitted
infections by occupying cellular binding sites and competing for nutrients. In addition, the production of lactic acid by members of
the microbiota provides an acidic environment within the vagina that also serves as a defense against infections. For the majority of
women, the lactic-acid–producing bacteria in the vagina are dominated by a variety of species of Lactobacillus. For women who
lack sufficient lactobacilli in their vagina, lactic acid production comes primarily from other species of bacteria such as
Leptotrichia spp., Megasphaera spp., and Atopobium vaginae. Lactobacillus spp. use glycogen from vaginal epithelial cells for
metabolism and production of lactic acid. This process is tightly regulated by the hormone estrogen. Increased levels of estrogen
correlate with increased levels of vaginal glycogen, increased production of lactic acid, and a lower vaginal pH. Therefore,
decreases in estrogen during the menstrual cycle and with menopause are associated with decreased levels of vaginal glycogen and

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lactic acid, and a higher pH. In addition to producing lactic acid, Lactobacillus spp. also contribute to the defenses against
infectious disease through their production of hydrogen peroxide and bacteriocins (antibacterial peptides).
A longitudinal section of the female reproductive and urinary systems. A tube labeled ureter leads to the urinary bladder which leads to the urethra. The urinary bladder sits just internal to the pubic bone. Just above
and behind the urinary bladder is the uterus (an oval shaped structure with a thick wall). Above the uterus is a tube labeled fallopian tube which connects to a small oval ovary. The opening in the uterus is the cervix
which leads to the vagina. Behhind this is the rectum which leads to the anus. The external flaps of skin are labeled labium minora and majora. A longitudinal section of the male reproductive and urinary system. A tube
at the top is labeled ureter which connects to the urinary bladder, which sits just behind the pubic bone. The bladder leads to a long tube labeled urethra which is in the center of the penis. The vas deferens also feeds into
the urethra. The testis has a structure on the top called epididymis which becomes the vas deferens. The seminal vesicles connect to the vas deferens just before it goes through the prostate gland (a structure just below
the urinary bladder). The vas deferens connects to the urethra after it passes through the prostate gland. The rectum and anus are behind all of these structures.
Figure 12.1.5 : Microbes in the urogenital tract include ones found on the skin in moist areas. The mucus membranes have microbes
generally associated with the GI tract, except during certain times of life.

Key Concepts and Summary

Normal flora lives in a mutualistic relationship with humans. Humans benefit from microbes being there and microbes benefit
too.
What genera of microbes are represented in each area are highly diverse and dependent on defenses and resources are available
in each area.

Footnotes
1. 1 Belkaid, Y., and J.A. Segre. “Dialogue Between Skin Microbiota and Immunity,” Science 346 (2014) 6212:954–959.
2. 2 Foulongne, Vincent, et al. “Human Skin Microbiota: High Diversity of DNA Viruses Identified on the Human Skin by High
Throughput Sequencing.” PLoS ONE (2012) 7(6): e38499. doi: 10.1371/journal.pone.0038499.
3. 3 Robinson, C.M., and J.K. Pfeiffer. “Viruses and the Microbiota.” Annual Review of Virology (2014) 1:55–59. doi:
10.1146/annurev-virology-031413-085550.
4. 4 Abelson, M.B., Lane, K., and Slocum, C.. “The Secrets of Ocular Microbiomes.” Review of Ophthalmology June 8, 2015.
www.reviewofophthalmology.com...isease/c/55178. Accessed Sept 14, 2016.
5. 5 Shaikh-Lesko, R. “Visualizing the Ocular Microbiome.” The Scientist May 12, 2014. http://www.the-scientist.com/?
articl...lar-Microbiome. Accessed Sept 14, 2016.
6. 6 J. Kluytmans et al. “Nasal Carriage of Staphylococcus aureus: Epidemiology, Underlying Mechanisms, and Associated
Risks.” Clinical Microbiology Reviews 10 no. 3 (1997):505–520.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

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12.2: Characteristics and Steps of Infectious Diseases
Learning Objectives
Distinguish between signs and symptoms of disease
Explain the difference between a communicable disease and a noncommunicable disease
Identify and describe the stages of an acute infectious disease in terms of number of pathogens present and severity of signs
and symptoms
Explain the concept of pathogenicity (virulence) in terms of infectious and lethal dose
Distinguish between primary and opportunistic pathogens and identify specific examples of each
Summarize the stages of pathogenesis
Explain the roles of portals of entry and exit in the transmission of disease and identify specific examples of these portals

A disease is any condition in which the normal structure or functions of the body are damaged or impaired. Physical injuries or
disabilities are not classified as disease, but there can be several causes for disease, including infection by a pathogen, genetics (as
in many cancers or deficiencies), noninfectious environmental causes, or inappropriate immune responses. Our focus in this chapter
will be on infectious diseases, although when diagnosing infectious diseases, it is always important to consider possible
noninfectious causes.

Signs and Symptoms of Disease

An infection is the successful colonization of a host by a microorganism. Microorganisms that can cause disease are known as
pathogens. Infections can lead to disease, which causes signs and symptoms resulting in a deviation from the normal structure or
functioning of the host. The signs of disease are objective and measurable, and can be directly observed by a clinician. Vital signs,
which are used to measure the body’s basic functions, include body temperature (normally 37 °C [98.6 °F]), heart rate (normally
60–100 beats per minute), breathing rate (normally 12–18 breaths per minute), and blood pressure (normally between 90/60 and
120/80 mm Hg). Changes in any of the body’s vital signs may be indicative of disease. For example, having a fever (a body
temperature significantly higher than 37 °C or 98.6 °F) is a sign of disease because it can be measured.
In addition to changes in vital signs, other observable conditions may be considered signs of disease. For example, the presence of
antibodies in a patient’s serum (the liquid portion of blood that lacks clotting factors) can be observed and measured through blood
tests and, therefore, can be considered a sign. However, it is important to note that the presence of antibodies is not always a sign of
an active disease. Antibodies can remain in the body long after an infection has resolved; also, they may develop in response to a
pathogen that is in the body but not currently causing disease.
Unlike signs, symptoms of disease are subjective. Symptoms are felt or experienced by the patient, but they cannot be clinically
confirmed or objectively measured. Examples of symptoms include nausea, loss of appetite, and pain. Such symptoms are
important to consider when diagnosing disease, but they are subject to memory bias and are difficult to measure precisely. Some
clinicians attempt to quantify symptoms by asking patients to assign a numerical value to their symptoms. For example, the Wong-
Baker Faces pain-rating scale asks patients to rate their pain on a scale of 0–10. An alternative method of quantifying pain is
measuring skin conductance fluctuations. These fluctuations reflect sweating due to skin sympathetic nerve activity resulting from
the stressor of pain.1
A specific group of signs and symptoms characteristic of a particular disease is called a syndrome. Many syndromes are named
using a nomenclature based on signs and symptoms or the location of the disease. Table 12.2.1 lists some of the prefixes and
suffixes commonly used in naming syndromes.
Table 12.2.1 : Nomenclature of Symptoms
Affix Meaning Example

cytopenia: reduction in the number of blood

cyto- cell
cells

hepat- of the liver hepatitis: inflammation of the liver

-pathy disease neuropathy: a disease affecting nerves

-emia of the blood bacteremia: presence of bacteria in blood

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 Affix Meaning Example

-itis inflammation colitis: inflammation of the colon

-lysis destruction hemolysis: destruction of red blood cells

-oma tumor lymphoma: cancer of the lymphatic system

leukocytosis: abnormally high number of white

-osis diseased or abnormal condition
blood cells

-derma of the skin keratoderma: a thickening of the skin

Clinicians must rely on signs and on asking questions about symptoms, medical history, and the patient’s recent activities to
identify a particular disease and the potential causative agent. Diagnosis is complicated by the fact that different microorganisms
can cause similar signs and symptoms in a patient. For example, an individual presenting with symptoms of diarrhea may have
been infected by one of a wide variety of pathogenic microorganisms. Bacterial pathogens associated with diarrheal disease include
Vibrio cholerae, Listeria monocytogenes, Campylobacter jejuni, and enteropathogenic Escherichia coli (EPEC). Viral pathogens
associated with diarrheal disease include norovirus and rotavirus. Parasitic pathogens associated with diarrhea include Giardia
lamblia and Cryptosporidium parvum. Likewise, fever is indicative of many types of infection, from the common cold to the
deadly Ebola hemorrhagic fever.
Finally, some diseases may be asymptomatic or subclinical, meaning they do not present any noticeable signs or symptoms. For
example, most individual infected with herpes simplex virus remain asymptomatic and are unaware that they have been infected.

Exercise 12.2.1

Explain the difference between signs and symptoms.

Periods of Disease

Figure 12.2.1 : The progression of an infectious disease can be divided into five periods, which are related to the number of
pathogen particles (red) and the severity of signs and symptoms (blue).
The five periods of disease (sometimes referred to as stages or phases) include the incubation, prodromal, illness, decline, and
convalescence periods (Figure 12.2.1). The incubation period occurs in an acute disease after the initial entry of the pathogen into
the host (patient). It is during this time the pathogen begins multiplying in the host. However, there are insufficient numbers of
pathogen particles (cells or viruses) present to cause signs and symptoms of disease. Incubation periods can vary from a day or two
in acute disease to months or years in chronic disease, depending upon the pathogen. Factors involved in determining the length of
the incubation period are diverse, and can include strength of the pathogen, strength of the host immune defenses, site of infection,
type of infection, and the size infectious dose received. During this incubation period, the patient is unaware that a disease is
beginning to develop.

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The prodromal period occurs after the incubation period. During this phase, the pathogen continues to multiply and the host begins
to experience general signs and symptoms of illness, which typically result from activation of the immune system, such as fever,
pain, soreness, swelling, or inflammation. Usually, such signs and symptoms are too general to indicate a particular disease.
Following the prodromal period is the period of illness, during which the signs and symptoms of disease are most obvious, specific
and severe.
The period of illness is followed by the period of decline, during which the number of pathogen particles begins to decrease, and
the signs and symptoms of illness begin to decline. However, during the decline period, patients may become susceptible to
developing secondary infections because their immune systems have been weakened by the primary infection. The final period is
known as the period of convalescence. During this stage, the patient generally returns to normal functions, although some diseases
may inflict permanent damage that the body cannot fully repair.
Infectious diseases can be contagious during all five of the periods of disease. Which periods of disease are more likely to
associated with transmissibility of an infection depends upon the disease, the pathogen, and the mechanisms by which the disease
develops and progresses. For example, with meningitis (infection of the lining of brain), the periods of infectivity depend on the
type of pathogen causing the infection. Patients with bacterial meningitis are contagious during the incubation period for up to a
week before the onset of the prodromal period, whereas patients with viral meningitis become contagious when the first signs and
symptoms of the prodromal period appear. With many viral diseases associated with rashes (e.g., chickenpox, measles, rubella,
roseola), patients are contagious during the incubation period up to a week before the rash develops. In contrast, with many
respiratory infections (e.g., colds, influenza, diphtheria, strep throat, and pertussis) the patient becomes contagious with the onset of
the prodromal period. Depending upon the pathogen, the disease, and the individual infected, transmission can still occur during the
periods of decline, convalescence, and even long after signs and symptoms of the disease disappear. For example, an individual
recovering from a diarrheal disease may continue to carry and shed the pathogen in feces for some time, posing a risk of
transmission to others through direct contact or indirect contact (e.g., through contaminated objects or food).

Exercise 12.2.2

Name some of the factors that can affect the length of the incubation period of a particular disease.

Acute and Chronic Diseases

The duration of the period of illness can vary greatly, depending on the pathogen, effectiveness of the immune response in the host,
and any medical treatment received. For an acute disease, pathologic changes occur over a relatively short time (e.g., hours, days,
or a few weeks) and involve a rapid onset of disease conditions. For example, influenza (caused by Influenzavirus) is considered an
acute disease because the incubation period is approximately 1–2 days. Infected individuals can spread influenza to others for
approximately 5 days after becoming ill. After approximately 1 week, individuals enter the period of decline.
For a chronic disease, pathologic changes can occur over longer time spans (e.g., months, years, or a lifetime). For example,
chronic gastritis (inflammation of the lining of the stomach) is caused by the gram-negative bacterium Helicobacter pylori. H.
pylori is able to colonize the stomach and persist in its highly acidic environment by producing the enzyme urease, which modifies
the local acidity, allowing the bacteria to survive indefinitely.2 Consequently, H. pylori infections can recur indefinitely unless the
infection is cleared using antibiotics.3 Hepatitis B virus can cause a chronic infection in some patients who do not eliminate the
virus after the acute illness. A chronic infection with hepatitis B virus is characterized by the continued production of infectious
virus for 6 months or longer after the acute infection, as measured by the presence of viral antigen in blood samples.
In latent diseases, as opposed to chronic infections, the causal pathogen goes dormant for extended periods of time with no active
replication. Examples of diseases that go into a latent state after the acute infection include herpes (herpes simplex viruses [HSV-1
and HSV-2]), chickenpox (varicella-zoster virus [VZV]), and mononucleosis (Epstein-Barr virus [EBV]). HSV-1, HSV-2, and VZV
evade the host immune system by residing in a latent form within cells of the nervous system for long periods of time, but they can
reactivate to become active infections during times of stress and immunosuppression. For example, an initial infection by VZV
may result in a case of childhood chickenpox, followed by a long period of latency. The virus may reactivate decades later, causing
episodes of shingles in adulthood. EBV goes into latency in B cells of the immune system and possibly epithelial cells; it can
reactivate years later to produce B-cell lymphoma.

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 Exercise 12.2.3

Explain the difference between latent disease and chronic disease.

Pathogenicity and Virulence

The ability of a microbial agent to cause disease is called pathogenicity, and the degree to which an organism is pathogenic is called
virulence. Virulence is a continuum. On one end of the spectrum are organisms that are avirulent (not harmful) and on the other are
organisms that are highly virulent. Highly virulent pathogens will almost always lead to a disease state when introduced to the
body, and some may even cause multi-organ and body system failure in healthy individuals. Less virulent pathogens may cause an
initial infection, but may not always cause severe illness. Pathogens with low virulence would more likely result in mild signs and
symptoms of disease, such as low-grade fever, headache, or muscle aches. Some individuals might even be asymptomatic.
An example of a highly virulent microorganism is Bacillus anthracis, the pathogen responsible for anthrax. B. anthracis can
produce different forms of disease, depending on the route of transmission (e.g., cutaneous injection, inhalation, ingestion). The
most serious form of anthrax is inhalation anthrax. After B. anthracis spores are inhaled, they germinate. An active infection
develops and the bacteria release potent toxins that cause edema (fluid buildup in tissues), hypoxia (a condition preventing oxygen
from reaching tissues), and necrosis (cell death and inflammation). Signs and symptoms of inhalation anthrax include high fever,
difficulty breathing, vomiting and coughing up blood, and severe chest pains suggestive of a heart attack. With inhalation anthrax,
the toxins and bacteria enter the bloodstream, which can lead to multi-organ failure and death of the patient. If a gene (or genes)
involved in pathogenesis is inactivated, the bacteria become less virulent or nonpathogenic.

Figure 12.2.2 : A graph like this is used to determine LD50 by plotting pathogen concentration against the percent of infected test
animals that have died. In this example, the LD50 = 104 pathogenic particles.
Virulence of a pathogen can be quantified using controlled experiments with laboratory animals. Two important indicators of
virulence are the median infectious dose (ID50) and the median lethal dose (LD50), both of which are typically determined
experimentally using animal models. The ID50 is the number of pathogen cells or virions required to cause active infection in 50%
of inoculated animals. The LD50 is the number of pathogenic cells, virions, or amount of toxin required to kill 50% of infected
animals. To calculate these values, each group of animals is inoculated with one of a range of known numbers of pathogen cells or
virions. In graphs like the one shown in Figure 12.2.2, the percentage of animals that have been infected (for ID50) or killed (for
LD50) is plotted against the concentration of pathogen inoculated. Figure 12.2.2 represents data graphed from a hypothetical
experiment measuring the LD50 of a pathogen. Interpretation of the data from this graph indicates that the LD50 of the pathogen for
the test animals is 104 pathogen cells or virions (depending upon the pathogen studied).
Table 12.2.2 lists selected foodborne pathogens and their ID50 values in humans (as determined from epidemiologic data and
studies on human volunteers). Keep in mind that these are median values. The actual infective dose for an individual can vary
widely, depending on factors such as route of entry; the age, health, and immune status of the host; and environmental and
pathogen-specific factors such as susceptibility to the acidic pH of the stomach. It is also important to note that a pathogen’s

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infective dose does not necessarily correlate with disease severity. For example, just a single cell of Salmonella enterica serotype
typhimurium can result in an active infection. The resultant disease, Salmonella gastroenteritis or salmonellosis, can cause nausea,
vomiting, and diarrhea, but has a mortality rate of less than 1% in healthy adults. In contrast, S. enterica serotype Typhi has a much
higher ID50, typically requiring as many as 1,000 cells to produce infection. However, this serotype causes typhoid fever, a much
more systemic and severe disease that has a mortality rate as high as 10% in untreated individuals.
Table 12.2.2 : ID50 for Selected Foodborne Diseases1
Pathogen ID50

Viruses

Hepatitis A virus 10–100

Norovirus 1–10

Rotavirus 10–100

Bacteria

Escherichia coli, enterohemorrhagic (EHEC, serotype O157) 10–100

E. coli, enteroinvasive (EIEC) 200–5,000

E. coli, enteropathogenic (EPEC) 10,000,000–10,000,000,000

E. coli, enterotoxigenic (ETEC) 10,000,000–10,000,000,000

Salmonella enterica serovar Typhi <1,000>

S. enterica serovar Typhimurium ≥1

Shigella dysenteriae 10–200

Vibrio cholerae (serotypes O139, O1) 1,000,000

V. parahemolyticus 100,000,000

Protozoa

Giardia lamblia 1

Cryptosporidium parvum 10–100

Exercise 12.2.4

1. What is the difference between a pathogen’s infective dose and lethal dose?
2. Which is more closely related to the severity of a disease?

Primary Pathogens versus Opportunistic Pathogens

Pathogens can be classified as either primary pathogens or opportunistic pathogens. A primary pathogen can cause disease in a host
regardless of the host’s resident microbiota or immune system. An opportunistic pathogen, by contrast, can only cause disease in
situations that compromise the host’s defenses, such as the body’s protective barriers, immune system, or normal microbiota.
Individuals susceptible to opportunistic infections include the very young, the elderly, women who are pregnant, patients
undergoing chemotherapy, people with immunodeficiencies (such as acquired immunodeficiency syndrome [AIDS]), patients who
are recovering from surgery, and those who have had a breach of protective barriers (such as a severe wound or burn).
An example of a primary pathogen is enterohemorrhagic E. coli (EHEC), which produces a virulence factor known as Shiga toxin.
This toxin inhibits protein synthesis, leading to severe and bloody diarrhea, inflammation, and renal failure, even in patients with
healthy immune systems. Staphylococcus epidermidis, on the other hand, is an opportunistic pathogen that is among the most
frequent causes of nosocomial disease.6 S. epidermidis is a member of the normal microbiota of the skin, where it is generally
avirulent. However, in hospitals, it can also grow in biofilms that form on catheters, implants, or other devices that are inserted into
the body during surgical procedures. Once inside the body, S. epidermidis can cause serious infections such as endocarditis, and it
produces virulence factors that promote the persistence of such infections.

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Other members of the normal microbiota can also cause opportunistic infections under certain conditions. This often occurs when
microbes that reside harmlessly in one body location end up in a different body system, where they cause disease. For example, E.
coli normally found in the large intestine can cause a urinary tract infection if it enters the bladder. This is the leading cause of
urinary tract infections among women.
Members of the normal microbiota may also cause disease when a shift in the environment of the body leads to overgrowth of a
particular microorganism. For example, the yeast Candida is part of the normal microbiota of the skin, mouth, intestine, and
vagina, but its population is kept in check by other organisms of the microbiota. If an individual is taking antibacterial medications,
however, bacteria that would normally inhibit the growth of Candida can be killed off, leading to a sudden growth in the population
of Candida, which is not affected by antibacterial medications because it is a fungus. An overgrowth of Candida can manifest as
oral thrush (growth on mouth, throat, and tongue), a vaginal yeast infection, or cutaneous candidiasis. Other scenarios can also
provide opportunities for Candida infections. Untreated diabetes can result in a high concentration of glucose in the saliva, which
provides an optimal environment for the growth of Candida, resulting in thrush. Immunodeficiencies such as those seen in patients
with HIV, AIDS, and cancer also lead to higher incidence of thrush. Vaginal yeast infections can result from decreases in estrogen
levels during the menstruation or menopause. The amount of glycogen available to lactobacilli in the vagina is controlled by levels
of estrogen; when estrogen levels are low, lactobacilli produce less lactic acid. The resultant increase in vaginal pH allows
overgrowth of Candida in the vagina.

Exercise 12.2.5

1. Explain the difference between a primary pathogen and an opportunistic pathogen.

2. Describe some conditions under which an opportunistic infection can occur.

Stages of Pathogenesis
To cause disease, a pathogen must successfully achieve four steps or stages of pathogenesis: exposure (contact), adhesion
(colonization), invasion, and infection. The pathogen must be able to gain entry to the host, travel to the location where it can
establish an infection, evade or overcome the host’s immune response, and cause damage (i.e., disease) to the host. In many cases,
the cycle is completed when the pathogen exits the host and is transmitted to a new host.

Exposure
An encounter with a potential pathogen is known as exposure or contact. The food we eat and the objects we handle are all ways
that we can come into contact with potential pathogens. Yet, not all contacts result in infection and disease. For a pathogen to cause
disease, it needs to be able to gain access into host tissue. An anatomic site through which pathogens can pass into host tissue is
called a portal of entry. These are locations where the host cells are in direct contact with the external environment. Major portals
of entry are identified in Figure 12.2.3 and include the skin, mucous membranes, and parenteral routes.

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 Figure 12.2.3 : Shown are different portals of entry where pathogens can gain access into the body. With the exception of the
placenta, many of these locations are directly exposed to the external environment.
Mucosal surfaces are the most important portals of entry for microbes; these include the mucous membranes of the respiratory
tract, the gastrointestinal tract, and the genitourinary tract. Although most mucosal surfaces are in the interior of the body, some are
contiguous with the external skin at various body openings, including the eyes, nose, mouth, urethra, and anus.
Most pathogens are suited to a particular portal of entry. A pathogen’s portal specificity is determined by the organism’s
environmental adaptions and by the enzymes and toxins they secrete. The respiratory and gastrointestinal tracts are particularly
vulnerable portals of entry because particles that include microorganisms are constantly inhaled or ingested, respectively.
Pathogens can also enter through a breach in the protective barriers of the skin and mucous membranes. Pathogens that enter the
body in this way are said to enter by the parenteral route. For example, the skin is a good natural barrier to pathogens, but breaks in
the skin (e.g., wounds, insect bites, animal bites, needle pricks) can provide a parenteral portal of entry for microorganisms.
In pregnant women, the placenta normally prevents microorganisms from passing from the mother to the fetus. However, a few
pathogens are capable of crossing the blood-placental barrier. The gram-positive bacterium Listeria monocytogenes, which causes
the foodborne disease listeriosis, is one example that poses a serious risk to the fetus and can sometimes lead to spontaneous
abortion. Other pathogens that can pass the placental barrier to infect the fetus are known collectively by the acronym TORCH
(Table 12.2.3).
Transmission of infectious diseases from mother to baby is also a concern at the time of birth when the baby passes through the
birth canal. Babies whose mothers have active chlamydia or gonorrhea infections may be exposed to the causative pathogens in the

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vagina, which can result in eye infections that lead to blindness. To prevent this, it is standard practice to administer antibiotic
drops to infants’ eyes shortly after birth.
Table 12.2.3 : Pathogens Capable of Crossing the Placental Barrier (TORCH Infections)

Disease Pathogen

T Toxoplasmosis Toxoplasma gondii (protozoan)

Syphilis Treponema pallidum (bacterium)

Chickenpox Varicella-zoster virus (human herpesvirus 3)
O7 Hepatitis B Hepatitis B virus (hepadnavirus)
HIV Retrovirus
Fifth disease (erythema infectiosum) Parvovirus B19

R Rubella (German measles) Togavirus

C Cytomegalovirus Human herpesvirus 5

H Herpes Herpes simplex viruses (HSV) 1 and 2

Clinical Focus: part 2

At the clinic, a physician takes down Michael’s medical history and asks about his activities and diet over the past week. Upon
learning that Michael became sick the day after the party, the physician orders a blood test to check for pathogens associated
with foodborne diseases. After tests confirm that presence of a gram-positive rod in Michael’s blood, he is given an injection of
a broad-spectrum antibiotic and sent to a nearby hospital, where he is admitted as a patient. There he is to receive additional
intravenous antibiotic therapy and fluids.

Exercise 12.2.6
1. Is this bacterium in Michael’s blood part of normal microbiota?
2. What portal of entry did the bacteria use to cause this infection?

Adhesion
Following the initial exposure, the pathogen adheres at the portal of entry. The term adhesion refers to the capability of pathogenic
microbes to attach to the cells of the body using adhesion factors, and different pathogens use various mechanisms to adhere to the
cells of host tissues.
Molecules (either proteins or carbohydrates) called adhesins are found on the surface of certain pathogens and bind to specific
receptors (glycoproteins) on host cells. Adhesins are present on the fimbriae and flagella of bacteria, the cilia of protozoa, and the
capsids or membranes of viruses. Protozoans can also use hooks and barbs for adhesion; spike proteins on viruses also enhance
viral adhesion. The production of glycocalyces (slime layers and capsules) (Figure 12.2.4), with their high sugar and protein
content, can also allow certain bacterial pathogens to attach to cells.
Biofilm growth can also act as an adhesion factor. A biofilm is a community of bacteria that produce a glycocalyx, which
contributes to the extrapolymeric substances (EPS) that allows the biofilm to attach to a surface. Persistent Pseudomonas
aeruginosa infections are common in patients suffering from cystic fibrosis, burn wounds, and middle-ear infections (otitis media)
because P. aeruginosa produces a biofilm. The EPS allows the microbe to adhere to the host cells and makes it harder for the host
to physically remove the pathogen. The EPS not only allows for attachment but provides protection against the immune system and
antibiotic or antimicrobial treatments, preventing the medications from reaching the cells within the biofilm. In addition, not all
bacteria in a biofilm are rapidly growing; some are in stationary phase. Since antibiotics are most effective against rapidly growing
bacteria, portions of bacteria in a biofilm are protected against antibiotics.8

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 Figure 12.2.4 : Glycocalyx produced by bacteria in a biofilm allows the cells to adhere to host tissues and to medical devices such
as the catheter surface shown here. (credit: modification of work by Centers for Disease Control and Prevention)

Invasion
Once adhesion is successful, invasion can proceed. Invasion involves the dissemination of a pathogen throughout local tissues or
the body. Pathogens may produce exoenzymes or toxins, which serve as virulence factors that allow them to colonize and damage
host tissues as they spread deeper into the body. Pathogens may also produce virulence factors that protect them against immune
system defenses. A pathogen’s specific virulence factors determine the degree of tissue damage that occurs. Figure 12.2.5 shows
the invasion of H. pylori into the tissues of the stomach, causing damage as it progresses.

Figure 12.2.5 : H. pylori is able to invade the lining of the stomach by producing virulence factors that enable it pass through the
mucin layer covering epithelial cells. (credit: modification of work by Zina Deretsky, National Science Foundation)
Intracellular pathogens achieve invasion by entering the host’s cells and reproducing. Some are obligate intracellular pathogens
(meaning they can only reproduce inside of host cells) and others are facultative intracellular pathogens (meaning they can
reproduce either inside or outside of host cells). By entering the host cells, intracellular pathogens are able to evade some
mechanisms of the immune system while also exploiting the nutrients in the host cell.
Entry to a cell can occur by endocytosis. For most kinds of host cells, pathogens use one of two different mechanisms for
endocytosis and entry. One mechanism relies on effector proteins secreted by the pathogen; these effector proteins trigger entry into
the host cell. This is the method that Salmonella and Shigella use when invading intestinal epithelial cells. When these pathogens
come in contact with epithelial cells in the intestine, they secrete effector molecules that cause protrusions of membrane ruffles that

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bring the bacterial cell in. This process is called membrane ruffling. The second mechanism relies on surface proteins expressed on
the pathogen that bind to receptors on the host cell, resulting in entry. For example, Yersinia pseudotuberculosis produces a surface
protein known as invasin that binds to beta-1 integrins expressed on the surface of host cells.
Some host cells, such as white blood cells and other phagocytes of the immune system, actively endocytose pathogens in a process
called phagocytosis. Although phagocytosis allows the pathogen to gain entry to the host cell, in most cases, the host cell kills and
degrades the pathogen by using digestive enzymes. Normally, when a pathogen is ingested by a phagocyte, it is enclosed within a
phagosome in the cytoplasm; the phagosome fuses with a lysosome to form a phagolysosome, where digestive enzymes kill the
pathogen. However, some intracellular pathogens have the ability to survive and multiply within phagocytes. Examples include
Listeria monocytogenes and Shigella; these bacteria produce proteins that lyse the phagosome before it fuses with the lysosome,
allowing the bacteria to escape into the phagocyte’s cytoplasm where they can multiply. Bacteria such as Mycobacterium
tuberculosis, Legionella pneumophila, and Salmonella species use a slightly different mechanism to evade being digested by the
phagocyte. These bacteria prevent the fusion of the phagosome with the lysosome, thus remaining alive and dividing within the
phagosome.

Infection
Following invasion, successful multiplication of the pathogen leads to infection. Infections can be described as local, focal, or
systemic, depending on the extent of the infection. A local infection is confined to a small area of the body, typically near the portal
of entry. For example, a hair follicle infected by Staphylococcus aureus infection may result in a boil around the site of infection,
but the bacterium is largely contained to this small location. Other examples of local infections that involve more extensive tissue
involvement include urinary tract infections confined to the bladder or pneumonia confined to the lungs.
In a focal infection, a localized pathogen, or the toxins it produces, can spread to a secondary location. For example, a dental
hygienist nicking the gum with a sharp tool can lead to a local infection in the gum by Streptococcus bacteria of the normal oral
microbiota. These Streptococcus spp. may then gain access to the bloodstream and make their way to other locations in the body,
resulting in a secondary infection.
When an infection becomes disseminated throughout the body, we call it a systemic infection. For example, infection by the
varicella-zoster virus typically gains entry through a mucous membrane of the upper respiratory system. It then spreads throughout
the body, resulting in the classic red skin lesions associated with chickenpox. Since these lesions are not sites of initial infection,
they are signs of a systemic infection.
Sometimes a primary infection, the initial infection caused by one pathogen, can lead to a secondary infection by another pathogen.
For example, the immune system of a patient with a primary infection by HIV becomes compromised, making the patient more
susceptible to secondary diseases like oral thrush and others caused by opportunistic pathogens. Similarly, a primary infection by
Influenzavirus damages and decreases the defense mechanisms of the lungs, making patients more susceptible to a secondary
pneumonia by a bacterial pathogen like Haemophilus influenzae or Streptococcus pneumoniae. Some secondary infections can
even develop as a result of treatment for a primary infection. Antibiotic therapy targeting the primary pathogen can cause collateral
damage to the normal microbiota, creating an opening for opportunistic pathogens (see Case in Point: A Secondary Yeast Infection
below).

A Secondary Yeast Infection

Anita, a 36-year-old mother of three, goes to an urgent care center complaining of pelvic pressure, frequent and painful
urination, abdominal cramps, and occasional blood-tinged urine. Suspecting a urinary tract infection (UTI), the physician
requests a urine sample and sends it to the lab for a urinalysis. Since it will take approximately 24 hours to get the results of the
culturing, the physician immediately starts Anita on the antibiotic ciprofloxacin. The next day, the microbiology lab confirms
the presence of E. coli in Anita’s urine, which is consistent with the presumptive diagnosis. However, the antimicrobial
susceptibility test indicates that ciprofloxacin would not effectively treat Anita’s UTI, so the physician prescribes a different
antibiotic.
After taking her antibiotics for 1 week, Anita returns to the clinic complaining that the prescription is not working. Although
the painful urination has subsided, she is now experiencing vaginal itching, burning, and discharge. After a brief examination,
the physician explains to Anita that the antibiotics were likely successful in killing the E. coli responsible for her UTI;
however, in the process, they also wiped out many of the “good” bacteria in Anita’s normal microbiota. The new symptoms

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 that Anita has reported are consistent with a secondary yeast infection by Candida albicans, an opportunistic fungus that
normally resides in the vagina but is inhibited by the bacteria that normally reside in the same environment.
To confirm this diagnosis, a microscope slide of a direct vaginal smear is prepared from the discharge to check for the presence
of yeast. A sample of the discharge accompanies this slide to the microbiology lab to determine if there has been an increase in
the population of yeast causing vaginitis. After the microbiology lab confirms the diagnosis, the physician prescribes an
antifungal drug for Anita to use to eliminate her secondary yeast infection.

Exercise 12.2.7

1. Why was Candida not killed by the antibiotics prescribed for the UTI?
2. List three conditions that could lead to a secondary infection.

Transmission of Disease
For a pathogen to persist, it must put itself in a position to be transmitted to a new host, leaving the infected host through a portal of
exit (Figure 12.2.6). As with portals of entry, many pathogens are adapted to use a particular portal of exit. Similar to portals of
entry, the most common portals of exit include the skin and the respiratory, urogenital, and gastrointestinal tracts. Coughing and
sneezing can expel pathogens from the respiratory tract. A single sneeze can send thousands of virus particles into the air.
Secretions and excretions can transport pathogens out of other portals of exit. Feces, urine, semen, vaginal secretions, tears, sweat,
and shed skin cells can all serve as vehicles for a pathogen to leave the body. Pathogens that rely on insect vectors for transmission
exit the body in the blood extracted by a biting insect. Similarly, some pathogens exit the body in blood extracted by needles.

Figure 12.2.6 : Pathogens leave the body of an infected host through various portals of exit to infect new hosts.

Key Concepts and Summary

In an infection, a microorganism enters a host and begins to multiply. Some infections cause disease, which is any deviation
from the normal function or structure of the host.
Signs of a disease are objective and are measured. Symptoms of a disease are subjective and are reported by the patient.

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 Diseases can either be noninfectious (due to genetics and environment) or infectious (due to pathogens). Some infectious
diseases are communicable (transmissible between individuals) or contagious (easily transmissible between individuals);
others are noncommunicable, but may be contracted via contact with environmental reservoirs or animals (zoonoses)
Nosocomial diseases are contracted in hospital settings, whereas iatrogenic disease are the direct result of a medical procedure
An acute disease is short in duration, whereas a chronic disease lasts for months or years. Latent diseases last for years, but
are distinguished from chronic diseases by the lack of active replication during extended dormant periods.
The periods of disease include the incubation period, the prodromal period, the period of illness, the period of decline, and
the period of convalescence. These periods are marked by changes in the number of infectious agents and the severity of signs
and symptoms.
Virulence, the degree to which a pathogen can cause disease, can be quantified by calculating either the ID50 or LD50 of a
pathogen on a given population.
Primary pathogens are capable of causing pathological changes associated with disease in a healthy individual, whereas
opportunistic pathogens can only cause disease when the individual is compromised by a break in protective barriers or
immunosuppression.
Infections and disease can be caused by pathogens in the environment or microbes in an individual’s resident microbiota.
Infections can be classified as local, focal, or systemic depending on the extent to which the pathogen spreads in the body.
A secondary infection can sometimes occur after the host’s defenses or normal microbiota are compromised by a primary
infection or antibiotic treatment.
Pathogens enter the body through portals of entry and leave through portals of exit. The stages of pathogenesis include
exposure, adhesion, invasion, infection, and transmission.

Footnotes
1. F. Savino et al. “Pain Assessment in Children Undergoing Venipuncture: The Wong–Baker Faces Scale Versus Skin
Conductance Fluctuations.” PeerJ 1 (2013):e37; https://peerj.com/articles/37/
2. J.G. Kusters et al. Pathogenesis of Helicobacter pylori Infection. Clinical Microbiology Reviews 19 no. 3 (2006):449–490.
3. N.R. Salama et al. “Life in the Human Stomach: Persistence Strategies of the Bacterial Pathogen Helicobacter pylori.” Nature
Reviews Microbiology 11 (2013):385–399.
4. C. Owens. “P. aeruginosa survives in sinks 10 years after hospital outbreak.” 2015. www.healio.com/infectious-dis...pital-
outbreak
5. Food and Drug Administration. “Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins.” 2nd ed. Silver
Spring, MD: US Food and Drug Administration; 2012.
6. M. Otto. “Staphylococcus epidermidis--The ‘Accidental’ Pathogen.” Nature Reviews Microbiology 7 no. 8 (2009):555–567.
7. The O in TORCH stands for “other.”
8. D. Davies. “Understanding Biofilm Resistance to Antibacterial Agents.” Nature Reviews Drug Discovery 2 (2003):114–122.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 12.2: Characteristics and Steps of Infectious Diseases is shared under a CC BY license and was authored, remixed, and/or curated
by OpenStax.

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12.3: Virulence Factors in Infection
Learning Objectives
Explain how virulence factors contribute to signs and symptoms of infectious disease
Differentiate between endotoxins and exotoxins
Describe and differentiate between various types of exotoxins
Describe virulence factors unique to fungi and parasites
Compare virulence factors of fungi and bacteria
Explain the difference between protozoan parasites and helminths
Describe how helminths evade the host immune system
Describe the mechanisms viruses use for adhesion and antigenic variation

In the previous section, we explained that some pathogens are more virulent than others. This is due to the unique virulence factors
produced by individual pathogens, which determine the extent and severity of disease they may cause. A pathogen’s virulence
factors are encoded by genes that can be identified. When genes encoding virulence factors are inactivated, virulence in the
pathogen is diminished. In this section, we examine various types and specific examples of virulence factors and how they
contribute to each step of pathogenesis.

Virulence Factors for Adhesion

As discussed in the previous section, the first two steps in pathogenesis are exposure and adhesion. Recall that an adhesin is a
structure, such as a protein or glycoprotein, found on the surface of a pathogen that attaches to receptors on the host cell. Adhesins
are found on bacterial, viral, fungal, and protozoan pathogens. One example of a bacterial adhesin is type 1 fimbrial adhesin, a
molecule found on the tips of fimbriae of enterotoxigenic E. coli (ETEC). Recall that fimbriae are hairlike protein bristles on the
cell surface. Type 1 fimbrial adhesin allows the fimbriae of ETEC cells to attach to the mannose glycans expressed on intestinal
epithelial cells. Table 12.3.1 lists common adhesins found in some of the pathogens we have discussed or will be seeing later in
this chapter.
Table 12.3.1 : Some Bacterial Adhesins and Their Host Attachment Sites
Pathogen Disease Adhesin Attachment Site

Streptococcus pyogenes Strep throat Protein F Respiratory epithelial cells

Streptococcus mutans Dental caries Adhesin P1 Teeth

Neisseria gonorrhoeae Gonorrhea Type IV pili Urethral epithelial cells

Enterotoxigenic E. coli
Traveler’s diarrhea Type 1 fimbriae Intestinal epithelial cells
(ETEC)

N-methylphenylalanine
Vibrio cholerae Cholera Intestinal epithelial cells
pili

Clinical Focus: part 3

The presence of bacteria in Michael’s blood is a sign of infection, since blood is normally sterile. There is no indication that the
bacteria entered the blood through an injury. Instead, it appears the portal of entry was the gastrointestinal route. Based on
Michael’s symptoms, the results of his blood test, and the fact that Michael was the only one in the family to partake of the hot
dogs, the physician suspects that Michael is suffering from a case of listeriosis.
Listeria monocytogenes, the facultative intracellular pathogen that causes listeriosis, is a common contaminant in ready-to-eat
foods such as lunch meats and dairy products. Once ingested, these bacteria invade intestinal epithelial cells and translocate to
the liver, where they grow inside hepatic cells. Listeriosis is fatal in about one in five normal healthy people, and mortality
rates are slightly higher in patients with pre-existing conditions that weaken the immune response. A cluster of virulence genes
encoded on a pathogenicity island is responsible for the pathogenicity of L. monocytogenes. These genes are regulated by a
transcriptional factor known as peptide chain release factor 1 (PrfA). One of the genes regulated by PrfA is hyl, which encodes
a toxin known as listeriolysin O (LLO), which allows the bacterium to escape vacuoles upon entry into a host cell. A second
gene regulated by PrfA is actA, which encodes for a surface protein known as actin assembly-inducing protein (ActA). ActA is

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 expressed on the surface of Listeria and polymerizes host actin. This enables the bacterium to produce actin tails, move around
the cell’s cytoplasm, and spread from cell to cell without exiting into the extracellular compartment.
Michael’s condition has begun to worsen. He is now experiencing a stiff neck and hemiparesis (weakness of one side of the
body). Concerned that the infection is spreading, the physician decides to conduct additional tests to determine what is causing
these new symptoms.

Exercise 12.3.1

1. What kind of pathogen causes listeriosis, and what virulence factors contribute to the signs and symptoms Michael is
experiencing?
2. Is it likely that the infection will spread from Michael’s blood? If so, how might this explain his new symptoms?

Bacterial Exoenzymes and Toxins as Virulence Factors

After exposure and adhesion, the next step in pathogenesis is invasion, which can involve enzymes and toxins. Many pathogens
achieve invasion by entering the bloodstream, an effective means of dissemination because blood vessels pass close to every cell in
the body. The downside of this mechanism of dispersal is that the blood also includes numerous elements of the immune system.
Various terms ending in –emia are used to describe the presence of pathogens in the bloodstream. The presence of bacteria in blood
is called bacteremia. Bacteremia involving pyogens (pus-forming bacteria) is called pyemia. When viruses are found in the blood,
it is called viremia. The term toxemia describes the condition when toxins are found in the blood. If bacteria are both present and
multiplying in the blood, this condition is called septicemia.
Patients with septicemia are described as septic, which can lead to shock, a life-threatening decrease in blood pressure (systolic
pressure <90 mm Hg) that prevents cells and organs from receiving enough oxygen and nutrients. Some bacteria can cause shock
through the release of toxins (virulence factors that can cause tissue damage) and lead to low blood pressure. Gram-negative
bacteria are engulfed by immune system phagocytes, which then release tumor necrosis factor, a molecule involved in
inflammation and fever. Tumor necrosis factor binds to blood capillaries to increase their permeability, allowing fluids to pass out
of blood vessels and into tissues, causing swelling, or edema(Figure 12.3.1). With high concentrations of tumor necrosis factor, the
inflammatory reaction is severe and enough fluid is lost from the circulatory system that blood pressure decreases to dangerously
low levels. This can have dire consequences because the heart, lungs, and kidneys rely on normal blood pressure for proper
function; thus, multi-organ failure, shock, and death can occur.

Figure 12.3.1 : This patient has edema in the tissue of the right hand. Such swelling can occur when bacteria cause the release of
pro-inflammatory molecules from immune cells and these molecules cause an increased permeability of blood vessels, allowing
fluid to escape the bloodstream and enter tissue.

Exoenzymes
Some pathogens produce extracellular enzymes, or exoenzymes, that enable them to invade host cells and deeper tissues.
Exoenzymes have a wide variety of targets. Some general classes of exoenzymes and associated pathogens are listed in Table

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12.3.2. Each of these exoenzymes functions in the context of a particular tissue structure to facilitate invasion or support its own
growth and defend against the immune system. For example, hyaluronidase S, an enzyme produced by pathogens like
Staphylococcus aureus, Streptococcus pyogenes, and Clostridium perfringens, degrades the glycoside hylauronan (hyaluronic acid),
which acts as an intercellular cement between adjacent cells in connective tissue (Figure 12.3.2). This allows the pathogen to pass
through the tissue layers at the portal of entry and disseminate elsewhere in the body (Figure 12.3.2).
Table 12.3.2 : Some Classes of Exoenzymes and Their Targets
Class Example Function

Degrades hyaluronic acid that cements cells

Glycohydrolases Hyaluronidase S in Staphylococcus aureus
together to promote spreading through tissues
Degrades DNA released by dying cells
Nucleases DNAse produced by S. aureus (bacteria and host cells) that can trap the
bacteria, thus promoting spread
Degrades phospholipid bilayer of host cells,
causing cellular lysis, and degrade membrane
Phospholipases Phospholipase C of Bacillus anthracis
of phagosomes to enable escape into the
cytoplasm
Degrades collagen in connective tissue to
Proteases Collagenase in Clostridium perfringens
promote spread

Figure 12.3.2 : (a) Hyaluronan is a polymer found in the layers of epidermis that connect adjacent cells. (b) Hyaluronidase
produced by bacteria degrades this adhesive polymer in the extracellular matrix, allowing passage between cells that would
otherwise be blocked.
Pathogen-produced nucleases, such as DNAse produced by S. aureus, degrade extracellular DNA as a means of escape and
spreading through tissue. As bacterial and host cells die at the site of infection, they lyse and release their intracellular contents.
The DNA chromosome is the largest of the intracellular molecules, and masses of extracellular DNA can trap bacteria and prevent
their spread. S. aureus produces a DNAse to degrade the mesh of extracellular DNA so it can escape and spread to adjacent tissues.
This strategy is also used by S. aureus and other pathogens to degrade and escape webs of extracellular DNA produced by immune
system phagocytes to trap the bacteria.
Enzymes that degrade the phospholipids of cell membranes are called phospholipases. Their actions are specific in regard to the
type of phospholipids they act upon and where they enzymatically cleave the molecules. The pathogen responsible for anthrax, B.
anthracis, produces phospholipase C. When B. anthracis is ingested by phagocytic cells of the immune system, phospholipase C
degrades the membrane of the phagosome before it can fuse with the lysosome, allowing the pathogen to escape into the cytoplasm
and multiply. Phospholipases can also target the membrane that encloses the phagosome within phagocytic cells. As described
earlier in this chapter, this is the mechanism used by intracellular pathogens such as L. monocytogenes and Rickettsia to escape the
phagosome and multiply within the cytoplasm of phagocytic cells. The role of phospholipases in bacterial virulence is not restricted
to phagosomal escape. Many pathogens produce phospholipases that act to degrade cell membranes and cause lysis of target cells.
These phospholipases are involved in lysis of red blood cells, white blood cells, and tissue cells.
Bacterial pathogens also produce various protein-digesting enzymes, or proteases. Proteases can be classified according to their
substrate target (e.g., serine proteases target proteins with the amino acid serine) or if they contain metals in their active site (e.g.,

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zinc metalloproteases contain a zinc ion, which is necessary for enzymatic activity).
One example of a protease that contains a metal ion is the exoenzyme collagenase. Collagenase digests collagen, the dominant
protein in connective tissue. Collagen can be found in the extracellular matrix, especially near mucosal membranes, blood vessels,
nerves, and in the layers of the skin. Similar to hyaluronidase, collagenase allows the pathogen to penetrate and spread through the
host tissue by digesting this connective tissue protein. The collagenase produced by the gram-positive bacterium Clostridium
perfringens, for example, allows the bacterium to make its way through the tissue layers and subsequently enter and multiply in the
blood (septicemia). C. perfringens then uses toxins and a phospholipase to cause cellular lysis and necrosis. Once the host cells
have died, the bacterium produces gas by fermenting the muscle carbohydrates. The widespread necrosis of tissue and
accompanying gas are characteristic of the condition known as gas gangrene (Figure 12.3.3).

Figure 12.3.3 : The illustration depicts a blood vessel with a single layer of endothelial cells surrounding the lumen and dense
connective tissue (shown in red) surrounding the endothelial cell layer. Collagenase produced by C. perfringens degrades the
collagen between the endothelial cells, allowing the bacteria to enter the bloodstream. (credit illustration: modification of work by
Bruce Blaus; credit micrograph: Micrograph provided by the Regents of University of Michigan Medical School © 2012)

Toxins
In addition to exoenzymes, certain pathogens are able to produce toxins, biological poisons that assist in their ability to invade and
cause damage to tissues. The ability of a pathogen to produce toxins to cause damage to host cells is called toxigenicity.
Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-
negative bacteria is called endotoxin (Figure 12.3.4). During infection and disease, gram-negative bacterial pathogens release
endotoxin either when the cell dies, resulting in the disintegration of the membrane, or when the bacterium undergoes binary
fission. The lipid component of endotoxin, lipid A, is responsible for the toxic properties of the LPS molecule. Lipid A is relatively
conserved across different genera of gram-negative bacteria; therefore, the toxic properties of lipid A are similar regardless of the
gram-negative pathogen. In a manner similar to that of tumor necrosis factor, lipid A triggers the immune system’s inflammatory
response. If the concentration of endotoxin in the body is low, the inflammatory response may provide the host an effective defense
against infection; on the other hand, high concentrations of endotoxin in the blood can cause an excessive inflammatory response,
leading to a severe drop in blood pressure, multi-organ failure, and death.

Figure 12.3.4 : Lipopolysaccharide is composed of lipid A, a core glycolipid, and an O-specific polysaccharide side chain. Lipid A
is the toxic component that promotes inflammation and fever.
A classic method of detecting endotoxin is by using the Limulus amebocyte lysate (LAL) test. In this procedure, the blood cells
(amebocytes) of the horseshoe crab (Limulus polyphemus) is mixed with a patient’s serum. The amebocytes will react to the
presence of any endotoxin. This reaction can be observed either chromogenically (color) or by looking for coagulation (clotting

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reaction) to occur within the serum. An alternative method that has been used is an enzyme-linked immunosorbent assay (ELISA)
that uses antibodies to detect the presence of endotoxin.
Unlike the toxic lipid A of endotoxin, exotoxins are protein molecules that are produced by a wide variety of living pathogenic
bacteria. Although some gram-negative pathogens produce exotoxins, the majority are produced by gram-positive pathogens.
Exotoxins differ from endotoxin in several other key characteristics, summarized in Table 12.3.3. In contrast to endotoxin, which
stimulates a general systemic inflammatory response when released, exotoxins are much more specific in their action and the cells
they interact with. Each exotoxin targets specific receptors on specific cells and damages those cells through unique molecular
mechanisms. Endotoxin remains stable at high temperatures, and requires heating at 121 °C (250 °F) for 45 minutes to inactivate.
By contrast, most exotoxins are heat labile because of their protein structure, and many are denatured (inactivated) at temperatures
above 41 °C (106 °F). As discussed earlier, endotoxin can stimulate a lethal inflammatory response at very high concentrations and
has a measured LD50 of 0.24 mg/kg. By contrast, very small concentrations of exotoxins can be lethal. For example, botulinum
toxin, which causes botulism, has an LD50 of 0.000001 mg/kg (240,000 times more lethal than endotoxin).
Table 12.3.3 : Comparison of Endotoxin and Exotoxins Produced by Bacteria
Characteristic Endotoxin Exotoxin

Gram-positive (primarily) and gram-negative

Source Gram-negative bacteria
bacteria

Composition Lipid A component of lipopolysaccharide Protein

Specific damage to cells dependent upon

General systemic symptoms of inflammation
Effect on host receptor-mediated targeting of cells and
and fever
specific mechanisms of action

Heat stability Heat stable Most are heat labile, but some are heat stable

LD50 High Low

The exotoxins can be grouped into three categories based on their target: intracellular targeting, membrane disrupting, and
superantigens. Table 12.3.4 provides examples of well-characterized toxins within each of these three categories.
Table 12.3.4 : Some Common Exotoxins and Associated Bacterial Pathogens
Category Example Pathogen Mechanism and Disease

Activation of adenylate cyclase in

intestinal cells, causing increased
levels of cyclic adenosine
Cholera toxin Vibrio cholerae
monophosphate (cAMP) and
secretion of fluids and electrolytes
out of cell, causing diarrhea
Inhibits the release of inhibitory
neurotransmitters in the central
Intracellular-targeting toxins Tetanus toxin Clostridium tetani
nervous system, causing spastic
paralysis
Inhibits release of the
neurotransmitter acetylcholine
Botulinum toxin Clostridium botulinum
from neurons, resulting in flaccid
paralysis
Inhibition of protein synthesis,
Diphtheria toxin Corynebacterium diphtheriae
causing cellular death

Membrane-disrupting toxins Streptolysin Streptococcus pyogenes

Proteins that assemble into pores
Pneumolysin Streptococcus pneumoniae in cell membranes, disrupting their
function and killing the cell
Alpha-toxin Staphylococcus aureus

Alpha-toxin Clostridium perfringens Phospholipases that degrade cell

membrane phospholipids,
Phospholipase C Pseudomonas aeruginosa
disrupting membrane function and
killing the cell

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 Category Example Pathogen Mechanism and Disease

Beta-toxin Staphylococcus aureus

Toxic shock syndrome toxin Staphylococcus aureus Stimulates excessive activation of

immune system cells and release
of cytokines (chemical mediators)
Superantigens Streptococcal mitogenic exotoxin Streptococcus pyogenes
from immune system cells. Life-
threatening fever, inflammation,
Streptococcal pyrogenic toxins Streptococcus pyogenes
and shock are the result.

The intracellular targeting toxins comprise two components: A for activity and B for binding. Thus, these types of toxins are known
as A-B exotoxins (Figure 12.3.5). The B component is responsible for the cellular specificity of the toxin and mediates the initial
attachment of the toxin to specific cell surface receptors. Once the A-B toxin binds to the host cell, it is brought into the cell by
endocytosis and entrapped in a vacuole. The A and B subunits separate as the vacuole acidifies. The A subunit then enters the cell
cytoplasm and interferes with the specific internal cellular function that it targets.

Figure 12.3.5 : (a) In A-B toxins, the B component binds to the host cell through its interaction with specific cell surface receptors.
(b) The toxin is brought in through endocytosis. (c) Once inside the vacuole, the A component (active component) separates from
the B component and the A component gains access to the cytoplasm. (credit: modification of work by “Biology Discussion
Forum”/YouTube)
Four unique examples of A-B toxins are the diphtheria, cholera, botulinum, and tetanus toxins. The diphtheria toxin is produced by
the gram-positive bacterium Corynebacterium diphtheriae, the causative agent of nasopharyngeal and cutaneous diphtheria. After
the A subunit of the diphtheria toxin separates and gains access to the cytoplasm, it facilitates the transfer of adenosine diphosphate
(ADP)-ribose onto an elongation-factor protein (EF-2) that is needed for protein synthesis. Hence, diphtheria toxin inhibits protein
synthesis in the host cell, ultimately killing the cell (Figure 12.3.6).

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 Figure 12.3.6 : The mechanism of the diphtheria toxin inhibiting protein synthesis. The A subunit inactivates elongation factor 2 by
transferring an ADP-ribose. This stops protein elongation, inhibiting protein synthesis and killing the cell.
Cholera toxin is an enterotoxin produced by the gram-negative bacterium Vibrio cholerae and is composed of one A subunit and
five B subunits. The mechanism of action of the cholera toxin is complex. The B subunits bind to receptors on the intestinal
epithelial cell of the small intestine. After gaining entry into the cytoplasm of the epithelial cell, the A subunit activates an
intracellular G protein. The activated G protein, in turn, leads to the activation of the enzyme adenyl cyclase, which begins to
produce an increase in the concentration of cyclic AMP (a secondary messenger molecule). The increased cAMP disrupts the
normal physiology of the intestinal epithelial cells and causes them to secrete excessive amounts of fluid and electrolytes into the
lumen of the intestinal tract, resulting in severe “rice-water stool” diarrhea characteristic of cholera.
Botulinum toxin (also known as botox) is a neurotoxin produced by the gram-positive bacterium Clostridium botulinum. It is the
most acutely toxic substance known to date. The toxin is composed of a light A subunit and heavy protein chain B subunit. The B
subunit binds to neurons to allow botulinum toxin to enter the neurons at the neuromuscular junction. The A subunit acts as a
protease, cleaving proteins involved in the neuron’s release of acetylcholine, a neurotransmitter molecule. Normally, neurons
release acetylcholine to induce muscle fiber contractions. The toxin’s ability to block acetylcholine release results in the inhibition
of muscle contractions, leading to muscle relaxation. This has the potential to stop breathing and cause death. Because of its action,
low concentrations of botox are used for cosmetic and medical procedures, including the removal of wrinkles and treatment of
overactive bladder.
Another neurotoxin is tetanus toxin, which is produced by the gram-positive bacterium Clostridium tetani. This toxin also has a
light A subunit and heavy protein chain B subunit. Unlike botulinum toxin, tetanus toxin binds to inhibitory interneurons, which
are responsible for release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA). Normally, these
neurotransmitters bind to neurons at the neuromuscular junction, resulting in the inhibition of acetylcholine release. Tetanus toxin
inhibits the release of glycine and GABA from the interneuron, resulting in permanent muscle contraction. The first symptom is
typically stiffness of the jaw (lockjaw). Violent muscle spasms in other parts of the body follow, typically culminating with
respiratory failure and death. Figure 12.3.7 shows the actions of both botulinum and tetanus toxins.

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 Figure 12.3.7 : Mechanisms of botulinum and tetanus toxins. (credit micrographs: modification of work by Centers for Disease
Control and Prevention)
Membrane-disrupting toxins affect cell membrane function either by forming pores or by disrupting the phospholipid bilayer in
host cell membranes. Two types of membrane-disrupting exotoxins are hemolysins and leukocidins, which form pores in cell
membranes, causing leakage of the cytoplasmic contents and cell lysis. These toxins were originally thought to target red blood
cells (erythrocytes) and white blood cells (leukocytes), respectively, but we now know they can affect other cells as well. The
gram-positive bacterium Streptococcus pyogenes produces streptolysins, water-soluble hemolysins that bind to the cholesterol
moieties in the host cell membrane to form a pore. The two types of streptolysins, O and S, are categorized by their ability to cause
hemolysis in erythrocytes in the absence or presence of oxygen. Streptolysin O is not active in the presence of oxygen, whereas
streptolysin S is active in the presence of oxygen. Other important pore-forming membrane-disrupting toxins include alpha toxin of
Staphylococcus aureus and pneumolysin of Streptococcus pneumoniae.
Bacterial phospholipases are membrane-disrupting toxins that degrade the phospholipid bilayer of cell membranes rather than
forming pores. We have already discussed the phospholipases associated with B. anthracis, L. pneumophila, and Rickettsia species
that enable these bacteria to effect the lysis of phagosomes. These same phospholipases are also hemolysins. Other phospholipases
that function as hemolysins include the alpha toxin of Clostridium perfringens, phospholipase C of P. aeruginosa, and beta toxin of
Staphylococcus aureus.
Some strains of S. aureus also produce a leukocidin called Panton-Valentine leukocidin (PVL). PVL consists of two subunits, S and
F. The S component acts like the B subunit of an A-B exotoxin in that it binds to glycolipids on the outer plasma membrane of
animal cells. The F-component acts like the A subunit of an A-B exotoxin and carries the enzymatic activity. The toxin inserts and
assembles into a pore in the membrane. Genes that encode PVL are more frequently present in S. aureus strains that cause skin
infections and pneumonia.1 PVL promotes skin infections by causing edema, erythema (reddening of the skin due to blood vessel
dilation), and skin necrosis. PVL has also been shown to cause necrotizing pneumonia. PVL promotes pro-inflammatory and

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cytotoxic effects on alveolar leukocytes. This results in the release of enzymes from the leukocytes, which, in turn, cause damage to
lung tissue.
The third class of exotoxins is the superantigens. These are exotoxins that trigger an excessive, nonspecific stimulation of immune
cells to secrete cytokines (chemical messengers). The excessive production of cytokines, often called a cytokine storm, elicits a
strong immune and inflammatory response that can cause life-threatening high fevers, low blood pressure, multi-organ failure,
shock, and death. The prototype superantigen is the toxic shock syndrome toxin of S. aureus. Most toxic shock syndrome cases are
associated with vaginal colonization by toxin-producing S. aureus in menstruating women; however, colonization of other body
sites can also occur. Some strains of Streptococcus pyogenes also produce superantigens; they are referred to as the streptococcal
mitogenic exotoxins and the streptococcal pyrogenic toxins.

Exercise 12.3.2
1. Describe how exoenzymes contribute to bacterial invasion.
2. Explain the difference between exotoxins and endotoxin.
3. Name the three classes of exotoxins.

Virulence Factors for Survival in the Host and Immune Evasion

Evading the immune system is also important to invasiveness. Bacteria use a variety of virulence factors to evade phagocytosis by
cells of the immune system. For example, many bacteria produce capsules, which are used in adhesion but also aid in immune
evasion by preventing ingestion by phagocytes. The composition of the capsule prevents immune cells from being able to adhere
and then phagocytose the cell. In addition, the capsule makes the bacterial cell much larger, making it harder for immune cells to
engulf the pathogen (Figure 12.3.8). A notable capsule-producing bacterium is the gram-positive pathogen Streptococcus
pneumoniae, which causes pneumococcal pneumonia, meningitis, septicemia, and other respiratory tract infections. Encapsulated
strains of S. pneumoniae are more virulent than nonencapsulated strains and are more likely to invade the bloodstream and cause
septicemia and meningitis.
Some pathogens can also produce proteases to protect themselves against phagocytosis. As will be discussed later, the human
immune system produces antibodies that bind to surface molecules found on specific bacteria (e.g., capsules, fimbriae, flagella,
LPS). This binding initiates phagocytosis and other mechanisms of antibacterial killing and clearance. Proteases combat antibody-
mediated killing and clearance by attacking and digesting the antibody molecules (Figure 12.3.8).
In addition to capsules and proteases, some bacterial pathogens produce other virulence factors that allow them to evade the
immune system. The fimbriae of certain species of Streptococcus contain M protein, which alters the surface of Streptococcus and
inhibits phagocytosis by blocking the binding of the complement molecules that assist phagocytes in ingesting bacterial pathogens.
The acid-fast bacterium Mycobacterium tuberculosis (the causative agent of tuberculosis) produces a waxy substance known as
mycolic acid in its cell envelope. When it is engulfed by phagocytes in the lung, the protective mycolic acid coat enables the
bacterium to resist some of the killing mechanisms within the phagolysosome.

Figure 12.3.8 : (a) A micrograph of capsules around bacterial cells. (b) Antibodies normally function by binding to antigens,
molecules on the surface of pathogenic bacteria. Phagocytes then bind to the antibody, initiating phagocytosis. (c) Some bacteria
also produce proteases, virulence factors that break down host antibodies to evade phagocytosis. (credit a: modification of work by
Centers for Disease Control and Prevention)

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Some bacteria produce virulence factors that promote infection by exploiting molecules naturally produced by the host. For
example, most strains of Staphylococcus aureus produce the exoenzyme coagulase, which exploits the natural mechanism of blood
clotting to evade the immune system. Normally, blood clotting is triggered in response to blood vessel damage; platelets begin to
plug the clot, and a cascade of reactions occurs in which fibrinogen, a soluble protein made by the liver, is cleaved into fibrin.
Fibrin is an insoluble, thread-like protein that binds to blood platelets, cross-links, and contracts to form a mesh of clumped
platelets and red blood cells. The resulting clot prevents further loss of blood from the damaged blood vessels. However, if bacteria
release coagulase into the bloodstream, the fibrinogen-to-fibrin cascade is triggered in the absence of blood vessel damage. The
resulting clot coats the bacteria in fibrin, protecting the bacteria from exposure to phagocytic immune cells circulating in the
bloodstream.
Whereas coagulase causes blood to clot, kinases have the opposite effect by triggering the conversion of plasminogen to plasmin,
which is involved in the digestion of fibrin clots. By digesting a clot, kinases allow pathogens trapped in the clot to escape and
spread, similar to the way that collagenase, hyaluronidase, and DNAse facilitate the spread of infection. Examples of kinases
include staphylokinases and streptokinases, produced by Staphylococcus aureusand Streptococcus pyogenes, respectively. It is
intriguing that S. aureus can produce both coagulase to promote clotting and staphylokinase to stimulate the digestion of clots. The
action of the coagulase provides an important protective barrier from the immune system, but when nutrient supplies are
diminished or other conditions signal a need for the pathogen to escape and spread, the production of staphylokinase can initiate
this process.
A final mechanism that pathogens can use to protect themselves against the immune system is called antigenic variation, which is
the alteration of surface proteins so that a pathogen is no longer recognized by the host’s immune system. For example, the
bacterium Borrelia burgdorferi, the causative agent of Lyme disease, contains a surface lipoprotein known as VlsE. Because of
genetic recombination during DNA replication and repair, this bacterial protein undergoes antigenic variation. Each time fever
occurs, the VlsE protein in B. burgdorferi can differ so much that antibodies against previous VlsE sequences are not effective. It is
believed that this variation in the VlsE contributes to the ability B. burgdorferi to cause chronic disease. Another important human
bacterial pathogen that uses antigenic variation to avoid the immune system is Neisseria gonorrhoeae, which causes the sexually
transmitted disease gonorrhea. This bacterium is well known for its ability to undergo antigenic variation of its type IV pili to avoid
immune defenses.

Exercise 12.3.3

1. Name at least two ways that a capsule provides protection from the immune system.
2. Besides capsules, name two other virulence factors used by bacteria to evade the immune system.

Eukaryote Virulence
Although fungi and parasites are important pathogens causing infectious diseases, their pathogenic mechanisms and virulence
factors are not as well characterized as those of bacteria. Despite the relative lack of detailed mechanisms, the stages of
pathogenesis and general mechanisms of virulence involved in disease production by these pathogens are similar to those of
bacteria.

Fungal Virulence
Pathogenic fungi can produce virulence factors that are similar to the bacterial virulence factors that have been discussed earlier in
this chapter. In this section, we will look at the virulence factors associated with species of Candida, Cryptococcus, Claviceps, and
Aspergillus.
Candida albicans is an opportunistic fungal pathogen and causative agent of oral thrush, vaginal yeast infections, and cutaneous
candidiasis. Candida produces adhesins (surface glycoproteins) that bind to the phospholipids of epithelial and endothelial cells. To
assist in spread and tissue invasion, Candida produces proteases and phospholipases (i.e., exoenzymes). One of these proteases
degrades keratin, a structural protein found on epithelial cells, enhancing the ability of the fungus to invade host tissue. In animal
studies, it has been shown that the addition of a protease inhibitor led to attenuation of Candida infection.2 Similarly, the
phospholipases can affect the integrity of host cell membranes to facilitate invasion.
The main virulence factor for Cryptococcus, a fungus that causes pneumonia and meningitis, is capsule production. The
polysaccharide glucuronoxylomannan is the principal constituent of the Cryptococcus capsule. Similar to encapsulated bacterial

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cells, encapsulated Cryptococcus cells are more resistant to phagocytosis than nonencapsulated Cryptococcus, which are effectively
phagocytosed and, therefore, less virulent.
Like some bacteria, many fungi produce exotoxins. Fungal toxins are called mycotoxins. Claviceps purpurea, a fungus that grows
on rye and related grains, produces a mycotoxin called ergot toxin, an alkaloid responsible for the disease known as ergotism.
There are two forms of ergotism: gangrenous and convulsive. In gangrenous ergotism, the ergot toxin causes vasoconstriction,
resulting in improper blood flow to the extremities, eventually leading to gangrene. A famous outbreak of gangrenous ergotism
occurred in Eastern Europe during the 5th century AD due to the consumption of rye contaminated with C. purpurea. In convulsive
ergotism, the toxin targets the central nervous system, causing mania and hallucinations.
The mycotoxin aflatoxin is a virulence factor produced by the fungus Aspergillus, an opportunistic pathogen that can enter the body
via contaminated food or by inhalation. Inhalation of the fungus can lead to the chronic pulmonary disease aspergillosis,
characterized by fever, bloody sputum, and/or asthma. Aflatoxin acts in the host as both a mutagen (a substance that causes
mutations in DNA) and a carcinogen (a substance involved in causing cancer), and has been associated with the development of
liver cancer. Aflatoxin has also been shown to cross the blood-placental barrier.3 A second mycotoxin produced by Aspergillus is
gliotoxin. This toxin promotes virulence by inducing host cells to self-destruct and by evading the host’s immune response by
inhibiting the function of phagocytic cells as well as the pro-inflammatory response. Like Candida, Aspergillus also produces
several proteases. One is elastase, which breaks down the protein elastin found in the connective tissue of the lung, leading to the
development of lung disease. Another is catalase, an enzyme that protects the fungus from hydrogen peroxide produced by the
immune system to destroy pathogens.

Exercise 12.3.4

1. List virulence factors common to bacteria and fungi.

2. What functions do mycotoxins perform to help fungi survive in the host?

Protozoan Virulence
Protozoan pathogens are unicellular eukaryotic parasites that have virulence factors and pathogenic mechanisms analogous to
prokaryotic and viral pathogens, including adhesins, toxins, antigenic variation, and the ability to survive inside phagocytic
vesicles.
Protozoans often have unique features for attaching to host cells. The protozoan Giardia lamblia, which causes the intestinal
disease giardiasis, uses a large adhesive disc composed of microtubules to attach to the intestinal mucosa. During adhesion, the
flagella of G. lamblia move in a manner that draws fluid out from under the disc, resulting in an area of lower pressure that
facilitates adhesion to epithelial cells. Giardia does not invade the intestinal cells but rather causes inflammation (possibly through
the release of cytopathic substances that cause damage to the cells) and shortens the intestinal villi, inhibiting absorption of
nutrients.
Some protozoans are capable of antigenic variation. The obligate intracellular pathogen Plasmodium falciparum (one of the
causative agents of malaria) resides inside red blood cells, where it produces an adhesin membrane protein known as PfEMP1. This
protein is expressed on the surface of the infected erythrocytes, causing blood cells to stick to each other and to the walls of blood
vessels. This process impedes blood flow, sometimes leading to organ failure, anemia, jaundice (yellowing of skin and sclera of the
eyes due to buildup of bilirubin from lysed red blood cells), and, subsequently, death. Although PfEMP1 can be recognized by the
host’s immune system, antigenic variations in the structure of the protein over time prevent it from being easily recognized and
eliminated. This allows malaria to persist as a chronic infection in many individuals.
The virulence factors of Trypanosoma brucei, the causative agent of African sleeping sickness, include the abilities to form
capsules and undergo antigenic variation. T. brucei evades phagocytosis by producing a dense glycoprotein coat that resembles a
bacterial capsule. Over time, host antibodies are produced that recognize this coat, but T. brucei is able to alter the structure of the
glycoprotein to evade recognition.

Exercise 12.3.5

Explain how antigenic variation by protozoan pathogens helps them survive in the host.

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Helminth Virulence
Helminths, or parasitic worms (in the animal kingdom), are multicellular eukaryotic parasites that depend heavily on virulence
factors that allow them to gain entry to host tissues. For example, the aquatic larval form of Schistosoma mansoni, which causes
schistosomiasis, penetrates intact skin with the aid of proteases that degrade skin proteins, including elastin.
To survive within the host long enough to perpetuate their often-complex life cycles, helminths need to evade the immune system.
Some helminths are so large that the immune system is ineffective against them. Others, such as adult roundworms (which cause
trichinosis, ascariasis, and other diseases), are protected by a tough outer cuticle.
Over the course of their life cycles, the surface characteristics of the parasites vary, which may help prevent an effective immune
response. Some helminths express polysaccharides called glycans on their external surface; because these glycans resemble
molecules produced by host cells, the immune system fails to recognize and attack the helminth as a foreign body. This “glycan
gimmickry,” as it has been called, serves as a protective cloak that allows the helminth to escape detection by the immune system.4
In addition to evading host defenses, helminths can actively suppress the immune system. S. mansoni, for example, degrades host
antibodies with proteases. Helminths produce many other substances that suppress elements of both innate nonspecific and
adaptive specific host defenses. They also release large amounts of material into the host that may locally overwhelm the immune
system or cause it to respond inappropriately.

Exercise 12.3.6

Describe how helminths avoid being destroyed by the host immune system.

Viral Virulence
Although viral pathogens are not similar to bacterial pathogens in terms of structure, some of the properties that contribute to their
virulence are similar. Viruses use adhesins to facilitate adhesion to host cells, and certain enveloped viruses rely on antigenic
variation to avoid the host immune defenses.

Viral Adhesins
One of the first steps in any viral infection is adhesion of the virus to specific receptors on the surface of cells. This process is
mediated by adhesins that are part of the viral capsid or membrane envelope. The interaction of viral adhesins with specific cell
receptors defines the tropism (preferential targeting) of viruses for specific cells, tissues, and organs in the body. The spike protein
hemagglutinin found on Influenzavirus is an example of a viral adhesin; it allows the virus to bind to the sialic acid on the
membrane of host respiratory and intestinal cells. Another viral adhesin is the glycoprotein gp20, found on HIV. For HIV to infect
cells of the immune system, it must interact with two receptors on the surface of cells. The first interaction involves binding
between gp120 and the CD4 cellular marker that is found on some essential immune system cells. However, before viral entry into
the cell can occur, a second interaction between gp120 and one of two chemokine receptors (CCR5 and CXCR4) must occur. Table
12.3.5 lists the adhesins for some common viral pathogens and the specific sites to which these adhesins allow viruses to attach.

Table 12.3.5 : Some Viral Adhesins and Their Host Attachment Sites
Pathogen Disease Adhesin Attachment Site

Sialic acid of respiratory and

Influenzavirus Influenza Hemagglutinin
intestinal cells
Heparan sulfate on mucosal
Herpes simplex virus I or II Oral herpes, genital herpes Glycoproteins gB, gC, gD
surfaces of the mouth and genitals
CD4 and CCR5 or CXCR4 of
Human immunodeficiency virus HIV/AIDS Glycoprotein gp120
immune system cells

Antigenic Variation in Viruses

Antigenic variation also occurs in certain types of enveloped viruses, including influenza viruses, which exhibit two forms of
antigenic variation: antigenic drift and antigenic shift (Figure 12.3.9). Antigenic drift is the result of point mutations causing slight
changes in the spike proteins hemagglutinin (H) and neuraminidase (N). On the other hand, antigenic shift is a major change in
spike proteins due to gene reassortment. This reassortment for antigenic shift occurs typically when two different influenza viruses
infect the same host.

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The rate of antigenic variation in influenza viruses is very high, making it difficult for the immune system to recognize the many
different strains of Influenzavirus. Although the body may develop immunity to one strain through natural exposure or vaccination,
antigenic variation results in the continual emergence of new strains that the immune system will not recognize. This is the main
reason that vaccines against Influenzavirus must be given annually. Each year’s influenza vaccine provides protection against the
most prevalent strains for that year, but new or different strains may be more prevalent the following year.

Figure 12.3.9 : Antigenic drift and antigenic shift in influenza viruses. (a) In antigenic drift, mutations in the genes for the surface
proteins neuraminidase and/or hemagglutinin result in small antigenic changes over time. (b) In antigenic shift, simultaneous
infection of a cell with two different influenza viruses results in mixing of the genes. The resultant virus possesses a mixture of the
proteins of the original viruses. Influenza pandemics can often be traced to antigenic shifts.
For another explanation of how antigenic shift and drift occur, watch this video.

Exercise 12.3.7

1. Describe the role of adhesins in viral tropism.

2. Explain the difference between antigenic drift and antigenic shift.

Key Concepts and Summary

Virulence factors contribute to a pathogen’s ability to cause disease.
Exoenzymes and toxins allow pathogens to invade host tissue and cause tissue damage. Exoenzymes are classified according to
the macromolecule they target and exotoxins are classified based on their mechanism of action. Bacterial toxins include
endotoxin and exotoxins.
Endotoxin is the lipid A component of the LPS of the gram-negative cell envelope. Exotoxins are proteins secreted mainly by
gram-positive bacteria, but also are secreted by gram-negative bacteria.
Bacterial pathogens may evade the host immune response by producing capsules to avoid phagocytosis, surviving the
intracellular environment of phagocytes, degrading antibodies, or through antigenic variation.
Fungal and parasitic pathogens use pathogenic mechanisms and virulence factors that are similar to those of bacterial pathogens
Fungi initiate infections through the interaction of adhesins with receptors on host cells. Some fungi produce toxins and
exoenzymes involved in disease production and capsules that provide protection of phagocytosis.
Protozoa adhere to target cells through complex mechanisms and can cause cellular damage through release of cytopathic
substances. Some protozoa avoid the immune system through antigenic variation and production of capsules.

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 Helminthic worms are able to avoid the immune system by coating their exteriors with glycan molecules that make them look
like host cells or by suppressing the immune system.
Viral pathogens use adhesins for initiating infections and antigenic variation to avoid immune defenses. Influenza viruses use
both antigenic drift and antigenic shift to avoid being recognized by the immune system.

Footnotes
1. V. Meka. “Panton-Valentine Leukocidin.” http://www.antimicrobe.org/h04c.file...L-S-aureus.asp
2. K. Fallon et al. “Role of Aspartic Proteases in Disseminated Candida albicans Infection in Mice.” Infection and Immunity 65
no. 2 (1997):551–556.
3. C.P. Wild et al. “In-utero exposure to aflatoxin in west Africa.” Lancet 337 no. 8757 (1991):1602.
4. I. van Die, R.D. Cummings. “Glycan Gimmickry by Parasitic Helminths: A Strategy for Modulating the Host Immune
Response?” Glycobiology 20 no. 1 (2010):2–12.

Contributor
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 12.3: Virulence Factors in Infection is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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12.4: How Diseases Spread
Learning Objectives
Describe the different types of disease reservoirs
Compare contact, vector, and vehicle modes of transmission
Identify important disease vectors
Compare different types of infectious diseases, including iatrogenic, nosocomial, and zoonotic diseases
Explain the prevalence of nosocomial infections

Understanding how infectious pathogens spread is critical to preventing infectious disease. Many pathogens require a living host to
survive, while others may be able to persist in a dormant state outside of a living host. But having infected one host, all pathogens
must also have a mechanism of transfer from one host to another or they will die when their host dies. Pathogens often have
elaborate adaptations to exploit host biology, behavior, and ecology to live in and move between hosts. Hosts have evolved
defenses against pathogens, but because their rates of evolution are typically slower than their pathogens (because their generation
times are longer), hosts are usually at an evolutionary disadvantage. This section will explore where pathogens survive—both
inside and outside hosts—and some of the many ways they move from one host to another.

Reservoirs and Carriers

For pathogens to persist over long periods of time they require reservoirs where they normally reside. Reservoirs can be living
organisms or nonliving sites. Nonliving reservoirs can include soil and water in the environment. These may naturally harbor the
organism because it may grow in that environment. These environments may also become contaminated with pathogens in human
feces, pathogens shed by intermediate hosts, or pathogens contained in the remains of intermediate hosts.
Pathogens may have mechanisms of dormancy or resilience that allow them to survive (but typically not to reproduce) for varying
periods of time in nonliving environments. For example, Clostridium tetani survives in the soil and in the presence of oxygen as a
resistant endospore. Although many viruses are soon destroyed once in contact with air, water, or other non-physiological
conditions, certain types are capable of persisting outside of a living cell for varying amounts of time. For example, a study that
looked at the ability of influenza viruses to infect a cell culture after varying amounts of time on a banknote showed survival times
from 48 hours to 17 days, depending on how they were deposited on the banknote.1 On the other hand, cold-causing rhinoviruses
are somewhat fragile, typically surviving less than a day outside of physiological fluids.
A human acting as a reservoir of a pathogen may or may not be capable of transmitting the pathogen, depending on the stage of
infection and the pathogen. To help prevent the spread of disease among school children, the CDC has developed guidelines based
on the risk of transmission during the course of the disease. For example, children with chickenpox are considered contagious for
five days from the start of the rash, whereas children with most gastrointestinal illnesses should be kept home for 24 hours after the
symptoms disappear.
An individual capable of transmitting a pathogen without displaying symptoms is referred to as a carrier. A passive carrier is
contaminated with the pathogen and can mechanically transmit it to another host; however, a passive carrier is not infected. For
example, a health-care professional who fails to wash his hands after seeing a patient harboring an infectious agent could become a
passive carrier, transmitting the pathogen to another patient who becomes infected.
By contrast, an active carrier is an infected individual who can transmit the disease to others. An active carrier may or may not
exhibit signs or symptoms of infection. For example, active carriers may transmit the disease during the incubation period (before
they show signs and symptoms) or the period of convalescence (after symptoms have subsided). Active carriers who do not present
signs or symptoms of disease despite infection are called asymptomatic carriers. Pathogens such as hepatitis B virus, herpes
simplex virus, and HIV are frequently transmitted by asymptomatic carriers. Mary Mallon, better known as Typhoid Mary, is a
famous historical example of an asymptomatic carrier. An Irish immigrant, Mallon worked as a cook for households in and around
New York City between 1900 and 1915. In each household, the residents developed typhoid fever (caused by Salmonella typhi) a
few weeks after Mallon started working. Later investigations determined that Mallon was responsible for at least 122 cases of
typhoid fever, five of which were fatal.2 See Eye on Ethics: Typhoid Mary for more about the Mallon case.
A pathogen may have more than one living reservoir. In zoonotic diseases, animals act as reservoirs of human disease and transmit
the infectious agent to humans through direct or indirect contact. In some cases, the disease also affects the animal, but in other

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cases the animal is asymptomatic.
In parasitic infections, the parasite’s preferred host is called the definitive host. In parasites with complex life cycles, the definitive
host is the host in which the parasite reaches sexual maturity. Some parasites may also infect one or more intermediate hosts in
which the parasite goes through several immature life cycle stages or reproduces asexually.
George Soper, the sanitary engineer who traced the typhoid outbreak to Mary Mallon, gives an account of his investigation, an
example of descriptive epidemiology, in “The Curious Career of Typhoid Mary.”

Exercise 12.4.1

1. List some nonliving reservoirs for pathogens.

2. Explain the difference between a passive carrier and an active carrier.

Transmission
Regardless of the reservoir, transmission must occur for an infection to spread. First, transmission from the reservoir to the
individual must occur. Then, the individual must transmit the infectious agent to other susceptible individuals, either directly or
indirectly. Pathogenic microorganisms employ diverse transmission mechanisms.

Contact Transmission
Contact transmission includes direct contact or indirect contact. Person-to-person transmission is a form of direct contact
transmission. Here the agent is transmitted by physical contact between two individuals (Figure 12.4.1) through actions such as
touching, kissing, sexual intercourse, or droplet sprays. Direct contact can be categorized as vertical, horizontal, or droplet
transmission. Vertical direct contact transmission occurs when pathogens are transmitted from mother to child during pregnancy,
birth, or breastfeeding. Other kinds of direct contact transmission are called horizontal direct contact transmission. Often, contact
between mucous membranes is required for entry of the pathogen into the new host, although skin-to-skin contact can lead to
mucous membrane contact if the new host subsequently touches a mucous membrane. Contact transmission may also be site-
specific; for example, some diseases can be transmitted by sexual contact but not by other forms of contact.
When an individual coughs or sneezes, small droplets of mucus that may contain pathogens are ejected. This leads to direct droplet
transmission, which refers to droplet transmission of a pathogen to a new host over distances of one meter or less. A wide variety
of diseases are transmitted by droplets, including influenza and many forms of pneumonia. Transmission over distances greater
than one meter is called airborne transmission.
Indirect contact transmission involves inanimate objects called fomites that become contaminated by pathogens from an infected
individual or reservoir (Figure 12.4.2). For example, an individual with the common cold may sneeze, causing droplets to land on a
fomite such as a tablecloth or carpet, or the individual may wipe her nose and then transfer mucus to a fomite such as a doorknob or
towel. Transmission occurs indirectly when a new susceptible host later touches the fomite and transfers the contaminated material
to a susceptible portal of entry. Fomites can also include objects used in clinical settings that are not properly sterilized, such as
syringes, needles, catheters, and surgical equipment. Pathogens transmitted indirectly via such fomites are a major cause of
healthcare-associated infections.

Figure 12.4.1 : Direct contact transmission of pathogens can occur through physical contact. Many pathogens require contact with a
mucous membrane to enter the body, but the host may transfer the pathogen from another point of contact (e.g., hand) to a mucous
membrane (e.g., mouth or eye). (credit left: modification of work by Lisa Doehnert)

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 Figure 12.4.2 : Fomites are nonliving objects that facilitate the indirect transmission of pathogens. Contaminated doorknobs,
towels, and syringes are all common examples of fomites. (credit left: modification of work by Kate Ter Haar; credit middle:
modification of work by Vernon Swanepoel; credit right: modification of work by “Zaldylmg”/Flickr)

Clinical Focus: Resolution

Based on Michael’s reported symptoms of stiff neck and hemiparesis, the physician suspects that the infection may have spread
to his nervous system. The physician decides to order a spinal tap to look for any bacteria that may have invaded the meninges
and cerebrospinal fluid (CSF), which would normally be sterile. To perform the spinal tap, Michael’s lower back is swabbed
with an iodine antiseptic and then covered with a sterile sheet. The needle is aseptically removed from the manufacturer’s
sealed plastic packaging by the clinician’s gloved hands. The needle is inserted and a small volume of fluid is drawn into an
attached sample tube. The tube is removed, capped and a prepared label with Michael’s data is affixed to it. This STAT (urgent
or immediate analysis required) specimen is divided into three separate sterile tubes, each with 1 mL of CSF. These tubes are
immediately taken to the hospital’s lab, where they are analyzed in the clinical chemistry, hematology, and microbiology
departments. The preliminary results from all three departments indicate there is a cerebrospinal infection occurring, with the
microbiology department reporting the presence of a gram-positive rod in Michael’s CSF.
These results confirm what his physician had suspected: Michael’s new symptoms are the result of meningitis, acute
inflammation of the membranes that protect the brain and spinal cord. Because meningitis can be life threatening and because
the first antibiotic therapy was not effective in preventing the spread of infection, Michael is prescribed an aggressive course of
two antibiotics, ampicillin and gentamicin, to be delivered intravenously. Michael remains in the hospital for several days for
supportive care and for observation. After a week, he is allowed to return home for bed rest and oral antibiotics. After 3 weeks
of this treatment, he makes a full recovery.

Vehicle Transmission
The term vehicle transmission refers to the transmission of pathogens through vehicles such as water, food, and air. Water
contamination through poor sanitation methods leads to waterborne transmission of disease. Waterborne disease remains a serious
problem in many regions throughout the world. The World Health Organization (WHO) estimates that contaminated drinking water
is responsible for more than 500,000 deaths each year.3 Similarly, food contaminated through poor handling or storage can lead to
foodborne transmission of disease (Figure 12.4.3).
Dust and fine particles known as aerosols, which can float in the air, can carry pathogens and facilitate the airborne transmission of
disease. For example, dust particles are the dominant mode of transmission of hantavirus to humans. Hantavirus is found in mouse
feces, urine, and saliva, but when these substances dry, they can disintegrate into fine particles that can become airborne when
disturbed; inhalation of these particles can lead to a serious and sometimes fatal respiratory infection.
Although droplet transmission over short distances is considered contact transmission as discussed above, longer distance
transmission of droplets through the air is considered vehicle transmission. Unlike larger particles that drop quickly out of the air
column, fine mucus droplets produced by coughs or sneezes can remain suspended for long periods of time, traveling considerable
distances. In certain conditions, droplets desiccate quickly to produce a droplet nucleus that is capable of transmitting pathogens;
air temperature and humidity can have an impact on effectiveness of airborne transmission.
Tuberculosis is often transmitted via airborne transmission when the causative agent, Mycobacterium tuberculosis, is released in
small particles with coughs. Because tuberculosis requires as few as 10 microbes to initiate a new infection, patients with
tuberculosis must be treated in rooms equipped with special ventilation, and anyone entering the room should wear a mask.

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 Figure 12.4.3 : Food is an important vehicle of transmission for pathogens, especially of the gastrointestinal and upper respiratory
systems. Notice the glass shield above the food trays, designed to prevent pathogens ejected in coughs and sneezes from entering
the food. (credit: Fort George G. Meade Public Affairs Office)

Vector Transmission
Diseases can also be transmitted by a mechanical or biological vector, an animal (typically an arthropod) that carries the disease
from one host to another. Mechanical transmission is facilitated by a mechanical vector, an animal that carries a pathogen from one
host to another without being infected itself. For example, a fly may land on fecal matter and later transmit bacteria from the feces
to food that it lands on; a human eating the food may then become infected by the bacteria, resulting in a case of diarrhea or
dysentery (Figure 12.4.4).
Biological transmission occurs when the pathogen reproduces within a biological vector that transmits the pathogen from one host
to another (Figure 12.4.4). Arthropods are the main vectors responsible for biological transmission (Figure 12.4.5). Most arthropod
vectors transmit the pathogen by biting the host, creating a wound that serves as a portal of entry. The pathogen may go through
part of its reproductive cycle in the gut or salivary glands of the arthropod to facilitate its transmission through the bite. For
example, hemipterans (called “kissing bugs” or “assassin bugs”) transmit Chagas disease to humans by defecating when they bite,
after which the human scratches or rubs the infected feces into a mucous membrane or break in the skin.
Biological insect vectors include mosquitoes, which transmit malaria and other diseases, and lice, which transmit typhus. Other
arthropod vectors can include arachnids, primarily ticks, which transmit Lyme disease and other diseases, and mites, which
transmit scrub typhus and rickettsial pox. Biological transmission, because it involves survival and reproduction within a
parasitized vector, complicates the biology of the pathogen and its transmission. There are also important non-arthropod vectors of
disease, including mammals and birds. Various species of mammals can transmit rabies to humans, usually by means of a bite that
transmits the rabies virus. Chickens and other domestic poultry can transmit avian influenza to humans through direct or indirect
contact with avian influenza virus A shed in the birds’ saliva, mucous, and feces.

Figure 12.4.4 : (a) A mechanical vector carries a pathogen on its body from one host to another, not as an infection. (b) A biological
vector carries a pathogen from one host to another after becoming infected itself.

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Figure 12.4.5 : (credit “Black fly”, “Tick”, “Tsetse fly”: modification of work by USDA; credit: “Flea”: modification of work by
Centers for Disease Control and Prevention; credit: “Louse”, “Mosquito”, “Sand fly”: modification of work by James Gathany,
Centers for Disease Control and Prevention; credit “Kissing bug”: modification of work by Glenn Seplak; credit “Mite”:
modification of work by Michael Wunderli)

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Exercise 12.4.2

1. Describe how diseases can be transmitted through the air.

2. Explain the difference between a mechanical vector and a biological vector.

Using GMOs to Stop the Spread of Zika

In 2016, an epidemic of the Zika virus was linked to a high incidence of birth defects in South America and Central America.
As winter turned to spring in the northern hemisphere, health officials correctly predicted the virus would spread to North
America, coinciding with the breeding season of its major vector, the Aedes aegypti mosquito.
The range of the A. aegypti mosquito extends well into the southern United States (Figure 12.4.6). Because these same
mosquitoes serve as vectors for other problematic diseases (dengue fever, yellow fever, and others), various methods of
mosquito control have been proposed as solutions. Chemical pesticides have been used effectively in the past, and are likely to
be used again; but because chemical pesticides can have negative impacts on the environment, some scientists have proposed
an alternative that involves genetically engineering A. aegypti so that it cannot reproduce. This method, however, has been the
subject of some controversy.
One method that has worked in the past to control pests, with little apparent downside, has been sterile male introductions. This
method controlled the screw-worm fly pest in the southwest United States and fruit fly pests of fruit crops. In this method,
males of the target species are reared in the lab, sterilized with radiation, and released into the environment where they mate
with wild females, who subsequently bear no live offspring. Repeated releases shrink the pest population.
A similar method, taking advantage of recombinant DNA technology, 4 introduces a dominant lethal allele into male
mosquitoes that is suppressed in the presence of tetracycline (an antibiotic) during laboratory rearing. The males are released
into the environment and mate with female mosquitoes. Unlike the sterile male method, these matings produce offspring, but
they die as larvae from the lethal gene in the absence of tetracycline in the environment. As of 2016, this method has yet to be
implemented in the United States, but a UK company tested the method in Piracicaba, Brazil, and found an 82% reduction in
wild A. aegypti larvae and a 91% reduction in dengue cases in the treated area. 5 In August 2016, amid news of Zika
infections in several Florida communities, the FDA gave the UK company permission to test this same mosquito control
method in Key West, Florida, pending compliance with local and state regulations and a referendum in the affected
communities.
The use of genetically modified organisms (GMOs) to control a disease vector has its advocates as well as its opponents. In
theory, the system could be used to drive the A. aegypti mosquito extinct—a noble goal according to some, given the damage
they do to human populations. 6 But opponents of the idea are concerned that the gene could escape the species boundary of
A. aegypti and cause problems in other species, leading to unforeseen ecological consequences. Opponents are also wary of the
program because it is being administered by a for-profit corporation, creating the potential for conflicts of interest that would
have to be tightly regulated; and it is not clear how any unintended consequences of the program could be reversed.
There are other epidemiological considerations as well. Aedes aegypti is apparently not the only vector for the Zika virus.
Aedes albopictus, the Asian tiger mosquito, is also a vector for the Zika virus. 7 A. albopictus is now widespread around the
planet including much of the United States (Figure 12.4.6). Many other mosquitoes have been found to harbor Zika virus,
though their capacity to act as vectors is unknown.8 Genetically modified strains of A. aegypti will not control the other species
of vectors. Finally, the Zika virus can apparently be transmitted sexually between human hosts, from mother to child, and
possibly through blood transfusion. All of these factors must be considered in any approach to controlling the spread of the
virus.
Clearly there are risks and unknowns involved in conducting an open-environment experiment of an as-yet poorly understood
technology. But allowing the Zika virus to spread unchecked is also risky. Does the threat of a Zika epidemic justify the
ecological risk of genetically engineering mosquitos? Are current methods of mosquito control sufficiently ineffective or
harmful that we need to try untested alternatives? These are the questions being put to public health officials now.

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 Figure 12.4.6 : The Zika virus is an enveloped virus transmitted by mosquitoes, especially Aedes aegypti. The range of this
mosquito includes much of the United States, from the Southwest and Southeast to as far north as the Mid-Atlantic. The range
of A. albopictus, another vector, extends even farther north to New England and parts of the Midwest. (credit micrograph:
modification of work by Cynthia Goldsmith, Centers for Disease Control and Prevention; credit photo: modification of work
by James Gathany, Centers for Disease Control and Prevention; credit map: modification of work by Centers for Disease
Control and Prevention)

Quarantining
Individuals suspected or known to have been exposed to certain contagious pathogens may be quarantined, or isolated to prevent
transmission of the disease to others. Hospitals and other health-care facilities generally set up special wards to isolate patients with
particularly hazardous diseases such as tuberculosis or Ebola (Figure 12.4.7). Depending on the setting, these wards may be
equipped with special air-handling methods, and personnel may implement special protocols to limit the risk of transmission, such
as personal protective equipment or the use of chemical disinfectant sprays upon entry and exit of medical personnel.
The duration of the quarantine depends on factors such as the incubation period of the disease and the evidence suggestive of an
infection. The patient may be released if signs and symptoms fail to materialize when expected or if preventive treatment can be
administered in order to limit the risk of transmission. If the infection is confirmed, the patient may be compelled to remain in
isolation until the disease is no longer considered contagious.
In the United States, public health authorities may only quarantine patients for certain diseases, such as cholera, diphtheria,
infectious tuberculosis, and strains of influenza capable of causing a pandemic. Individuals entering the United States or moving
between states may be quarantined by the CDC if they are suspected of having been exposed to one of these diseases. Although the
CDC routinely monitors entry points to the United States for crew or passengers displaying illness, quarantine is rarely
implemented.

Figure 12.4.7 : (a) The Aeromedical Biological Containment System (ABCS) is a module designed by the CDC and Department of
Defense specifically for transporting highly contagious patients by air. (b) An isolation ward for Ebola patients in Lagos, Nigeria.
(credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by CDC Global)

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Classifications of Disease
The World Health Organization’s (WHO) International Classification of Diseases (ICD) is used in clinical fields to classify diseases
and monitor morbidity (the number of cases of a disease) and mortality (the number of deaths due to a disease). In this section, we
will introduce terminology used by the ICD (and in health-care professions in general) to describe and categorize various types of
disease.
An infectious disease is any disease caused by the direct effect of a pathogen. A pathogen may be cellular (bacteria, parasites, and
fungi) or acellular (viruses, viroids, and prions). Some infectious diseases are also communicable, meaning they are capable of
being spread from person to person through either direct or indirect mechanisms. Some infectious communicable diseases are also
considered contagious diseases, meaning they are easily spread from person to person. Not all contagious diseases are equally so;
the degree to which a disease is contagious usually depends on how the pathogen is transmitted. For example, measles is a highly
contagious viral disease that can be transmitted when an infected person coughs or sneezes and an uninfected person breathes in
droplets containing the virus. Gonorrhea is not as contagious as measles because transmission of the pathogen (Neisseria
gonorrhoeae) requires close intimate contact (usually sexual) between an infected person and an uninfected person.
Diseases that are contracted as the result of a medical procedure are known as iatrogenic diseases. Iatrogenic diseases can occur
after procedures involving wound treatments, catheterization, or surgery if the wound or surgical site becomes contaminated. For
example, an individual treated for a skin wound might acquire necrotizing fasciitis (an aggressive, “flesh-eating” disease) if
bandages or other dressings became contaminated by Clostridium perfringens or one of several other bacteria that can cause this
condition.
Diseases acquired in hospital settings are known as nosocomial diseases. Several factors contribute to the prevalence and severity
of nosocomial diseases. First, sick patients bring numerous pathogens into hospitals, and some of these pathogens can be
transmitted easily via improperly sterilized medical equipment, bed sheets, call buttons, door handles, or by clinicians, nurses, or
therapists who do not wash their hands before touching a patient. Second, many hospital patients have weakened immune systems,
making them more susceptible to infections. Compounding this, the prevalence of antibiotics in hospital settings can select for
drug-resistant bacteria that can cause very serious infections that are difficult to treat.
Certain infectious diseases are not transmitted between humans directly but can be transmitted from animals to humans. Such a
disease is called zoonotic disease (or zoonosis). According to WHO, a zoonosis is a disease that occurs when a pathogen is
transferred from a vertebrate animal to a human; however, sometimes the term is defined more broadly to include diseases
transmitted by all animals (including invertebrates). For example, rabies is a viral zoonotic disease spread from animals to humans
through bites and contact with infected saliva. Many other zoonotic diseases rely on insects or other arthropods for transmission.
Examples include yellow fever (transmitted through the bite of mosquitoes infected with yellow fever virus) and Rocky Mountain
spotted fever (transmitted through the bite of ticks infected with Rickettsia rickettsii).
In contrast to communicable infectious diseases, a noncommunicable infectious disease is not spread from one person to another.
One example is tetanus, caused by Clostridium tetani, a bacterium that produces endospores that can survive in the soil for many
years. This disease is typically only transmitted through contact with a skin wound; it cannot be passed from an infected person to
another person. Similarly, Legionnaires disease is caused by Legionella pneumophila, a bacterium that lives within amoebae in
moist locations like water-cooling towers. An individual may contract Legionnaires disease via contact with the contaminated
water, but once infected, the individual cannot pass the pathogen to other individuals.
In addition to the wide variety of noncommunicable infectious diseases, noninfectious diseases (those not caused by pathogens) are
an important cause of morbidity and mortality worldwide. Noninfectious diseases can be caused by a wide variety factors,
including genetics, the environment, or immune system dysfunction, to name a few. For example, sickle cell anemia is an inherited
disease caused by a genetic mutation that can be passed from parent to offspring (Figure 12.4.8). Other types of noninfectious
diseases are listed in Table 12.4.2.
Table 12.4.2 : Types of Noninfectious Diseases
Type Definition Example

Inherited A genetic disease Sickle cell anemia

Congenital Disease that is present at or before birth Down syndrome

Parkinson disease (affecting central nervous

Degenerative Progressive, irreversible loss of function
system)

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 Type Definition Example

Impaired body function due to lack of

Nutritional deficiency Scurvy (vitamin C deficiency)
nutrients

Hypothyroidism – thyroid does not produce

Disease involving malfunction of glands that
Endocrine enough thyroid hormone, which is important
release hormones to regulate body functions
for metabolism

Neoplastic Abnormal growth (benign or malignant) Some forms of cancer

Idiopathic juxtafoveal retinal telangiectasia

Idiopathic Disease for which the cause is unknown (dilated, twisted blood vessels in the retina of
the eye)

Figure 12.4.8 : Blood smears showing two diseases of the blood. (a) Malaria is an infectious, zoonotic disease caused by the
protozoan pathogen Plasmodium falciparum (shown here) and several other species of the genus Plasmodium. It is transmitted by
mosquitoes to humans. (b) Sickle cell disease is a noninfectious genetic disorder that results in abnormally shaped red blood cells,
which can stick together and obstruct the flow of blood through the circulatory system. It is not caused by a pathogen, but rather a
genetic mutation. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by
Ed Uthman)
Lists of common infectious diseases can be found at the following Centers for Disease Control and Prevention (CDC), World
Health Organization (WHO), and International Classification of Diseases websites.

Exercise 12.4.3

1. Describe how a disease can be infectious but not contagious.

2. Explain the difference between iatrogenic disease and nosocomial disease.

Healthcare-Associated (Nosocomial) Infections

Hospitals, retirement homes, and prisons attract the attention of epidemiologists because these settings are associated with
increased incidence of certain diseases. Higher rates of transmission may be caused by characteristics of the environment itself,
characteristics of the population, or both. Consequently, special efforts must be taken to limit the risks of infection in these settings.
Infections acquired in health-care facilities, including hospitals, are called nosocomial infections or healthcare-associated infections
(HAI). HAIs are often connected with surgery or other invasive procedures that provide the pathogen with access to the portal of
infection. For an infection to be classified as an HAI, the patient must have been admitted to the health-care facility for a reason
other than the infection. In these settings, patients suffering from primary disease are often afflicted with compromised immunity
and are more susceptible to secondary infection and opportunistic pathogens.
In 2011, more than 720,000 HAIs occurred in hospitals in the United States, according to the CDC. About 22% of these HAIs
occurred at a surgical site, and cases of pneumonia accounted for another 22%; urinary tract infectionsaccounted for an additional
13%, and primary bloodstream infections 10%.9 Such HAIs often occur when pathogens are introduced to patients’ bodies through

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contaminated surgical or medical equipment, such as catheters and respiratory ventilators. Health-care facilities seek to limit
nosocomial infections through training and hygiene protocols such as those described in previous chapters.

Exercise 12.4.4

Give some reasons why HAIs occur.

Key Concepts and Summary

Reservoirs of human disease can include the human and animal populations, soil, water, and inanimate objects or materials.
Contact transmission can be direct or indirect through physical contact with either an infected host (direct) or contact with a
fomite that an infected host has made contact with previously (indirect).
Vector transmission occurs when a living organism carries an infectious agent on its body (mechanical) or as an infection host
itself (biological), to a new host.
Vehicle transmission occurs when a substance, such as soil, water, or air, carries an infectious agent to a new host.
Healthcare-associated infections (HAI), or nosocomial infections, are acquired in a clinical setting. Transmission is
facilitated by medical interventions and the high concentration of susceptible, immunocompromised individuals in clinical
settings.
Footnotes
1. 1 Yves Thomas, Guido Vogel, Werner Wunderli, Patricia Suter, Mark Witschi, Daniel Koch, Caroline Tapparel, and Laurent
Kaiser. “Survival of Influenza Virus on Banknotes.” Applied and Environmental Microbiology 74, no. 10 (2008): 3002–3007.
2. 2 Filio Marineli, Gregory Tsoucalas, Marianna Karamanou, and George Androutsos. “Mary Mallon (1869–1938) and the
History of Typhoid Fever.” Annals of Gastroenterology 26 (2013): 132–134. www.ncbi.nlm.nih.gov/pmc/arti...rol-26-132.pdf.
3. 3 World Health Organization. Fact sheet No. 391—Drinking Water. June 2005. www.who.int/mediacentre/factsheets/fs391/en.
4. 4 Blandine Massonnet-Bruneel, Nicole Corre-Catelin, Renaud Lacroix, Rosemary S. Lees, Kim Phuc Hoang, Derric Nimmo,
Luke Alphey, and Paul Reiter. “Fitness of Transgenic Mosquito Aedes aegypti Males Carrying a Dominant Lethal Genetic
System.” PLOS ONE 8, no. 5 (2013): e62711.
5. 5 Richard Levine. “Cases of Dengue Drop 91 Percent Due to Genetically Modified Mosquitoes.” Entomology Today.
entomologytoday.org/2016/07/...ied-mosquitoes.
6. 6 Olivia Judson. “A Bug’s Death.” The New York Times, September 25, 2003. www.nytimes.com/2003/09/25/op...g-s-
death.html.
7. Gilda Grard, Mélanie Caron, Illich Manfred Mombo, Dieudonné Nkoghe, Statiana Mboui Ondo, Davy Jiolle, Didier Fontenille,
Christophe Paupy, and Eric Maurice Leroy. “Zika Virus in Gabon (Central Africa)–2007: A New Threat from Aedes
albopictus?” PLOS Neglected Tropical Diseases 8, no. 2 (2014): e2681.
8. Constância F.J. Ayres. “Identification of Zika Virus Vectors and Implications for Control.” The Lancet Infectious Diseases 16,
no. 3 (2016): 278–279.
9. Centers for Disease Control and Prevention. “HAI Data and Statistics.” 2016. http://www.cdc.gov/hai/surveillance. Accessed
Jan 2, 2016.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 12.4: How Diseases Spread is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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12.5: The Language of Epidemiologists
Learning Objectives
Explain the difference between prevalence and incidence of disease
Distinguish the characteristics of sporadic, endemic, epidemic, and pandemic diseases
Explain the use of Koch’s postulates and their modifications to determine the etiology of disease
Summarize Koch’s postulates and molecular Koch’s postulates, respectively, and explain their significance and limitations
Explain the relationship between epidemiology and public health
Describe the entities involved in international public health and their activities
Identify and differentiate between emerging and reemerging infectious diseases

The field of epidemiology concerns the geographical distribution and timing of infectious disease occurrences and how they are
transmitted and maintained in nature, with the goal of recognizing and controlling outbreaks. The science of epidemiology includes
etiology (the study of the causes of disease) and investigation of disease transmission (mechanisms by which a disease is spread).

Analyzing Disease in a Population

Epidemiological analyses are always carried out with reference to a population, which is the group of individuals that are at risk for
the disease or condition. The population can be defined geographically, but if only a portion of the individuals in that area are
susceptible, additional criteria may be required. Susceptible individuals may be defined by particular behaviors, such as
intravenous drug use, owning particular pets, or membership in an institution, such as a college. Being able to define the population
is important because most measures of interest in epidemiology are made with reference to the size of the population.
The state of being diseased is called morbidity. Morbidity in a population can be expressed in a few different ways. Morbidity or
total morbidity is expressed in numbers of individuals without reference to the size of the population. The morbidity rate can be
expressed as the number of diseased individuals out of a standard number of individuals in the population, such as 100,000, or as a
percent of the population.
There are two aspects of morbidity that are relevant to an epidemiologist: a disease’s prevalence and its incidence. Prevalence is the
number, or proportion, of individuals with a particular illness in a given population at a point in time. For example, the Centers for
Disease Control and Prevention (CDC) estimated that in 2012, there were about 1.2 million people 13 years and older with an
active human immunodeficiency virus (HIV) infection. Expressed as a proportion, or rate, this is a prevalence of 467 infected
persons per 100,000 in the population.1 On the other hand, incidence is the number or proportion of new cases in a period of time.
For the same year and population, the CDC estimates that there were 43,165 newly diagnosed cases of HIV infection, which is an
incidence of 13.7 new cases per 100,000 in the population.2 The relationship between incidence and prevalence can be seen in
Figure 12.5.1. For a chronic disease like HIV infection, prevalence will generally be higher than incidence because it represents the
cumulative number of new cases over many years minus the number of cases that are no longer active (e.g., because the patient
died or was cured).
In addition to morbidity rates, the incidence and prevalence of mortality (death) may also be reported. A mortality rate can be
expressed as the percentage of the population that has died from a disease or as the number of deaths per 100,000 persons (or other
suitable standard number).

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 Figure 12.5.1 : This graph compares the incidence of HIV (the number of new cases reported each year) with the prevalence (the
total number of cases each year). Prevalence and incidence can also be expressed as a rate or proportion for a given population.

Exercise 12.5.1

1. Explain the difference between incidence and prevalence.

2. Describe how morbidity and mortality rates are expressed.

Patterns of Incidence
Diseases that are seen only occasionally, and usually without geographic concentration, are called sporadic diseases. Examples of
sporadic diseases include tetanus, rabies, and plague. In the United States, Clostridium tetani, the bacterium that causes tetanus, is
ubiquitous in the soil environment, but incidences of infection occur only rarely and in scattered locations because most individuals
are vaccinated, clean wounds appropriately, or are only rarely in a situation that would cause infection.3 Likewise in the United
States there are a few scattered cases of plague each year, usually contracted from rodents in rural areas in the western states.4
Diseases that are constantly present (often at a low level) in a population within a particular geographic region are called endemic
diseases. For example, malaria is endemic to some regions of Brazil, but is not endemic to the United States.
Diseases for which a larger than expected number of cases occurs in a short time within a geographic region are called epidemic
diseases. Influenza is a good example of a commonly epidemic disease. Incidence patterns of influenza tend to rise each winter in
the northern hemisphere. These seasonal increases are expected, so it would not be accurate to say that influenza is epidemic every
winter; however, some winters have an usually large number of seasonal influenza cases in particular regions, and such situations
would qualify as epidemics (Figure 12.5.2 and Figure 12.5.3).
An epidemic disease signals the breakdown of an equilibrium in disease frequency, often resulting from some change in
environmental conditions or in the population. In the case of influenza, the disruption can be due to antigenic shift or drift, which
allows influenza virus strains to circumvent the acquired immunity of their human hosts.
An epidemic that occurs on a worldwide scale is called a pandemic disease. For example, HIV/AIDS is a pandemic disease and
novel influenza virus strains often become pandemic.

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 Figure 12.5.2 : The 2007–2008 influenza season in the United States saw much higher than normal numbers of visits to emergency
departments for influenza-like symptoms as compared to the previous and the following years. (credit: modification of work by
Centers for Disease Control and Prevention)

Figure 12.5.3 : The seasonal epidemic threshold (blue curve) is set by the CDC-based data from the previous five years. When
actual mortality rates exceed this threshold, a disease is considered to be epidemic. As this graph shows, pneumonia- and influenza-
related mortality saw pronounced epidemics during the winters of 2003–2004, 2005, and 2008. (credit: modification of work by
Centers for Disease Control and Prevention)

Exercise 12.5.2

1. Explain the difference between sporadic and endemic disease.

2. Explain the difference between endemic and epidemic disease.

Etiology
When studying an epidemic, an epidemiologist’s first task is to determinate the cause of the disease, called the etiologic agent or
causative agent. Connecting a disease to a specific pathogen can be challenging because of the extra effort typically required to
demonstrate direct causation as opposed to a simple association. It is not enough to observe an association between a disease and a
suspected pathogen; controlled experiments are needed to eliminate other possible causes. In addition, pathogens are typically

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difficult to detect when there is no immediate clue as to what is causing the outbreak. Signs and symptoms of disease are also
commonly nonspecific, meaning that many different agents can give rise to the same set of signs and symptoms. This complicates
diagnosis even when a causative agent is familiar to scientists.
Robert Koch was the first scientist to specifically demonstrate the causative agent of a disease (anthrax) in the late 1800s. Koch
developed four criteria, now known as Koch’s postulates, which had to be met in order to positively link a disease with a
pathogenic microbe. Without Koch’s postulates, the Golden Age of Microbiology would not have occurred. Between 1876 and
1905, many common diseases were linked with their etiologic agents, including cholera, diphtheria, gonorrhea, meningitis, plague,
syphilis, tetanus, and tuberculosis. Today, we use the molecular Koch’s postulates, a variation of Koch’s original postulates that can
be used to establish a link between the disease state and virulence traits unique to a pathogenic strain of a microbe.

Koch’s Postulates
In 1884, Koch published four postulates that summarized his method for determining whether a particular microorganism was the
cause of a particular disease. Each of Koch’s postulates represents a criterion that must be met before a disease can be positively
linked with a pathogen. In order to determine whether the criteria are met, tests are performed on laboratory animals and cultures
from healthy and diseased animals are compared (Figure 12.5.4).

Koch’s Postulates
1. The suspected pathogen must be found in every case of disease and not be found in healthy individuals.
2. The suspected pathogen can be isolated and grown in pure culture.
3. A healthy test subject infected with the suspected pathogen must develop the same signs and symptoms of disease as seen
in postulate
4. The pathogen must be re-isolated from the new host and must be identical to the pathogen from postulate 2.

Figure 12.5.4 : The steps for confirming that a pathogen is the cause of a particular disease using Koch’s postulates.
In many ways, Koch’s postulates are still central to our current understanding of the causes of disease. However, advances in
microbiology have revealed some important limitations in Koch’s criteria. Koch made several assumptions that we now know are
untrue in many cases. The first relates to postulate 1, which assumes that pathogens are only found in diseased, not healthy,

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individuals. This is not true for many pathogens. For example, H. pylori, described earlier in this chapter as a pathogen causing
chronic gastritis, is also part of the normal microbiota of the stomach in many healthy humans who never develop gastritis. It is
estimated that upwards of 50% of the human population acquires H. pylori early in life, with most maintaining it as part of the
normal microbiota for the rest of their life without ever developing disease.
Koch’s second faulty assumption was that all healthy test subjects are equally susceptible to disease. We now know that individuals
are not equally susceptible to disease. Individuals are unique in terms of their microbiota and the state of their immune system at
any given time. The makeup of the resident microbiota can influence an individual’s susceptibility to an infection. Members of the
normal microbiota play an important role in immunity by inhibiting the growth of transient pathogens. In some cases, the
microbiota may prevent a pathogen from establishing an infection; in others, it may not prevent an infection altogether but may
influence the severity or type of signs and symptoms. As a result, two individuals with the same disease may not always present
with the same signs and symptoms. In addition, some individuals have stronger immune systems than others. Individuals with
immune systems weakened by age or an unrelated illness are much more susceptible to certain infections than individuals with
strong immune systems.
Koch also assumed that all pathogens are microorganisms that can be grown in pure culture (postulate 2) and that animals could
serve as reliable models for human disease. However, we now know that not all pathogens can be grown in pure culture, and many
human diseases cannot be reliably replicated in animal hosts. Viruses and certain bacteria, including Rickettsia and Chlamydia, are
obligate intracellular pathogens that can grow only when inside a host cell. If a microbe cannot be cultured, a researcher cannot
move past postulate 2. Likewise, without a suitable nonhuman host, a researcher cannot evaluate postulate 2 without deliberately
infecting humans, which presents obvious ethical concerns. AIDS is an example of such a disease because the human
immunodeficiency virus (HIV) only causes disease in humans.

Exercise 12.5.3

Briefly summarize the limitations of Koch’s postulates.

Molecular Koch’s Postulates

In 1988, Stanley Falkow (1934–) proposed a revised form of Koch’s postulates known as molecular Koch’s postulates. These are
listed in the left column of Table 12.5.1. The premise for molecular Koch’s postulates is not in the ability to isolate a particular
pathogen but rather to identify a gene that may cause the organism to be pathogenic.
Falkow’s modifications to Koch’s original postulates explain not only infections caused by intracellular pathogens but also the
existence of pathogenic strains of organisms that are usually nonpathogenic. For example, the predominant form of the bacterium
Escherichia coli is a member of the normal microbiota of the human intestine and is generally considered harmless. However, there
are pathogenic strains of E. coli such as enterotoxigenic E. coli (ETEC) and enterohemorrhagic E. coli (O157:H7) (EHEC). We
now know ETEC and EHEC exist because of the acquisition of new genes by the once-harmless E. coli, which, in the form of these
pathogenic strains, is now capable of producing toxins and causing illness. The pathogenic forms resulted from minor genetic
changes. The right-side column of Table 12.5.1 illustrates how molecular Koch’s postulates can be applied to identify EHEC as a
pathogenic bacterium.
Table 12.5.1 : Molecular Koch’s Postulates Applied to EHEC
Molecular Koch’s Postulates Application to EHEC

(1) The phenotype (sign or symptom of disease) should be associated EHEC causes intestinal inflammation and diarrhea, whereas
only with pathogenic strains of a species. nonpathogenic strains of E. coli do not.

One of the genes in EHEC encodes for Shiga toxin, a bacterial toxin
(2) Inactivation of the suspected gene(s) associated with pathogenicity
(poison) that inhibits protein synthesis. Inactivating this gene reduces
should result in a measurable loss of pathogenicity.
the bacteria’s ability to cause disease.
By adding the gene that encodes the toxin back into the genome (e.g.,
(3) Reversion of the inactive gene should restore the disease phenotype.
with a phage or plasmid), EHEC’s ability to cause disease is restored.

As with Koch’s original postulates, the molecular Koch’s postulates have limitations. For example, genetic manipulation of some
pathogens is not possible using current methods of molecular genetics. In a similar vein, some diseases do not have suitable animal
models, which limits the utility of both the original and molecular postulates.

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 Exercise 12.5.4

Explain the differences between Koch’s original postulates and the molecular Koch’s postulates.
List some challenges to determining the causative agent of a disease outbreak.

The Role of Public Health Organizations

The main national public health agency in the United States is the Centers for Disease Control and Prevention (CDC), an agency of
the Department of Health and Human Services. The CDC is charged with protecting the public from disease and injury. One way
that the CDC carries out this mission is by overseeing the National Notifiable Disease Surveillance System (NNDSS) in
cooperation with regional, state, and territorial public health departments. The NNDSS monitors diseases considered to be of public
health importance on a national scale. Such diseases are called notifiable diseases or reportable diseases because all cases must be
reported to the CDC. A physician treating a patient with a notifiable disease is legally required to submit a report on the case.
Notifiable diseases include HIV infection, measles, West Nile virus infections, and many others. Some states have their own lists of
notifiable diseases that include diseases beyond those on the CDC’s list.
Notifiable diseases are tracked by epidemiological studies and the data is used to inform health-care providers and the public about
possible risks. The CDC publishes the Morbidity and Mortality Weekly Report (MMWR), which provides physicians and health-
care workers with updates on public health issues and the latest data pertaining to notifiable diseases. Table 12.5.2 is an example of
the kind of data contained in the MMWR.
Table 12.5.2: Incidence of Four Notifiable Diseases in the United States, Week Ending January 2, 2016

Median of Previous 52 Maximum of Previous 52

Disease Current Week (Jan 2, 2016) Cumulative Cases 2015
Weeks Weeks

Campylobacteriosis 406 869 1,385 46,618

Chlamydia trachomatis
11,024 28,562 31,089 1,425,303
infection

Giardiasis 115 230 335 11,870

Gonorrhea 3,207 7,155 8,283 369,926

The current Morbidity and Mortality Weekly Report is available online.

Exercise 12.5.5

Describe how health agencies obtain data about the incidence of diseases of public health importance.

The World Health Organization (WHO)

A large number of international programs and agencies are involved in efforts to promote global public health. Among their goals
are developing infrastructure in health care, public sanitation, and public health capacity; monitoring infectious disease occurrences
around the world; coordinating communications between national public health agencies in various countries; and coordinating
international responses to major health crises. In large part, these international efforts are necessary because disease-causing
microorganisms know no national boundaries.
International public health issues are coordinated by the World Health Organization (WHO), an agency of the United Nations. Of
its roughly $4 billion budget for 2015–165, about $1 billion was funded by member states and the remaining $3 billion by voluntary
contributions. In addition to monitoring and reporting on infectious disease, WHO also develops and implements strategies for their
control and prevention. WHO has had a number of successful international public health campaigns. For example, its vaccination
program against smallpox, begun in the mid-1960s, resulted in the global eradication of the disease by 1980. WHO continues to be
involved in infectious disease control, primarily in the developing world, with programs targeting malaria, HIV/AIDS, and
tuberculosis, among others. It also runs programs to reduce illness and mortality that occur as a result of violence, accidents,
lifestyle-associated illnesses such as diabetes, and poor health-care infrastructure.
WHO maintains a global alert and response system that coordinates information from member nations. In the event of a public
health emergency or epidemic, it provides logistical support and coordinates international response to the emergency. The United

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States contributes to this effort through the CDC. The CDC carries out international monitoring and public health efforts, mainly in
the service of protecting US public health in an increasingly connected world. Similarly, the European Union maintains a Health
Security Committee that monitors disease outbreaks within its member countries and internationally, coordinating with WHO.

Exercise 12.5.6

Name the organizations that participate in international public health monitoring.

Emerging and Reemerging Infectious Diseases

Both WHO and some national public health agencies such as the CDC monitor and prepare for emerging infectious diseases. An
emerging infectious disease is either new to the human population or has shown an increase in prevalence in the previous twenty
years. Whether the disease is new or conditions have changed to cause an increase in frequency, its status as emerging implies the
need to apply resources to understand and control its growing impact.
Emerging diseases may change their frequency gradually over time, or they may experience sudden epidemic growth. The
importance of vigilance was made clear during the Ebola hemorrhagic fever epidemic in western Africa through 2014–2015.
Although health experts had been aware of the Ebola virus since the 1970s, an outbreak on such a large scale had never happened
before (Figure 12.5.5). Previous human epidemics had been small, isolated, and contained. Indeed, the gorilla and chimpanzee
populations of western Africa had suffered far worse from Ebola than the human population. The pattern of small isolated human
epidemics changed in 2014. Its high transmission rate, coupled with cultural practices for treatment of the dead and perhaps its
emergence in an urban setting, caused the disease to spread rapidly, and thousands of people died. The international public health
community responded with a large emergency effort to treat patients and contain the epidemic.
Emerging diseases are found in all countries, both developed and developing (Table 12.5.3). Some nations are better equipped to
deal with them. National and international public health agencies watch for epidemics like the Ebola outbreak in developing
countries because those countries rarely have the health-care infrastructure and expertise to deal with large outbreaks effectively.
Even with the support of international agencies, the systems in western Africa struggled to identify and care for the sick and control
spread. In addition to the altruistic goal of saving lives and assisting nations lacking in resources, the global nature of transportation
means that an outbreak anywhere can spread quickly to every corner of the planet. Managing an epidemic in one location—its
source—is far easier than fighting it on many fronts.
Ebola is not the only disease that needs to be monitored in the global environment. In 2015, WHO set priorities on several
emerging diseases that had a high probability of causing epidemics and that were poorly understood (and thus urgently required
research and development efforts).
A reemerging infectious disease is a disease that is increasing in frequency after a previous period of decline. Its reemergence may
be a result of changing conditions or old prevention regimes that are no longer working. Examples of such diseases are drug-
resistant forms of tuberculosis, bacterial pneumonia, and malaria. Drug-resistant strains of the bacteria causing gonorrhea and
syphilis are also becoming more widespread, raising concerns of untreatable infections.

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 Figure 12.5.5 : Even before the Ebola epidemic of 2014–15, Ebola was considered an emerging disease because of several smaller
outbreaks between the mid-1990s and 2000s.

Table 12.5.3: Some Emerging and Reemerging Infectious Diseases

Disease Pathogen Year Discovered Affected Regions Transmission

Contact with infected body

AIDS HIV 1981 Worldwide
fluids

Africa, Asia, India;

Chikungunya fever Chikungunya virus 1952 spreading to Europe and Mosquito-borne
the Americas

Contact with infected body

Ebola virus disease Ebola virus 1976 Central and Western Africa
fluids

H1N1 Influenza (swine

H1N1 virus 2009 Worldwide Droplet transmission
flu)

Borrelia burgdorferi From mammal reservoirs to

Lyme disease 1981 Northern hemisphere
bacterium humans by tick vectors

Africa, Australia, Canada

West Nile virus disease West Nile virus 1937 to Venezuela, Europe, Mosquito-borne
Middle East, Western Asia

Exercise 12.5.7

1. Explain why it is important to monitor emerging infectious diseases.

2. Explain how a bacterial disease could reemerge, even if it had previously been successfully treated and controlled.

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SARS Outbreak and Identification
On November 16, 2002, the first case of a SARS outbreak was reported in Guangdong Province, China. The patient exhibited
influenza-like symptoms such as fever, cough, myalgia, sore throat, and shortness of breath. As the number of cases grew, the
Chinese government was reluctant to openly communicate information about the epidemic with the World Health Organization
(WHO) and the international community. The slow reaction of Chinese public health officials to this new disease contributed to
the spread of the epidemic within and later outside China. In April 2003, the Chinese government finally responded with a
huge public health effort involving quarantines, medical checkpoints, and massive cleaning projects. Over 18,000 people were
quarantined in Beijing alone. Large funding initiatives were created to improve health-care facilities, and dedicated outbreak
teams were created to coordinate the response. By August 16, 2003, the last SARS patients were released from a hospital in
Beijing nine months after the first case was reported in China.
In the meantime, SARS spread to other countries on its way to becoming a global pandemic. Though the infectious agent had
yet to be identified, it was thought to be an influenza virus. The disease was named SARS, an acronym for severe acute
respiratory syndrome, until the etiologic agent could be identified. Travel restrictions to Southeast Asia were enforced by many
countries. By the end of the outbreak, there were 8,098 cases and 774 deaths worldwide. China and Hong Kong were hit
hardest by the epidemic, but Taiwan, Singapore, and Toronto, Canada, also saw significant numbers of cases (Figure 12.5.6).
Fortunately, timely public health responses in many countries effectively suppressed the outbreak and led to its eventual
containment. For example, the disease was introduced to Canada in February 2003 by an infected traveler from Hong Kong,
who died shortly after being hospitalized. By the end of March, hospital isolation and home quarantine procedures were in
place in the Toronto area, stringent anti-infection protocols were introduced in hospitals, and the media were actively reporting
on the disease. Public health officials tracked down contacts of infected individuals and quarantined them. A total of 25,000
individuals were quarantined in the city. Thanks to the vigorous response of the Canadian public health community, SARS was
brought under control in Toronto by June, a mere four months after it was introduced.
In 2003, WHO established a collaborative effort to identify the causative agent of SARS, which has now been identified as a
coronavirus that was associated with horseshoe bats. The genome of the SARS virus was sequenced and published by
researchers at the CDC and in Canada in May 2003, and in the same month researchers in the Netherlands confirmed the
etiology of the disease by fulfilling Koch’s postulates for the SARS coronavirus. The last known case of SARS worldwide was
reported in 2004.

Figure 12.5.6 : This map shows the spread of SARS as of March 28, 2003. (credit: modification of work by Central Intelligence
Agency)

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This database of reports chronicles outbreaks of infectious disease around the world. It was on this system that the first information
about the SARS outbreak in China emerged.
The CDC publishes Emerging Infectious Diseases, a monthly journal available online.

Key Concepts and Summary

Epidemiology is the science underlying public health.
Morbidity means being in a state of illness, whereas mortality refers to death; both morbidity rates and mortality rates are
of interest to epidemiologists.
Incidence is the number of new cases (morbidity or mortality), usually expressed as a proportion, during a specified time
period; prevalence is the total number affected in the population, again usually expressed as a proportion.
Sporadic diseases only occur rarely and largely without a geographic focus. Endemic diseases occur at a constant (and often
low) level within a population. Epidemic diseases and pandemic diseases occur when an outbreak occurs on a significantly
larger than expected level, either locally or globally, respectively.
Koch’s postulates specify the procedure for confirming a particular pathogen as the etiologic agent of a particular disease.
Koch’s postulates have limitations in application if the microbe cannot be isolated and cultured or if there is no animal host for
the microbe. In this case, molecular Koch’s postulates would be utilized.
In the United States, the Centers for Disease Control and Prevention monitors notifiable diseases and publishes weekly
updates in the Morbidity and Mortality Weekly Report.
The World Health Organization (WHO) is an agency of the United Nations that collects and analyzes data on disease
occurrence from member nations. WHO also coordinates public health programs and responses to international health
emergencies.
Emerging diseases are those that are new to human populations or that have been increasing in the past two decades.
Reemerging diseases are those that are making a resurgence in susceptible populations after previously having been controlled
in some geographic areas.

Footnotes
1. H. Irene Hall, Qian An, Tian Tang, Ruiguang Song, Mi Chen, Timothy Green, and Jian Kang. “Prevalence of Diagnosed and
Undiagnosed HIV Infection—United States, 2008–2012.” Morbidity and Mortality Weekly Report 64, no. 24 (2015): 657–662.
2. Centers for Disease Control and Prevention. “Diagnoses of HIV Infection in the United States and Dependent Areas, 2014.”
HIV Surveillance Report 26 (2015).
3. Centers for Disease Control and Prevention. “Tetanus Surveillance—United States, 2001–2008.” Morbidity and Mortality
Weekly Report 60, no. 12 (2011): 365.
4. Centers for Disease Control and Prevention. “Plague in the United States.” 2015. http://www.cdc.gov/plague/maps. Accessed
June 1, 2016.
5. World Health Organization. “Programme Budget 2014–2015.” www.who.int/about/finances-ac...lity/budget/en.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 12.5: The Language of Epidemiologists is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

Access for free at OpenStax 12.5.10 https://bio.libretexts.org/@go/page/31871

12.6: Tracking Infectious Diseases
Learning Objectives
Explain the research approaches used by the pioneers of epidemiology
Explain how descriptive, analytical, and experimental epidemiological studies go about determining the cause of morbidity
and mortality

Epidemiology has its roots in the work of physicians who looked for patterns in disease occurrence as a way to understand how to
prevent it. The idea that disease could be transmitted was an important precursor to making sense of some of the patterns. In 1546,
Girolamo Fracastoro first proposed the germ theory of disease in his essay De Contagione et Contagiosis Morbis, but this theory
remained in competition with other theories, such as the miasma hypothesis, for many years. Uncertainty about the cause of disease
was not an absolute barrier to obtaining useful knowledge from patterns of disease. Some important researchers, such as Florence
Nightingale, subscribed to the miasma hypothesis. The transition to acceptance of the germ theory during the 19th century provided
a solid mechanistic grounding to the study of disease patterns. The studies of 19th century physicians and researchers such as John
Snow, Florence Nightingale, Ignaz Semmelweis, Joseph Lister, Robert Koch, Louis Pasteur, and others sowed the seeds of modern
epidemiology.

Pioneers of Epidemiology
John Snow (Figure 12.6.1) was a British physician known as the father of epidemiology for determining the source of the 1854
Broad Street cholera epidemic in London. Based on observations he had made during an earlier cholera outbreak (1848–1849),
Snow proposed that cholera was spread through a fecal-oral route of transmission and that a microbe was the infectious agent. He
investigated the 1854 cholera epidemic in two ways. First, suspecting that contaminated water was the source of the epidemic,
Snow identified the source of water for those infected. He found a high frequency of cholera cases among individuals who obtained
their water from the River Thames downstream from London. This water contained the refuse and sewage from London and
settlements upstream. He also noted that brewery workers did not contract cholera and on investigation found the owners provided
the workers with beer to drink and stated that they likely did not drink water.1 Second, he also painstakingly mapped the incidence
of cholera and found a high frequency among those individuals using a particular water pump located on Broad Street. In response
to Snow’s advice, local officials removed the pump’s handle,2 resulting in the containment of the Broad Street cholera epidemic.

Figure 12.6.1 : (a) John Snow (1813–1858), British physician and father of epidemiology. (b) Snow’s detailed mapping of cholera
incidence led to the discovery of the contaminated water pump on Broad street (red square) responsible for the 1854 cholera
epidemic. (credit a: modification of work by “Rsabbatini”/Wikimedia Commons)
Snow’s work represents an early epidemiological study and it resulted in the first known public health response to an epidemic.
Snow’s meticulous case-tracking methods are now common practice in studying disease outbreaks and in associating new diseases
with their causes. His work further shed light on unsanitary sewage practices and the effects of waste dumping in the Thames.

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Additionally, his work supported the germ theory of disease, which argued disease could be transmitted through contaminated
items, including water contaminated with fecal matter.
Snow’s work illustrated what is referred to today as a common source spread of infectious disease, in which there is a single source
for all of the individuals infected. In this case, the single source was the contaminated well below the Broad Street pump. Types of
common source spread include point source spread, continuous common source spread, and intermittent common source spread. In
point source spread of infectious disease, the common source operates for a short time period—less than the incubation period of
the pathogen. An example of point source spread is a single contaminated potato salad at a group picnic. In continuous common
source spread, the infection occurs for an extended period of time, longer than the incubation period. An example of continuous
common source spread would be the source of London water taken downstream of the city, which was continuously contaminated
with sewage from upstream. Finally, with intermittent common source spread, infections occur for a period, stop, and then begin
again. This might be seen in infections from a well that was contaminated only after large rainfalls and that cleared itself of
contamination after a short period.
In contrast to common source spread, propagated spread occurs through direct or indirect person-to-person contact. With
propagated spread, there is no single source for infection; each infected individual becomes a source for one or more subsequent
infections. With propagated spread, unless the spread is stopped immediately, infections occur for longer than the incubation
period. Although point sources often lead to large-scale but localized outbreaks of short duration, propagated spread typically
results in longer duration outbreaks that can vary from small to large, depending on the population and the disease (Figure 12.6.2).
In addition, because of person-to-person transmission, propagated spread cannot be easily stopped at a single source like point
source spread.

Figure 12.6.2 : (a) Outbreaks that can be attributed to point source spread often have a short duration. (b) Outbreaks attributed to
propagated spread can have a more extended duration. (credit a, b: modification of work by Centers for Disease Control and
Prevention)
Florence Nightingale’s work is another example of an early epidemiological study. In 1854, Nightingale was part of a contingent of
nurses dispatched by the British military to care for wounded soldiers during the Crimean War. Nightingale kept meticulous records
regarding the causes of illness and death during the war. Her recordkeeping was a fundamental task of what would later become the
science of epidemiology. Her analysis of the data she collected was published in 1858. In this book, she presented monthly
frequency data on causes of death in a wedge chart histogram (Figure 12.6.3). This graphical presentation of data, unusual at the
time, powerfully illustrated that the vast majority of casualties during the war occurred not due to wounds sustained in action but to
what Nightingale deemed preventable infectious diseases. Often these diseases occurred because of poor sanitation and lack of
access to hospital facilities. Nightingale’s findings led to many reforms in the British military’s system of medical care.
Joseph Lister provided early epidemiological evidence leading to good public health practices in clinics and hospitals. These
settings were notorious in the mid-1800s for fatal infections of surgical wounds at a time when the germ theory of disease was not
yet widely accepted. Most physicians did not wash their hands between patient visits or clean and sterilize their surgical tools.
Lister, however, discovered the disinfecting properties of carbolic acid, also known as phenol. He introduced several disinfection
protocols that dramatically lowered post-surgical infection rates.3 He demanded that surgeons who worked for him use a 5%

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carbolic acid solution to clean their surgical tools between patients, and even went so far as to spray the solution onto bandages and
over the surgical site during operations (Figure 12.6.4). He also took precautions not to introduce sources of infection from his skin
or clothing by removing his coat, rolling up his sleeves, and washing his hands in a dilute solution of carbolic acid before and
during the surgery.

Figure 12.6.3 : (a) Florence Nightingale reported on the data she collected as a nurse in the Crimean War. (b) Nightingale’s diagram
shows the number of fatalities in soldiers by month of the conflict from various causes. The total number dead in a particular month
is equal to the area of the wedge for that month. The colored sections of the wedge represent different causes of death: wounds
(pink), preventable infectious diseases (gray), and all other causes (brown).

Figure 12.6.4 : Joseph Lister initiated the use of a carbolic acid (phenol) during surgeries. This illustration of a surgery shows a
pressurized canister of carbolic acid being sprayed over the surgical site.
Visit the website for The Ghost Map, a book about Snow’s work related to the Broad Street pump cholera outbreak.
John Snow’s own account of his work has additional links and information.
This CDC resource further breaks down the pattern expected from a point-source outbreak.
Learn more about Nightingale’s wedge chart here.

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 Exercise 12.6.1
1. Explain the difference between common source spread and propagated spread of disease.
2. Describe how the observations of John Snow, Florence Nightingale, and Joseph Lister led to improvements in public
health.

Types of Epidemiological Studies

Today, epidemiologists make use of study designs, the manner in which data are gathered to test a hypothesis, similar to those of
researchers studying other phenomena that occur in populations. These approaches can be divided into observational studies (in
which subjects are not manipulated) and experimental studies (in which subjects are manipulated). Collectively, these studies give
modern-day epidemiologists multiple tools for exploring the connections between infectious diseases and the populations of
susceptible individuals they might infect.
Observational Studies

In an observational study, data are gathered from study participants through measurements (such as physiological variables like
white blood cell count), or answers to questions in interviews (such as recent travel or exercise frequency). The subjects in an
observational study are typically chosen at random from a population of affected or unaffected individuals. However, the subjects
in an observational study are in no way manipulated by the researcher. Observational studies are typically easier to carry out than
experimental studies, and in certain situations they may be the only studies possible for ethical reasons.
Observational studies are only able to measure associations between disease occurrence and possible causative agents; they do not
necessarily prove a causal relationship. For example, suppose a study finds an association between heavy coffee drinking and lower
incidence of skin cancer. This might suggest that coffee prevents skin cancer, but there may be another unmeasured factor involved,
such as the amount of sun exposure the participants receive. If it turns out that coffee drinkers work more in offices and spend less
time outside in the sun than those who drink less coffee, then it may be possible that the lower rate of skin cancer is due to less sun
exposure, not to coffee consumption. The observational study cannot distinguish between these two potential causes.
There are several useful approaches in observational studies. These include methods classified as descriptive epidemiology and
analytical epidemiology. Descriptive epidemiology gathers information about a disease outbreak, the affected individuals, and how
the disease has spread over time in an exploratory stage of study. This type of study will involve interviews with patients, their
contacts, and their family members; examination of samples and medical records; and even histories of food and beverages
consumed. Such a study might be conducted while the outbreak is still occurring. Descriptive studies might form the basis for
developing a hypothesis of causation that could be tested by more rigorous observational and experimental studies.
Analytical epidemiology employs carefully selected groups of individuals in an attempt to more convincingly evaluate hypotheses
about potential causes for a disease outbreak. The selection of cases is generally made at random, so the results are not biased
because of some common characteristic of the study participants. Analytical studies may gather their data by going back in time
(retrospective studies), or as events unfold forward in time (prospective studies).
Retrospective studies gather data from the past on present-day cases. Data can include things like the medical history, age, gender,
or occupational history of the affected individuals. This type of study examines associations between factors chosen or available to
the researcher and disease occurrence.
Prospective studies follow individuals and monitor their disease state during the course of the study. Data on the characteristics of
the study subjects and their environments are gathered at the beginning and during the study so that subjects who become ill may
be compared with those who do not. Again, the researchers can look for associations between the disease state and variables that
were measured during the study to shed light on possible causes.
Analytical studies incorporate groups into their designs to assist in teasing out associations with disease. Approaches to group-
based analytical studies include cohort studies, case-control studies, and cross-sectional studies. The cohort method examines
groups of individuals (called cohorts) who share a particular characteristic. For example, a cohort might consist of individuals born
in the same year and the same place; or it might consist of people who practice or avoid a particular behavior, e.g., smokers or
nonsmokers. In a cohort study, cohorts can be followed prospectively or studied retrospectively. If only a single cohort is followed,
then the affected individuals are compared with the unaffected individuals in the same group. Disease outcomes are recorded and
analyzed to try to identify correlations between characteristics of individuals in the cohort and disease incidence. Cohort studies are
a useful way to determine the causes of a condition without violating the ethical prohibition of exposing subjects to a risk factor.

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Cohorts are typically identified and defined based on suspected risk factors to which individuals have already been exposed
through their own choices or circumstances.
Case-control studies are typically retrospective and compare a group of individuals with a disease to a similar group of individuals
without the disease. Case-control studies are far more efficient than cohort studies because researchers can deliberately select
subjects who are already affected with the disease as opposed to waiting to see which subjects from a random sample will develop
a disease.
A cross-sectional study analyzes randomly selected individuals in a population and compares individuals affected by a disease or
condition to those unaffected at a single point in time. Subjects are compared to look for associations between certain measurable
variables and the disease or condition. Cross-sectional studies are also used to determine the prevalence of a condition.
Experimental Studies
Experimental epidemiology uses laboratory or clinical studies in which the investigator manipulates the study subjects to study the
connections between diseases and potential causative agents or to assess treatments. Examples of treatments might be the
administration of a drug, the inclusion or exclusion of different dietary items, physical exercise, or a particular surgical procedure.
Animals or humans are used as test subjects. Because experimental studies involve manipulation of subjects, they are typically
more difficult and sometimes impossible for ethical reasons.
Koch’s postulates require experimental interventions to determine the causative agent for a disease. Unlike observational studies,
experimental studies can provide strong evidence supporting cause because other factors are typically held constant when the
researcher manipulates the subject. The outcomes for one group receiving the treatment are compared to outcomes for a group that
does not receive the treatment but is treated the same in every other way. For example, one group might receive a regimen of a drug
administered as a pill, while the untreated group receives a placebo (a pill that looks the same but has no active ingredient). Both
groups are treated as similarly as possible except for the administration of the drug. Because other variables are held constant in
both the treated and the untreated groups, the researcher is more certain that any change in the treated group is a result of the
specific manipulation.
Experimental studies provide the strongest evidence for the etiology of disease, but they must also be designed carefully to
eliminate subtle effects of bias. Typically, experimental studies with humans are conducted as double-blind studies, meaning
neither the subjects nor the researchers know who is a treatment case and who is not. This design removes a well-known cause of
bias in research called the placebo effect, in which knowledge of the treatment by either the subject or the researcher can influence
the outcomes.

Exercise 12.6.2

1. Describe the advantages and disadvantages of observational studies and experimental studies.
2. Explain the ways that groups of subjects can be selected for analytical studies.

Key Concepts and Summary

Early pioneers of epidemiology such as John Snow, Florence Nightingale, and Joseph Lister, studied disease at the population
level and used data to disrupt disease transmission.
Descriptive epidemiology studies rely on case analysis and patient histories to gain information about outbreaks, frequently
while they are still occurring.
Retrospective epidemiology studies use historical data to identify associations with the disease state of present cases.
Prospective epidemiology studies gather data and follow cases to find associations with future disease states.
Analytical epidemiology studies are observational studies that are carefully designed to compare groups and uncover
associations between environmental or genetic factors and disease.
Experimental epidemiology studies generate strong evidence of causation in disease or treatment by manipulating subjects and
comparing them with control subjects.
1. John Snow. On the Mode of Communication of Cholera. Second edition, Much Enlarged. John Churchill, 1855.
2. John Snow. “The Cholera near Golden-Wquare, and at Deptford.” Medical Times and Gazette 9 (1854): 321–322.
http://www.ph.ucla.edu/epi/snow/chol...densquare.html.
3. O.M. Lidwell. “Joseph Lister and Infection from the Air.” Epidemiology and Infection 99 (1987): 569–578.
www.ncbi.nlm.nih.gov/pmc/arti...00006-0004.pdf.

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Contributors and Attributions
Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 12.6: Tracking Infectious Diseases is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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Chapter 12 Exercises
Review Questions for Chapter 12
Multiple Choice
1) Which of the following would be a sign of an infection?
A. muscle aches
B. headache
C. fever
D. nausea

2) Which of the following is an example of a noncommunicable infectious disease?

A. infection with a respiratory virus
B. food poisoning due to a preformed bacterial toxin in food
C. skin infection acquired from a dog bite
D. infection acquired from the stick of a contaminated needle

3) During an oral surgery, the surgeon nicked the patient’s gum with a sharp instrument. This allowed Streptococcus, a bacterium
normally present in the mouth, to gain access to the blood. As a result, the patient developed bacterial endocarditis (an infection of
the heart). Which type of disease is this?
A. iatrogenic
B. nosocomial
C. vectors
D. zoonotic

4) Which period is the stage of disease during which the patient begins to present general signs and symptoms?
A. convalescence
B. incubation
C. illness
D. prodromal

5) A communicable disease that can be easily transmitted from person to person is which type of disease?
A. contagious
B. iatrogenic
C. acute
D. nosocomial

6) Which of the following is a pathogen that could not be identified by the original Koch’s postulates?
A. Staphylococcus aureus
B. Pseudomonas aeruginosa
C. Human immunodeficiency virus
D. Salmonella enterica serovar Typhimurium

7) Pathogen A has an ID50 of 50 particles, pathogen B has an ID50 of 1,000 particles, and pathogen C has an ID50 of 1 ×
106 particles. Which pathogen is most virulent?

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A. pathogen A
B. pathogen B
C. pathogen C

8) Which of the following choices lists the steps of pathogenesis in the correct order?
A. invasion, infection, adhesion, exposure
B. adhesion, exposure, infection, invasion
C. exposure, adhesion, invasion, infection
D. disease, infection, exposure, invasion

9) Which of the following would be a virulence factor of a pathogen?

A. a surface protein allowing the pathogen to bind to host cells
B. a secondary host the pathogen can infect
C. a surface protein the host immune system recognizes
D. the ability to form a provirus

10) You have recently identified a new toxin. It is produced by a gram-negative bacterium. It is composed mostly of protein, has
high toxicity, and is not heat stable. You also discover that it targets liver cells. Based on these characteristics, how would you
classify this toxin?
A. superantigen
B. endotoxin
C. exotoxin
D. leukocidin

11) Which of the following applies to hyaluronidase?

A. It acts as a spreading factor.
B. It promotes blood clotting.
C. It is an example of an adhesin.
D. It is produced by immune cells to target pathogens.

12) Phospholipases are enzymes that do which of the following?

A. degrade antibodies
B. promote pathogen spread through connective tissue.
C. degrade nucleic acid to promote spread of pathogen
D. degrade cell membranes to allow pathogens to escape phagosomes

13) Which of the following is a major virulence factor for the fungal pathogen Cryptococcus?
A. hemolysin
B. capsule
C. collagenase
D. fimbriae

14) Which of the following pathogens undergoes antigenic variation to avoid immune defenses?
A. Candida
B. Cryptococcus

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C. Plasmodium
D. Giardia

15) Which is the most common type of biological vector of human disease?
a. viruses
b. bacteria
c. mammals
d. arthropods

16) A mosquito bites a person who subsequently develops a fever and abdominal rash. What type of transmission would this be?
a. mechanical vector transmission
b. biological vector transmission
c. direct contact transmission
d. vehicle transmission

17) Cattle are allowed to pasture in a field that contains the farmhouse well, and the farmer’s family becomes ill with a
gastrointestinal pathogen after drinking the water. What type of transmission of infectious agents would this be?
a. biological vector transmission
b. direct contact transmission
c. indirect contact transmission
d. vehicle transmission

18) A blanket from a child with chickenpox is likely to be contaminated with the virus that causes chickenpox (Varicella-zoster
virus). What is the blanket called?
a. fomite
b. host
c. pathogen
d. vector

19) Which of the following would NOT be considered an emerging disease?

a. Ebola hemorrhagic fever
b. West Nile virus fever/encephalitis
c. Zika virus disease
d. Tuberculosis

20) Which of the following would NOT be considered a reemerging disease?

a. Drug-resistant tuberculosis
b. Drug-resistant gonorrhea
c. Malaria
d. West Nile virus fever/encephalitis

21) Which of the following factors can lead to reemergence of a disease?

a. A mutation that allows it to infect humans
b. A period of decline in vaccination rates
c. A change in disease reporting procedures

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 d. Better education on the signs and symptoms of the disease

22) Why are emerging diseases with very few cases the focus of intense scrutiny?
a. They tend to be more deadly
b. They are increasing and therefore not controlled
c. They naturally have higher transmission rates
d. They occur more in developed countries

Fill-in-the-Blanks
23) A difference between an acute disease and chronic disease is that chronic diseases have an extended period of __________.

24) A person steps on a rusty nail and develops tetanus. In this case, the person has acquired a(n) __________ disease.

25) A(n) __________ pathogen causes disease only when conditions are favorable for the microorganism because of transfer to an
inappropriate body site or weakened immunity in an individual.

26) The concentration of pathogen needed to kill 50% of an infected group of test animals is the __________.

27) A(n) __________ infection is a small region of infection from which a pathogen may move to another part of the body to
establish a second infection.

28) Cilia, fimbriae, and pili are all examples of structures used by microbes for __________.

29) The glycoprotein adhesion gp120 on HIV must interact with __________ on some immune cells as the first step in the process
of infecting the cell.

30) Adhesins are usually located on __________ of the pathogen and are composed mainly of __________ and __________.

31) The Shiga and diphtheria toxins target __________ in host cells.

32) Antigenic __________ is the result of reassortment of genes responsible for the production of influenza virus spike proteins
between different virus particles while in the same host, whereas antigenic __________ is the result of point mutations in the spike
proteins.

33) Candida can invade tissue by producing the exoenzymes __________ and __________.

34) The larval form of Schistosoma mansoni uses a __________ to help it gain entry through intact skin.

35) The ________ collects data and conducts epidemiologic studies in the United States.

36) ________occurs when an infected individual passes the infection on to other individuals, who pass it on to still others,
increasing the penetration of the infection into the susceptible population.

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37) A batch of food contaminated with botulism exotoxin, consumed at a family reunion by most of the members of a family,
would be an example of a ________ outbreak.

38) A patient in the hospital with a urinary catheter develops a bladder infection. This is an example of a(n) ________ infection.

39) A ________ is an animal that can transfer infectious pathogens from one host to another.

40) The ________ collects data and conducts epidemiologic studies at the global level.

Short Answer
41) Brian goes to the hospital after not feeling well for a week. He has a fever of 38 °C (100.4 °F) and complains of nausea and a
constant migraine. Distinguish between the signs and symptoms of disease in Brian’s case.

42) Describe the virulence factors associated with the fungal pathogen Aspergillus.

43) Explain how helminths evade the immune system.

44) During an epidemic, why might the prevalence of a disease at a particular time not be equal to the sum of the incidences of the
disease?

45) In what publication would you find data on emerging/reemerging diseases in the United States?

46) What activity did John Snow conduct, other than mapping, that contemporary epidemiologists also use when trying to
understand how to control a disease?

47) Differentiate between droplet vehicle transmission and airborne transmission.

Critical Thinking
48) Two periods of acute disease are the periods of illness and period of decline. (a) In what way are both of these periods similar?
(b) In terms of quantity of pathogen, in what way are these periods different? (c) What initiates the period of decline?

49) In July 2015, a report 1 was released indicating the gram-negative bacterium Pseudomonas aeruginosa was found on hospital
sinks 10 years after the initial outbreak in a neonatal intensive care unit. P. aeruginosa usually causes localized ear and eye
infections but can cause pneumonia or septicemia in vulnerable individuals like newborn babies. Explain how the current discovery
of the presence of this reported P. aeruginosa could lead to a recurrence of nosocomial disease.

50) Diseases that involve biofilm-producing bacteria are of serious concern. They are not as easily treated compared with those
involving free-floating (or planktonic) bacteria. Explain three reasons why biofilm formers are more pathogenic.

51) A microbiologist has identified a new gram-negative pathogen that causes liver disease in rats. She suspects that the
bacterium’s fimbriae are a virulence factor. Describe how molecular Koch’s postulates could be used to test this hypothesis.

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52) Acupuncture is a form of alternative medicine that is used for pain relief. Explain how acupuncture could facilitate exposure to
pathogens.

53) Two types of toxins are hemolysins and leukocidins. (a) How are these toxins similar? (b) How do they differ?

54) Imagine that a mutation in the gene encoding the cholera toxin was made. This mutation affects the A-subunit, preventing it
from interacting with any host protein. (a) Would the toxin be able to enter into the intestinal epithelial cell? (b) Would the toxin be
able to cause diarrhea?

55) Why might an epidemiological population in a state not be the same size as the number of people in a state? Use an example.

56) Many people find that they become ill with a cold after traveling by airplane. The air circulation systems of commercial aircraft
use HEPA filters that should remove any infectious agents that pass through them. What are the possible reasons for increased
incidence of colds after flights?

57) An Atlantic crossing by boat from England to New England took 60–80 days in the 18th century. In the late 19th century the
voyage took less than a week. How do you think these time differences for travel might have impacted the spread of infectious
diseases from Europe to the Americas, or vice versa?

Footnotes
1. C. Owens. “P. aeruginosa survives in sinks 10 years after hospital outbreak.” 2015. http://www.healio.com/infectious-dis...pital-
outbreak

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 CHAPTER OVERVIEW

13: Innate Nonspecific Host Defenses

13.1: First Line defense- Physical, Mechanical and Chemical Defenses
13.2: Second Line Defenses: Cells and Fluids
13.3: Pathogen Recognition and Phagocytosis
13.4: Inflammation and Fever
Chapter 13 Exercises

Thumbnail: Scanning electron micrograph of a phagocyte (yellow, right) phagocytosing anthrax bacilli (orange, left). (CC BY 2.5;
Volker Brinkmann via PLOS).

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1
13.1: First Line defense- Physical, Mechanical and Chemical Defenses
Learning Objectives
Describe the various physical barriers and mechanical defenses that protect the human body against infection and disease
Describe how enzymes in body fluids provide protection against infection or disease
Describe the role of microbiota as a first-line defense against infection and disease

Nonspecific innate immunity can be characterized as a multifaceted system of defenses that targets invading pathogens in a
nonspecific manner. In this chapter, we have divided the numerous defenses that make up this system into three categories: physical
defenses, chemical defenses, and cellular defenses. However, it is important to keep in mind that these defenses do not function
independently, and the categories often overlap. Table 13.1.1 provides an overview of the nonspecific defenses discussed in this
chapter.
Table 13.1.1 : Overview of Nonspecific Innate Immune Defenses

Overview of Nonspecific Innate Immune Defenses

Physical barriers
Physical defenses Mechanical defenses
Microbiome
Chemicals and enzymes in body fluids
Antimicrobial peptides
Chemical defenses Plasma protein mediators
Cytokines
Inflammation-eliciting mediators
Granulocytes
Cellular defenses
Agranulocytes

Clinical Focus: part 1

Angela, a 25-year-old female patient in the emergency department, is having some trouble communicating verbally because of
shortness of breath. A nurse observes constriction and swelling of the airway and labored breathing. The nurse asks Angela if
she has a history of asthma or allergies. Angela shakes her head no, but there is fear in her eyes. With some difficulty, she
explains that her father died suddenly at age 27, when she was just a little girl, of a similar respiratory attack. The underlying
cause had never been identified.

Exercise 13.1.1
1. What are some possible causes of constriction and swelling of the airway?
2. What causes swelling of body tissues in general?

Physical defenses provide the body’s most basic form of nonspecific defense. They include physical barriers to microbes, such as
the skin and mucous membranes, as well as mechanical defenses that physically remove microbes and debris from areas of the
body where they might cause harm or infection. In addition, the microbiome provides a measure of physical protection against
disease, as microbes of the normal microbiota compete with pathogens for nutrients and cellular binding sites necessary to cause
infection.

Physical Barriers
Physical barriers play an important role in preventing microbes from reaching tissues that are susceptible to infection. At the
cellular level, barriers consist of cells that are tightly joined to prevent invaders from crossing through to deeper tissue. For
example, the endothelial cells that line blood vessels have very tight cell-to-cell junctions, blocking microbes from gaining access
to the bloodstream. Cell junctions are generally composed of cell membrane proteins that may connect with the extracellular matrix

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or with complementary proteins from neighboring cells. Tissues in various parts of the body have different types of cell junctions.
These include tight junctions, desmosomes, and gap junctions, as illustrated in Figure 13.1.1. Invading microorganisms may
attempt to break down these substances chemically, using enzymes such as proteases that can cause structural damage to create a
point of entry for pathogens.

Figure 13.1.1 : There are multiple types of cell junctions in human tissue, three of which are shown here. Tight junctions rivet two
adjacent cells together, preventing or limiting material exchange through the spaces between them. Desmosomes have intermediate
fibers that act like shoelaces, tying two cells together, allowing small materials to pass through the resulting spaces. Gap junctions
are channels between two cells that permit their communication via signals. (credit: modification of work by Mariana Ruiz
Villareal)

The Skin Barrier

One of the body’s most important physical barriers is the skin barrier, which is composed of three layers of closely packed cells.
The thin upper layer is called the epidermis. A second, thicker layer, called the dermis, contains hair follicles, sweat glands, nerves,
and blood vessels. A layer of fatty tissue called the hypodermis lies beneath the dermis and contains blood and lymph vessels
(Figure 13.1.2).

Figure 13.1.2 : Human skin has three layers, the epidermis, the dermis, and the hypodermis, which provide a thick barrier between
microbes outside the body and deeper tissues. Dead skin cells on the surface of the epidermis are continually shed, taking with
them microbes on the skin’s surface. (credit: modification of work by National Institutes of Health)
The topmost layer of skin, the epidermis, consists of cells that are packed with keratin. These dead cells remain as a tightly
connected, dense layer of protein-filled cell husks on the surface of the skin. The keratin makes the skin’s surface mechanically
tough and resistant to degradation by bacterial enzymes. Keratin also helps to make the outer surface of the skin relatively
waterproof, this helps keep the surface of the skin dry, which reduces microbial growth. Fatty acids on the skin’s surface create a

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dry, salty, and acidic environment that inhibits the growth of some microbes and is highly resistant to breakdown by bacterial
enzymes. Sebum from the oil glands in hair follicles is an endogenous mediator, providing an additional layer of defense by
helping seal off the pore of the hair follicle, preventing bacteria on the skin’s surface from invading sweat glands and surrounding
tissue. Certain members of the microbiome, can use lipase enzymes to degrade sebum, using it as a food source. This produces
oleic acid, which creates a mildly acidic environment on the surface of the skin that is inhospitable to many pathogenic microbes.
Oleic acid is an example of an exogenously produced mediator because it is produced by resident microbes and not directly by
body cells. In addition, the dead cells of the epidermis are frequently shed, along with any microbes that may be clinging to them
(desquamation). Shed skin cells are continually replaced with new cells from below, providing a new barrier that will soon be shed
in the same way.
Perspiration (sweat) provides some moisture to the epidermis, which can increase the potential for microbial growth. For this
reason, more microbes are found on the regions of the skin that produce the most sweat, such as the skin of the underarms and
groin. However, in addition to water, sweat also contains substances that inhibit microbial growth, such as salts, lysozyme, and
antimicrobial peptides. Sebum also serves to protect the skin and reduce water loss. Although some of the lipids and fatty acids in
sebum inhibit microbial growth, sebum contains compounds that provide nutrition for certain microbes. In the ears, cerumen
(earwax) exhibits antimicrobial properties due to the presence of fatty acids, which lower the pH to between 3 and 5.
Infections can occur when the skin barrier is compromised or broken. A wound can serve as a point of entry for opportunistic
pathogens, which can infect the skin tissue surrounding the wound and possibly spread to deeper tissues.

Every Rose has its Thorn

Mike, a gardener from southern California, recently noticed a small red bump on his left forearm. Initially, he did not think
much of it, but soon it grew larger and then ulcerated (opened up), becoming a painful lesion that extended across a large part
of his forearm (Figure 13.1.3). He went to an urgent care facility, where a physician asked about his occupation. When he said
he was a landscaper, the physician immediately suspected a case of sporotrichosis, a type of fungal infection known as rose
gardener’s disease because it often afflicts landscapers and gardening enthusiasts.
Under most conditions, fungi cannot produce skin infections in healthy individuals. Fungi grow filaments known as hyphae,
which are not particularly invasive and can be easily kept at bay by the physical barriers of the skin and mucous membranes.
However, small wounds in the skin, such as those caused by thorns, can provide an opening for opportunistic pathogens like
Sporothrix schenkii, a soil-dwelling fungus and the causative agent of rose gardener’s disease. Once it breaches the skin barrier,
S. schenkii can infect the skin and underlying tissues, producing ulcerated lesions like Mike’s. Compounding matters, other
pathogens may enter the infected tissue, causing secondary bacterial infections.
Luckily, rose gardener’s disease is treatable. Mike’s physician wrote him a prescription for some antifungal drugs as well as a
course of antibiotics to combat secondary bacterial infections. His lesions eventually healed, and Mike returned to work with a
new appreciation for gloves and protective clothing.

Figure 13.1.3 : Rose gardener’s disease can occur when the fungus Sporothrix schenkii breaches the skin through small cuts,
such as might be inflicted by thorns. (credit left: modification of work by Elisa Self; credit right: modification of work by
Centers for Disease Control and Prevention)

Barriers in the Eye

Although the eye and skin have distinct anatomy, they are both in direct contact with the external environment. An important
component of the eye is the nasolacrimal drainage system, which serves as a conduit for the fluid of the eye, called tears. Tears
flow from the external eye to the nasal cavity by the lacrimal apparatus, which is composed of the structures involved in tear
production (Figure 13.1.4). The lacrimal gland, above the eye, secretes tears to keep the eye moist. There are two small openings,

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one on the inside edge of the upper eyelid and one on the inside edge of the lower eyelid, near the nose. Each of these openings is
called a lacrimal punctum. Together, these lacrimal puncta collect tears from the eye that are then conveyed through lacrimal ducts
to a reservoir for tears called the lacrimal sac, also known as the dacrocyst or tear sac.
From the sac, tear fluid flows via a nasolacrimal duct to the inner nose. Each nasolacrimal duct is located underneath the skin and
passes through the bones of the face into the nose. Chemicals in tears, such as defensins, lactoferrin, and lysozyme, help to prevent
colonization by pathogens. Lysozyme cleaves the bond between NAG and NAM in peptidoglycan, a component of the cell wall in
bacteria. It is more effective against gram-positive bacteria, which lack the protective outer membrane associated with gram-
negative bacteria. Lactoferrin inhibits microbial growth by chemically binding and sequestering iron. This effectually starves many
microbes that require iron for growth. In addition, mucins facilitate removal of microbes from the surface of the eye.

Figure 13.1.4 : The lacrimal apparatus includes the structures of the eye associated with tear production and drainage. (credit:
modification of work by “Evidence Based Medical Educator Inc.”/YouTube)

Mucous Membranes
The mucous membranes lining the nose, mouth, lungs, and urinary and digestive tracts provide another nonspecific barrier against
potential pathogens. Mucous membranes consist of a layer of epithelial cells bound by tight junctions. The epithelial cells secrete a
moist, sticky substance called mucus, which covers and protects the more fragile cell layers beneath it and traps debris and
particulate matter, including microbes. Mucus secretions also contain antimicrobial peptides.
In many regions of the body, mechanical actions serve to flush mucus (along with trapped or dead microbes) out of the body or
away from potential sites of infection. For example, in the respiratory system, inhalation can bring microbes, dust, mold spores, and
other small airborne debris into the body. The nasal cavity is lined with hairs that trap large particles, like dust and pollen, and
prevent their access to deeper tissues. The nasal cavity is also lined with a mucous membrane and Bowman’s glands that produce
mucus to help trap particles and microorganisms for removal, a layer known as the mucociliary blanket. The viscosity and acidity
of this secretion inhibits microbial attachment to the underlying cells. The upper respiratory system is under constant surveillance
by mucosa-associated lymphoid tissue (MALT), including the adenoids and tonsils. Other mucosal defenses include secreted
antibodies (IgA), lysozyme, surfactant, and antimicrobial peptides called defensins. The epithelial cells lining the upper parts of the
respiratory tract are called ciliated epithelial cells because they have hair-like appendages known as cilia. Movement of the cilia
propels debris-laden mucus out and away from the lungs. The expelled mucus is then swallowed and destroyed in the stomach, or
coughed up, or sneezed out (Figure 13.1.5). This system of removal is often called the mucociliary escalator.

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 Figure 13.1.5: This scanning electron micrograph shows ciliated and nonciliated epithelial cells from the human trachea. The
mucociliary escalator pushes mucus away from the lungs, along with any debris or microorganisms that may be trapped in the
sticky mucus, and the mucus moves up to the esophagus where it can be removed by swallowing.
The mucociliary escalator is such an effective barrier to microbes that the lungs, the lowermost (and most sensitive) portion of the
respiratory tract, were long considered to be a sterile environment in healthy individuals. Only recently has research suggested that
healthy lungs may have a small normal microbiota. Disruption of the mucociliary escalator by the damaging effects of smoking or
diseases such as cystic fibrosis can lead to increased colonization of bacteria in the lower respiratory tract and frequent infections,
which highlights the importance of this physical barrier to host defenses. Lastly, the outer surface of the lungs is protected with a
double-layered pleural membrane, which protects the lungs and provides lubrication to permit the lungs to move easily during
respiration. They also are protected by alveolar macrophages. These phagocytes efficiently kill any microbes that manage to evade
the other defenses.
Like the respiratory tract, the digestive tract is a portal of entry through which microbes enter the body, and the mucous membranes
lining the digestive tract provide a nonspecific physical barrier against ingested microbes. Several factors appear to work against
making the mouth hospitable to certain microbes. For example, chewing allows microbes to mix better with saliva so they can be
swallowed or spit out more easily. In the oral cavity, saliva contains mediators such as lactoperoxidase enzymes, and lysozyme,
which can damage microbial cells. Lysozyme is part of the first line of defense in the innate immune system and cleaves linkages
between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in bacterial peptidoglycan. Additionally, fluids containing
immunoglobulins and phagocytic cells are produced in the gingival spaces. The stomach is an extremely acidic environment (pH
1.5–3.5) due to the gastric juices that break down food and kill many ingested microbes; this helps prevent infection from
pathogens. Further down, the intestinal tract is lined with epithelial cells, interspersed with mucus-secreting goblet cells (Figure
13.1.6). This mucus mixes with material received from the stomach, trapping foodborne microbes and debris.

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 Figure 13.1.6 : Goblet cells produce and secrete mucus. The arrows in this micrograph point to the mucus-secreting goblet cells
(magnification 1600⨯) in the intestinal epithelium. (credit micrograph: Micrograph provided by the Regents of University of
Michigan Medical School © 2012)
Goblet cells, which are modified simple columnar epithelial cells, also line the GI tract (Figure 13.1.6). Goblet cells secrete a gel-
forming mucin, which is the major component of mucus. The production of a protective layer of mucus helps reduce the risk of
pathogens reaching deeper tissues. Small aggregates of underlying lymphoid tissue in the ileum, called Peyer’s patches, detect
pathogens in the intestines via microfold (M) cells, which transfer antigens from the lumen of the intestine to the lymphocytes on
Peyer’s patches to induce an immune response. The Peyer’s patches then secrete IgA and other pathogen-specific antibodies into
the intestinal lumen to help keep intestinal microbes at safe levels.
The mechanical action of peristalsis, a series of muscular contractions in the digestive tract, moves the sloughed mucus and other
material through the intestines, rectum, and anus, excreting the material in feces. In fact, feces are composed of approximately 25%
microbes, 25% sloughed epithelial cells, 25% mucus, and 25% digested or undigested food. Finally, the normal microbiota
provides an additional barrier to infection via a variety of mechanisms. For example, these organisms outcompete potential
pathogens for space and nutrients within the intestine. This is known as competitive exclusion. Members of the microbiota may
also secrete protein toxins known as bacteriocins that are able to bind to specific receptors on the surface of susceptible bacteria.

Endothelia
The epithelial cells lining the urogenital tract, blood vessels, lymphatic vessels, and certain other tissues are known as endothelia.
These tightly packed cells provide a particularly effective frontline barrier against invaders. The endothelia of the blood-brain
barrier, for example, protect the central nervous system (CNS), which consists of the brain and the spinal cord. The CNS is one of
the most sensitive and important areas of the body, as microbial infection of the CNS can quickly lead to serious and often fatal
inflammation. The cell junctions in the blood vessels traveling through the CNS are some of the tightest and toughest in the body,
preventing any transient microbes in the bloodstream from entering the CNS. This keeps the cerebrospinal fluid that surrounds and
bathes the brain and spinal cord sterile under normal conditions.
In both men and women, however, the kidneys are sterile. Although urine does contain some antibacterial components, bacteria
will grow in urine left out at room temperature. Therefore, it is primarily the flushing action that keeps the ureters and bladder free
of microbes. The female reproductive system employs lactate, an exogenously produced chemical mediator, to inhibit microbial

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growth. The cells and tissue layers composing the vagina also produce glycogen, a branched and more complex polymer of
glucose.

Exercise 13.1.2
1. Describe how the mucociliary escalator functions.
2. What other defenses do each of the body sites have in common?
3. Name two places you would find endothelia.

Antimicrobial Peptides
The antimicrobial peptides (AMPs) are a special class of nonspecific cell-derived mediators with broad-spectrum antimicrobial
properties. Some AMPs are produced routinely by the body, whereas others are primarily produced (or produced in greater
quantities) in response to the presence of an invading pathogen. Research has begun exploring how AMPs can be used in the
diagnosis and treatment of disease.
AMPs may induce cell damage in microorganisms in a variety of ways, including by inflicting damage to membranes, destroying
DNA and RNA, or interfering with cell-wall synthesis. Depending on the specific antimicrobial mechanism, a particular AMP may
inhibit only certain groups of microbes (e.g., gram-positive or gram-negative bacteria) or it may be more broadly effective against
bacteria, fungi, protozoa, and viruses. Many AMPs are found on the skin, but they can also be found in other regions of the body.
A family of AMPs called defensins can be produced by epithelial cells throughout the body as well as by cellular defenses such as
macrophages and neutrophils. Defensins may be secreted or act inside host cells; they combat microorganisms by damaging their
plasma membranes. AMPs called bacteriocins are produced exogenously by certain members of the resident microbiota within the
gastrointestinal tract. The genes coding for these types of AMPs are often carried on plasmids and can be passed between different
species within the resident microbiota through lateral or horizontal gene transfer. There are numerous other AMPs throughout the
body. The characteristics of a few of the more significant AMPs are summarized in Table 13.1.2.
Table 13.1.2 : Characteristics of Selected Antimicrobial Peptides (AMPs)
AMP Secreted by Body site Pathogens inhibited Mode of action

Bacteriocins Resident microbiota Gastrointestinal tract Bacteria Disrupt membrane

Epithelial cells,
Cathelicidin macrophages, and other Skin Bacteria and fungi Disrupts membrane
cell types

Epithelial cells, Fungi, bacteria, and many

Defensins Throughout the body Disrupt membrane
macrophages, neutrophils viruses

Disrupts membrane
Dermicidin Sweat glands Skin Bacteria and fungi
integrity and ion channels
Disrupt intracellular
Histatins Salivary glands Oral cavity Fungi
function

Exercise 13.1.3

Why are antimicrobial peptides (AMPs) considered nonspecific defenses?

Mechanical Defenses
In addition to physical barriers that keep microbes out, the body has a number of mechanical defenses that physically remove
pathogens from the body, preventing them from taking up residence. We have already discussed several examples of mechanical
defenses, including the shedding of skin cells, the expulsion of mucus via the mucociliary escalator, and the excretion of feces
through intestinal peristalsis. Other important examples of mechanical defenses include the flushing action of urine and tears,
which both serve to carry microbes away from the body. The flushing action of urine is largely responsible for the normally sterile
environment of the urinary tract, which includes the kidneys, ureters, and urinary bladder. Urine passing out of the body washes out
transient microorganisms, preventing them from taking up residence. The eyes also have physical barriers and mechanical
mechanisms for preventing infections. The eyelashes and eyelids prevent dust and airborne microorganisms from reaching the

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surface of the eye. Any microbes or debris that make it past these physical barriers may be flushed out by the mechanical action of
blinking, which bathes the eye in tears, washing debris away (Figure 13.1.7).
Figure 13.1.7 : Tears flush microbes away from the surface of the eye. Urine washes microbes out of the urinary tract as it passes
through; as a result, the urinary system is normally sterile.

Exercise 13.1.4

Name two mechanical defenses that protect the eyes.

Microbiome
In various regions of the body, resident microbiota serve as an important first-line defense against invading pathogens. Through
their occupation of cellular binding sites and competition for available nutrients, the resident microbiota prevent the critical early
steps of pathogen attachment and proliferation required for the establishment of an infection. For example, in the vagina, members
of the resident microbiota compete with opportunistic pathogens like the yeast Candida. This competition prevents infections by
limiting the availability of nutrients, thus inhibiting the growth of Candida, keeping its population in check. Similar competitions
occur between the microbiota and potential pathogens on the skin, in the upper respiratory tract, and in the gastrointestinal tract.
The resident microbiota also contribute to the chemical defenses of the innate nonspecific host defenses.
The importance of the normal microbiota in host defenses is highlighted by the increased susceptibility to infectious diseases when
the microbiota is disrupted or eliminated. Treatment with antibiotics can significantly deplete the normal microbiota of the
gastrointestinal tract, providing an advantage for pathogenic bacteria to colonize and cause diarrheal infection. In the case of
diarrhea caused by Clostridium difficile, the infection can be severe and potentially lethal. One strategy for treating C. difficile
infections is fecal transplantation, which involves the transfer of fecal material from a donor (screened for potential pathogens) into
the intestines of the recipient patient as a method of restoring the normal microbiota and combating C. difficile infections.
Table 13.1.3 provides a summary of the physical defenses discussed in this section.
Table 13.1.3 : Physical Defenses of Nonspecific Innate Immunity

Defense Examples Function

Cellular barriers Skin, mucous membranes, endothelial cells Deny entry to pathogens

Shedding of skin cells, mucociliary sweeping, Remove pathogens from potential sites of
Mechanical defenses
peristalsis, flushing action of urine and tears infection

Resident bacteria of the skin, upper respiratory

Compete with pathogens for cellular binding
Microbiome tract, gastrointestinal tract, and genitourinary
sites and nutrients
tract

Exercise 13.1.5

List two ways resident microbiota defend against pathogens.

Key Concepts and Summary

Nonspecific innate immunity provides a first line of defense against infection by nonspecifically blocking entry of microbes
and targeting them for destruction or removal from the body.
The physical defenses of innate immunity include physical barriers, mechanical actions that remove microbes and debris, and
the microbiome, which competes with and inhibits the growth of pathogens.
The skin, mucous membranes, and endothelia throughout the body serve as physical barriers that prevent microbes from
reaching potential sites of infection. Tight cell junctions in these tissues prevent microbes from passing through.
Microbes trapped in dead skin cells or mucus are removed from the body by mechanical actions such as shedding of skin cells,
mucociliary sweeping, coughing, peristalsis, and flushing of bodily fluids (e.g., urination, tears)
Numerous chemical mediators produced endogenously and exogenously exhibit nonspecific antimicrobial functions.
Many chemical mediators are found in body fluids such as sebum, saliva, mucus, gastric and intestinal fluids, urine, tears,
cerumen, and vaginal secretions.

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 Antimicrobial peptides (AMPs) found on the skin and in other areas of the body are largely produced in response to the
presence of pathogens. These include dermcidin, cathelicidin, defensins, histatins, and bacteriocins.
The resident microbiota provide a physical defense by occupying available cellular binding sites and competing with pathogens
for available nutrients.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 13.1: First Line defense- Physical, Mechanical and Chemical Defenses is shared under a CC BY license and was authored,
remixed, and/or curated by OpenStax.

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13.2: Second Line Defenses: Cells and Fluids
Learning Objectives
Identify and describe the components of blood
Explain the process by which the formed elements of blood are formed (hematopoiesis)
Describe the characteristics of formed elements found in peripheral blood, as well as their respective functions within the
innate immune system
Explain why the circulatory and lymphatic systems lack normal microbiota

Plasma Protein Mediators

Many nonspecific innate immune factors are found in plasma, the fluid portion of blood. Plasma contains electrolytes, sugars,
lipids, and proteins, each of which helps to maintain homeostasis (i.e., stable internal body functioning), and contains the proteins
involved in the clotting of blood. Additional proteins found in blood plasma, such as acute-phase proteins, complement proteins,
and cytokines, are involved in the nonspecific innate immune response.

Plasma versus Serum

There are two terms for the fluid portion of blood: plasma and serum. How do they differ if they are both fluid and
lack cells? The fluid portion of blood left over after coagulation (blood cell clotting) has taken place is serum.
Although molecules such as many vitamins, electrolytes, certain sugars, complement proteins, and antibodies are
still present in serum, clotting factors are largely depleted. Plasma, conversely, still contains all the clotting
elements. To obtain plasma from blood, an anticoagulant must be used to prevent clotting. Examples of
anticoagulants include heparin and ethylene diamine tetraacetic acid (EDTA). Because clotting is inhibited, once
obtained, the sample must be gently spun down in a centrifuge. The heavier, denser blood cells form a pellet at the
bottom of a centrifuge tube, while the fluid plasma portion, which is lighter and less dense, remains above the cell
pellet.

Acute-Phase Proteins
The acute-phase proteins are another class of antimicrobial mediators. Acute-phase proteins are primarily produced in the liver and
secreted into the blood in response to inflammatory molecules from the immune system. Examples of acute-phase proteins include
C-reactive protein, serum amyloid A, ferritin, transferrin, fibrinogen, and mannose-binding lectin. Each of these proteins has a
different chemical structure and inhibits or destroys microbes in some way (Table 13.2.1).
Table 13.2.1 : Some Acute-Phase Proteins and Their Functions

Some Acute-Phase Proteins and Their Functions

C-reactive protein Coats bacteria (opsonization), preparing them for ingestion by

Serum amyloid A phagocytes

Ferritin
Bind and sequester iron, thereby inhibiting the growth of pathogens
Transferrin

Fibrinogen Involved in formation of blood clots that trap bacterial pathogens

Mannose-binding lectin Activates complement cascade

The Complement System

The complement system is a group of plasma protein mediators that can act as an innate nonspecific defense while also serving to
connect innate and adaptive immunity (discussed in the next chapter). The complement system is composed of more than 30
proteins (including C1 through C9) that normally circulate as precursor proteins in blood. These precursor proteins become
activated when stimulated or triggered by a variety of factors, including the presence of microorganisms. Complement proteins are
considered part of innate nonspecific immunity because they are always present in the blood and tissue fluids, allowing them to be
activated quickly. Also, when activated through the alternative pathway (described later in this section), complement proteins target

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pathogens in a nonspecific manner. The process by which circulating complement precursors become functional is called
complement activation. This process is a cascade that can be triggered by one of three different mechanisms, known as the
alternative, classical, and lectin pathways.
The alternative pathway is initiated by the spontaneous activation of the complement protein C3. The hydrolysis of C3 produces
two products, C3a and C3b. When no invader microbes are present, C3b is very quickly degraded in a hydrolysis reaction using the
water in the blood. However, if invading microbes are present, C3b attaches to the surface of these microbes. Once attached, C3b
will recruit other complement proteins in a cascade (Figure 13.2.1).
The classical pathway provides a more efficient mechanism of activating the complement cascade, but it depends upon the
production of antibodies by the specific adaptive immune defenses. To initiate the classical pathway, a specific antibody must first
bind to the pathogen to form an antibody-antigen complex. This activates the first protein in the complement cascade, the C1
complex. The C1 complex is a multipart protein complex, and each component participates in the full activation of the overall
complex. Following recruitment and activation of the C1 complex, the remaining classical pathway complement proteins are
recruited and activated in a cascading sequence (Figure 13.2.1).
The lectin activation pathway is similar to the classical pathway, but it is triggered by the binding of mannose-binding lectin, an
acute-phase protein, to carbohydrates on the microbial surface. Like other acute-phase proteins, lectins are produced by liver cells
and are commonly upregulated in response to inflammatory signals received by the body during an infection (Figure 13.2.1).

Figure 13.2.1 : The three complement activation pathways have different triggers, as shown here, but all three result in the
activation of the complement protein C3, which produces C3a and C3b. The latter binds to the surface of the target cell and then
works with other complement proteins to cleave C5 into C5a and C5b. C5b also binds to the cell surface and then recruits C6
through C9; these molecules form a ring structure called the membrane attack complex (MAC), which punches through the cell
membrane of the invading pathogen, causing it to swell and burst.
Although each complement activation pathway is initiated in a different way, they all provide the same protective outcomes:
opsonization, inflammation, chemotaxis, and cytolysis. The term opsonization refers to the coating of a pathogen by a chemical
substance (called an opsonin) that allows phagocytic cells to recognize, engulf, and destroy it more easily. Opsonins from the
complement cascade include C1q, C3b, and C4b. Additional important opsonins include mannose-binding proteins and antibodies.
The complement fragments C3a and C5a are well-characterized anaphylatoxins with potent proinflammatory functions.
Anaphylatoxins activate mast cells, causing degranulation and the release of inflammatory chemical signals, including mediators
that cause vasodilation and increased vascular permeability. C5a is also one of the most potent chemoattractants for neutrophils and
other white blood cells, cellular defenses that will be discussed in the next section.
The complement proteins C6, C7, C8, and C9 assemble into a membrane attack complex (MAC), which allows C9 to polymerize
into pores in the membranes of gram-negative bacteria. These pores allow water, ions, and other molecules to move freely in and
out of the targeted cells, eventually leading to cell lysis and death of the pathogen (Figure 13.2.1). However, the MAC is only
effective against gram-negative bacteria; it cannot penetrate the thick layer of peptidoglycan associated with cell walls of gram-
positive bacteria. Since the MAC does not pose a lethal threat to gram-positive bacterial pathogens, complement-mediated
opsonization is more important for their clearance.

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Cytokines
Cytokines are soluble proteins that act as communication signals between cells. In a nonspecific innate immune response, various
cytokines may be released to stimulate production of chemical mediators or other cell functions, such as cell proliferation, cell
differentiation, inhibition of cell division, apoptosis, and chemotaxis.
When a cytokine binds to its target receptor, the effect can vary widely depending on the type of cytokine and the type of cell or
receptor to which it has bound. The function of a particular cytokine can be described as autocrine, paracrine, or endocrine (Figure
13.2.2). In autocrine function, the same cell that releases the cytokine is the recipient of the signal; in other words, autocrine

function is a form of self-stimulation by a cell. In contrast, paracrine function involves the release of cytokines from one cell to
other nearby cells, stimulating some response from the recipient cells. Last, endocrine function occurs when cells release cytokines
into the bloodstream to be carried to target cells much farther away.

Figure 13.2.2 : Autocrine, paracrine, and endocrine actions describe which cells are targeted by cytokines and how far the cytokines
must travel to bind to their intended target cells’ receptors.
Three important classes of cytokines are the interleukins, chemokines, and interferons. The interleukins were originally thought to
be produced only by leukocytes (white blood cells) and to only stimulate leukocytes, thus the reasons for their name. Although
interleukins are involved in modulating almost every function of the immune system, their role in the body is not restricted to
immunity. Interleukins are also produced by and stimulate a variety of cells unrelated to immune defenses.
The chemokines are chemotactic factors that recruit leukocytes to sites of infection, tissue damage, and inflammation. In contrast to
more general chemotactic factors, like complement factor C5a, chemokines are very specific in the subsets of leukocytes they
recruit.
Interferons are a diverse group of immune signaling molecules and are especially important in our defense against viruses. Type I
interferons (interferon-α and interferon-β) are produced and released by cells infected with virus. These interferons stimulate
nearby cells to stop production of mRNA, destroy RNA already produced, and reduce protein synthesis. These cellular changes
inhibit viral replication and production of mature virus, slowing the spread of the virus. Type I interferons also stimulate various
immune cells involved in viral clearance to more aggressively attack virus-infected cells. Type II interferon (interferon-γ) is an
important activator of immune cells (Figure 13.2.3).

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 Figure 13.2.3 : Interferons are cytokines released by a cell infected with a virus. Interferon-α and interferon-β signal uninfected
neighboring cells to inhibit mRNA synthesis, destroy RNA, and reduce protein synthesis (top arrow). Interferon-α and interferon-β
also promote apoptosis in cells infected with the virus (middle arrow). Interferon-γ alerts neighboring immune cells to an attack
(bottom arrow). Although interferons do not cure the cell releasing them or other infected cells, which will soon die, their release
may prevent additional cells from becoming infected, thus stemming the infection.

Inflammation-Eliciting Mediators
Many of the chemical mediators discussed in this section contribute in some way to inflammation and fever, which are nonspecific
immune responses. Cytokines stimulate the production of acute-phase proteins such as C-reactive protein and mannose-binding
lectin in the liver. These acute-phase proteins act as opsonins, activating complement cascades through the lectin pathway.
Some cytokines also bind mast cells and basophils, inducing them to release histamine, a proinflammatory compound. Histamine
receptors are found on a variety of cells and mediate proinflammatory events, such as bronchoconstriction (tightening of the
airways) and smooth muscle contraction.
In addition to histamine, mast cells may release other chemical mediators, such as leukotrienes. Leukotrienes are lipid-based
proinflammatory mediators that are produced from the metabolism of arachidonic acid in the cell membrane of leukocytes and
tissue cells. Compared with the proinflammatory effects of histamine, those of leukotrienes are more potent and longer lasting.
Together, these chemical mediators can induce coughing, vomiting, and diarrhea, which serve to expel pathogens from the body.
Certain cytokines also stimulate the production of prostaglandins, chemical mediators that promote the inflammatory effects of
kinins and histamines. Prostaglandins can also help to set the body temperature higher, leading to fever, which promotes the
activities of white blood cells and slightly inhibits the growth of pathogenic microbes.
Another inflammatory mediator, bradykinin, contributes to edema, which occurs when fluids and leukocytes leak out of the
bloodstream and into tissues. It binds to receptors on cells in the capillary walls, causing the capillaries to dilate and become more
permeable to fluids.

Exercise 13.2.1

1. What do the three complement activation pathways have in common?

2. Explain autocrine, paracrine, and endocrine signals.
3. Name two important inflammation-eliciting mediators.

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Non-specific Cells
The non-fluid portion of blood consists of various types of formed elements, so called because they are all formed from the same
stem cells found in bone marrow. The three major categories of formed elements are: red blood cells (RBCs), also called
erythrocytes; platelets, also called thrombocytes; and white blood cells (WBCs), also called leukocytes.
Red blood cells are primarily responsible for carrying oxygen to tissues. Platelets are cellular fragments that participate in blood
clot formation and tissue repair. Several different types of WBCs participate in various nonspecific mechanisms of innate and
adaptive immunity. In this section, we will focus primarily on the innate mechanisms of various types of WBCs.

Hematopoiesis
All of the formed elements of blood are derived from pluripotent hematopoietic stem cells (HSCs) in the bone marrow. As the
HSCs make copies of themselves in the bone marrow, individual cells receive different cues from the body that control how they
develop and mature. As a result, the HSCs differentiate into different types of blood cells that, once mature, circulate in peripheral
blood. This process of differentiation, called hematopoiesis, is shown in more detail in Figure 13.2.4.
In terms of sheer numbers, the vast majority of HSCs become erythrocytes. Much smaller numbers become leukocytes and
platelets. Leukocytes can be further subdivided into granulocytes, which are characterized by numerous granules visible in the
cytoplasm, and agranulocytes, which lack granules. Figure 13.2.5 provides an overview of the various types of formed elements,
including their relative numbers, primary function, and lifespans.

Figure 13.2.4 : All the formed elements of the blood arise by differentiation of hematopoietic stem cells in the bone marrow.

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Figure 13.2.5 : Formed elements of blood include erythrocytes (red blood cells), leukocytes (white blood cells), and platelets.

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Granulocytes
The various types of granulocytes can be distinguished from one another in a blood smear by the appearance of their nuclei and the
contents of their granules, which confer different traits, functions, and staining properties. The neutrophils, also called
polymorphonuclear neutrophils (PMNs), have a nucleus with three to five lobes and small, numerous, lilac-colored granules. Each
lobe of the nucleus is connected by a thin strand of material to the other lobes. The eosinophils have fewer lobes in the nucleus
(typically 2–3) and larger granules that stain reddish-orange. The basophils have a two-lobed nucleus and large granules that stain
dark blue or purple (Figure 13.2.6).

Figure 13.2.6 : Granulocytes can be distinguished by the number of lobes in their nuclei and the staining properties of their
granules. (credit “neutrophil” micrograph: modification of work by Ed Uthman)
Neutrophils (PMNs)
Neutrophils (PMNs) are frequently involved in the elimination and destruction of extracellular bacteria. They are capable of
migrating through the walls of blood vessels to areas of bacterial infection and tissue damage, where they seek out and kill
infectious bacteria. PMN granules contain a variety of defensins and hydrolytic enzymes that help them destroy bacteria through
phagocytosis. In addition, when many neutrophils are brought into an infected area, they can be stimulated to release toxic
molecules into the surrounding tissue to better clear infectious agents. This is called degranulation.
Another mechanism used by neutrophils is neutrophil extracellular traps (NETs), which are extruded meshes of chromatin that are
closely associated with antimicrobial granule proteins and components. Chromatin is DNA with associated proteins (usually
histone proteins, around which DNA wraps for organization and packing within a cell). By creating and releasing a mesh or lattice-
like structure of chromatin that is coupled with antimicrobial proteins, the neutrophils can mount a highly concentrated and
efficient attack against nearby pathogens. Proteins frequently associated with NETs include lactoferrin, gelatinase, cathepsin G, and
myeloperoxidase. Each has a different means of promoting antimicrobial activity, helping neutrophils eliminate pathogens. The
toxic proteins in NETs may kill some of the body’s own cells along with invading pathogens. However, this collateral damage can
be repaired after the danger of the infection has been eliminated.
As neutrophils fight an infection, a visible accumulation of leukocytes, cellular debris, and bacteria at the site of infection can be
observed. This buildup is what we call pus (also known as purulent or suppurative discharge or drainage). The presence of pus is a
sign that the immune defenses have been activated against an infection; historically, some physicians believed that inducing pus
formation could actually promote the healing of wounds. The practice of promoting “laudable pus” (by, for instance, wrapping a
wound in greasy wool soaked in wine) dates back to the ancient physician Galen in the 2nd century AD, and was practiced in

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variant forms until the 17th century (though it was not universally accepted). Today, this method is no longer practiced because we
now know that it is not effective. Although a small amount of pus formation can indicate a strong immune response, artificially
inducing pus formation does not promote recovery.
Eosinophils

Eosinophils are granulocytes that protect against protozoa and helminths; they also play a role in allergic reactions. The granules of
eosinophils, which readily absorb the acidic reddish dye eosin, contain histamine, degradative enzymes, and a compound known as
major basic protein (MBP) (Figure 13.2.3). MBP binds to the surface carbohydrates of parasites, and this binding is associated with
disruption of the cell membrane and membrane permeability.
Basophils
Basophils have cytoplasmic granules of varied size and are named for their granules’ ability to absorb the basic dye methylene blue
(Figure 13.2.3). Their stimulation and degranulation can result from multiple triggering events. Activated complement fragments
C3a and C5a, produced in the activation cascades of complement proteins, act as anaphylatoxins by inducing degranulation of
basophils and inflammatory responses. This cell type is important in allergic reactions and other responses that involve
inflammation. One of the most abundant components of basophil granules is histamine, which is released along with other chemical
factors when the basophil is stimulated. These chemicals can be chemotactic and can help to open the gaps between cells in the
blood vessels. Other mechanisms for basophil triggering require the assistance of antibodies.
Mast Cells

Hematopoiesis also gives rise to mast cells, which appear to be derived from the same common myeloid progenitor cell as
neutrophils, eosinophils, and basophils. Functionally, mast cells are very similar to basophils, containing many of the same
components in their granules (e.g., histamine) and playing a similar role in allergic responses and other inflammatory reactions.
However, unlike basophils, mast cells leave the circulating blood and are most frequently found residing in tissues. They are often
associated with blood vessels and nerves or found close to surfaces that interface with the external environment, such as the skin
and mucous membranes in various regions of the body (Figure 13.2.7).

Figure 13.2.7 : Mast cells function similarly to basophils by inducing and promoting inflammatory responses. (a) This figure shows
mast cells in blood. In a blood smear, they are difficult to differentiate from basophils (b). Unlike basophils, mast cells migrate
from the blood into various tissues. (credit right: modification of work by Greenland JR, Xu X, Sayah DM, Liu FC, Jones KD,
Looney MR, Caughey GH)

Exercise 13.2.2
1. Describe the granules and nuclei of neutrophils, eosinophils, basophils, and mast cells.
2. Name three antimicrobial mechanisms of neutrophils

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 Clinical Focus: part 2
To relieve the constriction of her airways, Angela is immediately treated with antihistamines and administered corticosteroids
through an inhaler, and then monitored for a period of time. Though her condition does not worsen, the drugs do not seem to be
alleviating her condition. She is admitted to the hospital for further observation, testing, and treatment.
Following admission, a clinician conducts allergy testing to try to determine if something in her environment might be
triggering an allergic inflammatory response. A doctor orders blood analysis to check for levels of particular cytokines. A
sputum sample is also taken and sent to the lab for microbial staining, culturing, and identification of pathogens that could be
causing an infection.

Exercise 13.2.3

1. Which aspects of the innate immune system could be contributing to Angela’s airway constriction?
2. Why was Angela treated with antihistamines?
3. Why would the doctor be interested in levels of cytokines in Angela’s blood?

Agranulocytes
As their name suggests, agranulocytes lack visible granules in the cytoplasm. Agranulocytes can be categorized as lymphocytes or
monocytes (Figure 13.2.2). Among the lymphocytes are natural killer cells, which play an important role in nonspecific innate
immune defenses. Lymphocytes also include the B cells and T cells, which are discussed in the next chapter because they are
central players in the specific adaptive immune defenses. The monocytes differentiate into macrophages and dendritic cells, which
are collectively referred to as the mononuclear phagocyte system.
Natural Killer Cells
Most lymphocytes are primarily involved in the specific adaptive immune response, and thus will be discussed in the following
chapter. An exception is the natural killer cells (NK cells); these mononuclear lymphocytes use nonspecific mechanisms to
recognize and destroy cells that are abnormal in some way. Cancer cells and cells infected with viruses are two examples of cellular
abnormalities that are targeted by NK cells. Recognition of such cells involves a complex process of identifying inhibitory and
activating molecular markers on the surface of the target cell. Molecular markers that make up the major histocompatibility
complex (MHC) are expressed by healthy cells as an indication of “self.” This will be covered in more detail in next chapter. NK
cells are able to recognize normal MHC markers on the surface of healthy cells, and these MHC markers serve as an inhibitory
signal preventing NK cell activation. However, cancer cells and virus-infected cells actively diminish or eliminate expression of
MHC markers on their surface. When these MHC markers are diminished or absent, the NK cell interprets this as an abnormality
and a cell in distress. This is one part of the NK cell activation process (Figure 13.2.8). NK cells are also activated by binding to
activating molecular molecules on the target cell. These activating molecular molecules include “altered self” or “nonself”
molecules. When a NK cell recognizes a decrease in inhibitory normal MHC molecules and an increase in activating molecules on
the surface of a cell, the NK cell will be activated to eliminate the cell in distress.
Once a cell has been recognized as a target, the NK cell can use several different mechanisms to kill its target. For example, it may
express cytotoxic membrane proteins and cytokines that stimulate the target cell to undergo apoptosis, or controlled cell suicide.
NK cells may also use perforin-mediated cytotoxicity to induce apoptosis in target cells. This mechanism relies on two toxins
released from granules in the cytoplasm of the NK cell: perforin, a protein that creates pores in the target cell, and granzymes,
proteases that enter through the pores into the target cell’s cytoplasm, where they trigger a cascade of protein activation that leads
to apoptosis. The NK cell binds to the abnormal target cell, releases its destructive payload, and detaches from the target cell. While
the target cell undergoes apoptosis, the NK cell synthesizes more perforin and proteases to use on its next target.

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 Figure 13.2.8 : Natural killer (NK) cells are inhibited by the presence of the major histocompatibility cell (MHC) receptor on
healthy cells. Cancer cells and virus-infected cells have reduced expression of MHC and increased expression of activating
molecules. When a NK cell recognizes decreased MHC and increased activating molecules, it will kill the abnormal cell.
NK cells contain these toxic compounds in granules in their cytoplasm. When stained, the granules are azurophilic and can be
visualized under a light microscope (Figure 13.2.9). Even though they have granules, NK cells are not considered granulocytes
because their granules are far less numerous than those found in true granulocytes. Furthermore, NK cells have a different lineage
than granulocytes, arising from lymphoid rather than myeloid stem cells (Figure 13.2.9).

Figure 13.2.9: Natural killer cell with perforin-containing granules. (credit: modification of work by Rolstad B)

Monocytes

The largest of the white blood cells, monocytes have a nucleus that lacks lobes, and they also lack granules in the cytoplasm
(Figure 13.2.10). Nevertheless, they are effective phagocytes, engulfing pathogens and apoptotic cells to help fight infection.
When monocytes leave the bloodstream and enter a specific body tissue, they differentiate into tissue-specific phagocytes called
macrophages and dendritic cells. They are particularly important residents of lymphoid tissue, as well as nonlymphoid sites and

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organs. Macrophages and dendritic cells can reside in body tissues for significant lengths of time. Macrophages in specific body
tissues develop characteristics suited to the particular tissue. Not only do they provide immune protection for the tissue in which
they reside but they also support normal function of their neighboring tissue cells through the production of cytokines.
Macrophages are given tissue-specific names, and a few examples of tissue-specific macrophages are listed in Table 13.2.2.
Dendritic cells are important sentinels residing in the skin and mucous membranes, which are portals of entry for many pathogens.
Monocytes, macrophages, and dendritic cells are all highly phagocytic and important promoters of the immune response through
their production and release of cytokines. These cells provide an essential bridge between innate and adaptive immune responses,
as discussed in the next section as well as the next chapter.

Figure 13.2.10: Monocytes are large, agranular white blood cells with a nucleus that lacks lobes. When monocytes leave the
bloodstream, they differentiate and become macrophages with tissue-specific properties. (credit left: modification of work by
Armed Forces Institute of Pathology; credit right: modification of work by Centers for Disease Control and Prevention)
Table 13.2.2 : Macrophages Found in Various Body Tissues

Tissue Macrophage

Brain and central nervous system Microglial cells

Liver Kupffer cells

Lungs Alveolar macrophages (dust cells)

Peritoneal cavity Peritoneal macrophages

Exercise 13.2.4
1. Describe the signals that activate natural killer cells.
2. What is the difference between monocytes and macrophages?

The Circulatory System

The proteins, fluids and cells of the second line defense travel via two systems. The main system being the circulatory (or
cardiovascular) system is a closed network of organs and vessels that moves blood around the body (Figure 13.2.11). The primary
purposes of the circulatory system are to deliver nutrients, immune factors, and oxygen to tissues and to carry away waste products
for elimination. The heart is a four-chambered pump that propels the blood throughout the body. When the heart contracts, the
blood from the right ventricle is pumped through the pulmonary arteries to the lungs. There, the blood is oxygenated at the alveoli
and returns to the heart through the pulmonary veins. When the heart contracts, the oxygenated blood is pumped throughout the
body via a series of thick-walled vessels called arteries. The first and largest artery is called the aorta. The arteries sequentially
branch and decrease in size (and are called arterioles) until they end in a network of smaller vessels called capillaries. The capillary
beds are located in the interstitial spaces within tissues and release nutrients, immune factors, and oxygen to those tissues. The
capillaries connect to a series of vessels called venules, which increase in size to form the veins. The veins join together into larger

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vessels as they transfer blood back to the heart. The largest veins, the superior and inferior vena cava, return the blood to the right
atrium.

Figure 13.2.11: The major components of the human circulatory system include the heart, arteries, veins, and capillaries. This
network delivers blood to the body’s organs and tissues. (credit top left: modification of work by Mariana Ruiz Villareal; credit
bottom right: modification of work by Bruce Blaus)
Other organs play important roles in the circulatory system as well. The kidneys filter the blood, removing waste products and
eliminating them in the urine. The liver also filters the blood and removes damaged or defective red blood cells. The spleen filters
and stores blood, removes damaged red blood cells, and is a reservoir for immune factors. All of these filtering structures serve as
sites for entrapment of microorganisms and help maintain an environment free of microorganisms in the blood.

The Lymphatic System

The lymphatic system is also a network of vessels that run throughout the body (Figure 13.2.12). However, these vessels do not
form a full circulating system and are not pressurized by the heart. Rather, the lymphatic system is an open system with the fluid
moving in one direction from the extremities toward two drainage points into veins just above the heart. Lymphatic fluids move
more slowly than blood because they are not pressurized. Small lymph capillaries interact with blood capillaries in the interstitial
spaces in tissues. Fluids from the tissues enter the lymph capillaries and are drained away. These fluids, termed lymph, also contain
large numbers of white blood cells.

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 Figure 13.2.12: The essential components of the human lymphatic system drain fluid away from tissues.

The lymphatic system contains two types of lymphoid tissues. The primary lymphoid tissue includes bone marrow (containing the
hematopoietic stem cells) and the thymus. The secondary lymphoid tissues include the spleen, lymph nodes, and several areas of
diffuse lymphoid tissues underlying epithelial membranes. The spleen, an encapsulated structure, filters blood and captures
pathogens and antigens that pass into it (Figure 13.2.13). The spleen contains specialized macrophages and dendritic cells that are
crucial for antigen presentation, a mechanism critical for activation of T lymphocytes and B lymphocytes in the third line of
defense. Lymph nodes are bean-shaped organs situated throughout the body. These structures contain areas called germinal centers
that are rich in B and T lymphocytes. The lymph nodes also contain macrophages and dendritic cells for antigen presentation.
Lymph from nearby tissues enters the lymph node through afferent lymphatic vessels and encounters these lymphocytes as it passes
through; the lymph exits the lymph node through the efferent lymphatic vessels (Figure 13.2.13).

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 Figure 13.2.13: (a) The spleen is a lymphatic organ located in the upper left quadrant of the abdomen near the stomach and left
kidney. It contains numerous phagocytes and lymphocytes that combat and prevent circulatory infections by killing and removing
pathogens from the blood. (b) Lymph nodes are masses of lymphatic tissue located along the larger lymph vessels. They contain
numerous lymphocytes that kill and remove pathogens from lymphatic fluid that drains from surrounding tissues.
The lymphatic system filters fluids that have accumulated in tissues before they are returned to the blood. A brief overview of this
process is provided at this website.

Exercise 13.2.5

What is the main function of the lymphatic system?

Key Concepts and Summary

Plasma contains various proteins that serve as chemical mediators, including acute-phase proteins, complement proteins, and
cytokines.
The complement system involves numerous precursor proteins that circulate in plasma. These proteins become activated in a
cascading sequence in the presence of microbes, resulting in the opsonization of pathogens, chemoattraction of leukocytes,
induction of inflammation, and cytolysis through the formation of a membrane attack complex (MAC).
Cytokines are proteins that facilitate various nonspecific responses by innate immune cells, including production of other
chemical mediators, cell proliferation, cell death, and differentiation.
Cytokines play a key role in the inflammatory response, triggering production of inflammation-eliciting mediators such as
acute-phase proteins, histamine, leukotrienes, prostaglandins, and bradykinin.
The formed elements of the blood include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets
(thrombocytes). Of these, leukocytes are primarily involved in the immune response.
All formed elements originate in the bone marrow as stem cells (HSCs) that differentiate through hematopoiesis.
Granulocytes are leukocytes characterized by a lobed nucleus and granules in the cytoplasm. These include neutrophils
(PMNs), eosinophils, and basophils.
Neutrophils are the leukocytes found in the largest numbers in the bloodstream and they primarily fight bacterial infections.
Eosinophils target parasitic infections. Eosinophils and basophils are involved in allergic reactions. Both release histamine and
other proinflammatory compounds from their granules upon stimulation.
Mast cells function similarly to basophils but can be found in tissues outside the bloodstream.
Natural killer (NK) cells are lymphocytes that recognize and kill abnormal or infected cells by releasing proteins that trigger
apoptosis.
Monocytes are large, mononuclear leukocytes that circulate in the bloodstream. They may leave the bloodstream and take up
residence in body tissues, where they differentiate and become tissue-specific macrophages and dendritic cells.

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 The circulatory system and the lymphatic system provide different but connected transport systems for them proteins and cells
to be able to reach (almost) the entire body.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 13.2: Second Line Defenses: Cells and Fluids is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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13.3: Pathogen Recognition and Phagocytosis
Learning Objectives
Explain how leukocytes migrate from peripheral blood into infected tissues
Explain the mechanisms by which leukocytes recognize pathogens
Explain the process of phagocytosis and the mechanisms by which phagocytes destroy and degrade pathogens

Several of the cell types discussed in the previous section can be described as phagocytes—cells whose main function is to seek,
ingest, and kill pathogens. This process, called phagocytosis, was first observed in starfish in the 1880s by Nobel Prize-winning
zoologist Ilya Metchnikoff (1845–1916), who made the connection to white blood cells (WBCs) in humans and other animals. At
the time, Pasteur and other scientists believed that WBCs were spreading pathogens rather than killing them (which is true for some
diseases, such as tuberculosis). But in most cases, phagocytes provide a strong, swift, and effective defense against a broad range of
microbes, making them a critical component of innate nonspecific immunity. This section will focus on the mechanisms by which
phagocytes are able to seek, recognize, and destroy pathogens.

Extravasation (Diapedesis) of Leukocytes

Some phagocytes are leukocytes (WBCs) that normally circulate in the bloodstream. To reach pathogens located in infected tissue,
leukocytes must pass through the walls of small capillary blood vessels within tissues. This process, called extravasation, or
diapedesis, is initiated by complement factor C5a, as well as cytokines released into the immediate vicinity by resident
macrophages and tissue cells responding to the presence of the infectious agent (Figure 13.3.1). Similar to C5a, many of these
cytokines are proinflammatory and chemotactic, and they bind to cells of small capillary blood vessels, initiating a response in the
endothelial cells lining the inside of the blood vessel walls. This response involves the upregulation and expression of various
cellular adhesion molecules and receptors. Leukocytes passing through will stick slightly to the adhesion molecules, slowing down
and rolling along the blood vessel walls near the infected area. When they reach a cellular junction, they will bind to even more of
these adhesion molecules, flattening out and squeezing through the cellular junction in a process known as transendothelial
migration. This mechanism of “rolling adhesion” allows leukocytes to exit the bloodstream and enter the infected areas, where they
can begin phagocytosing the invading pathogens.
Note that extravasation does not occur in arteries or veins. These blood vessels are surrounded by thicker, multilayer protective
walls, in contrast to the thin single-cell-layer walls of capillaries. Furthermore, the blood flow in arteries is too turbulent to allow
for rolling adhesion. Also, some leukocytes tend to respond to an infection more quickly than others. The first to arrive typically
are neutrophils, often within hours of a bacterial infection. By contract, monocytes may take several days to leave the bloodstream
and differentiate into macrophages.

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 Figure 13.3.1 : Damaged cells and macrophages that have ingested pathogens release cytokines that are proinflammatory and
chemotactic for leukocytes. In addition, activation of complement at the site of infection results in production of the chemotactic
and proinflammatory C5a. Leukocytes exit the blood vessel and follow the chemoattractant signal of cytokines and C5a to the site
of infection. Granulocytes such as neutrophils release chemicals that destroy pathogens. They are also capable of phagocytosis and
intracellular killing of bacterial pathogens.
Watch the following videos on leukocyte extravasation and leukocyte rolling to learn more.

Exercise 13.3.1

Explain the role of adhesion molecules in the process of extravasation.

Pathogen Recognition
Opsonization (coating by signalling proteins) of pathogens by antibody; complement factors C1q, C3b, and C4b; and lectins can
assist phagocytic cells in recognition of pathogens and attachment to initiate phagocytosis. However, not all pathogen recognition is
opsonin dependent. Phagocytes can also recognize molecular structures that are common to many groups of pathogenic microbes.
Such structures are called pathogen-associated molecular patterns (PAMPs). Common PAMPs include the following:
peptidoglycan, found in bacterial cell walls;
flagellin, a protein found in bacterial flagella;
lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria;
lipopeptides, molecules expressed by most bacteria; and
nucleic acids such as viral DNA or RNA.

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Like numerous other PAMPs, these substances are integral to the structure of broad classes of microbes.
The structures that allow phagocytic cells to detect PAMPs are called pattern recognition receptors (PRRs). One group of PRRs is
the toll-like receptors (TLRs), which bind to various PAMPs and communicate with the nucleus of the phagocyte to elicit a
response. Many TLRs (and other PRRs) are located on the surface of a phagocyte, but some can also be found embedded in the
membranes of interior compartments and organelles (Figure 13.3.2). These interior PRRs can be useful for the binding and
recognition of intracellular pathogens that may have gained access to the inside of the cell before phagocytosis could take place.
Viral nucleic acids, for example, might encounter an interior PRR, triggering production of the antiviral cytokine interferon.
In addition to providing the first step of pathogen recognition, the interaction between PAMPs and PRRs on macrophages provides
an intracellular signal that activates the phagocyte, causing it to transition from a dormant state of readiness and slow proliferation
to a state of hyperactivity, proliferation, production/secretion of cytokines, and enhanced intracellular killing. PRRs on
macrophages also respond to chemical distress signals from damaged or stressed cells. This allows macrophages to extend their
responses beyond protection from infectious diseases to a broader role in the inflammatory response initiated from injuries or other
diseases.

Figure 13.3.2 : Phagocytic cells contain pattern recognition receptors (PRRs) capable of recognizing various pathogen-associated
molecular patterns (PAMPs). These PRRs can be found on the plasma membrane or in internal phagosomes. When a PRR
recognizes a PAMP, it sends a signal to the nucleus that activates genes involved in phagocytosis, cellular proliferation, production
and secretion of antiviral interferons and proinflammatory cytokines, and enhanced intracellular killing.

Exercise 13.3.2

1. Name four pathogen-associated molecular patterns (PAMPs).

2. Describe the process of phagocyte activation.

Clinical Focus: Resolution

Given her father’s premature death, Angela’s doctor suspects that she has hereditary angioedema, a genetic disorder that
compromises the function of C1 inhibitor protein. Patients with this genetic abnormality may have occasional episodes of
swelling in various parts of the body. In Angela’s case, the swelling has occurred in the respiratory tract, leading to difficulty
breathing. Swelling may also occur in the gastrointestinal tract, causing abdominal cramping, diarrhea, and vomiting, or in the
muscles of the face or limbs. This swelling may be nonresponsive to steroid treatment and is often misdiagnosed as an allergy.
Because there are three types of hereditary angioedema, the doctor orders a more specific blood test to look for levels of C1-
INH, as well as a functional assay of Angela’s C1 inhibitors. The results suggest that Angela has type I hereditary angioedema,

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 which accounts for 80%–85% of all cases. This form of the disorder is caused by a deficiency in C1 esterase inhibitors, the
proteins that normally help suppress activation of the complement system. When these proteins are deficient or nonfunctional,
overstimulation of the system can lead to production of inflammatory anaphylatoxins, which results in swelling and fluid
buildup in tissues.
There is no cure for hereditary angioedema, but timely treatment with purified and concentrated C1-INH from blood donors
can be effective, preventing tragic outcomes like the one suffered by Angela’s father. A number of therapeutic drugs, either
currently approved or in late-stage human trials, may also be considered as options for treatment in the near future. These drugs
work by inhibiting inflammatory molecules or the receptors for inflammatory molecules.
Thankfully, Angela’s condition was quickly diagnosed and treated. Although she may experience additional episodes in the
future, her prognosis is good and she can expect to live a relatively normal life provided she seeks treatment at the onset of
symptoms.

Pathogen Degradation
Once pathogen recognition and attachment occurs, the pathogen is engulfed in a vesicle and brought into the internal compartment
of the phagocyte in a process called phagocytosis (Figure 13.3.3). PRRs can aid in phagocytosis by first binding to the pathogen’s
surface, but phagocytes are also capable of engulfing nearby items even if they are not bound to specific receptors. To engulf the
pathogen, the phagocyte forms a pseudopod that wraps around the pathogen and then pinches it off into a membrane vesicle called
a phagosome. Acidification of the phagosome (pH decreases to the range of 4–5) provides an important early antibacterial
mechanism. The phagosome containing the pathogen fuses with one or more lysosomes, forming a phagolysosome. Formation of
the phagolysosome enhances the acidification, which is essential for activation of pH-dependent digestive lysosomal enzymes and
production of hydrogen peroxide and toxic reactive oxygen species. Lysosomal enzymes such as lysozyme, phospholipase, and
proteases digest the pathogen. Other enzymes are involved a respiratory burst. During the respiratory burst, phagocytes will
increase their uptake and consumption of oxygen, but not for energy production. The increased oxygen consumption is focused on
the production of superoxide anion, hydrogen peroxide, hydroxyl radicals, and other reactive oxygen species that are antibacterial.
In addition to the reactive oxygen species produced by the respiratory burst, reactive nitrogen compounds with cytotoxic (cell-
killing) potential can also form. For example, nitric oxide can react with superoxide to form peroxynitrite, a highly reactive
nitrogen compound with degrading capabilities similar to those of the reactive oxygen species. Some phagocytes even contain an
internal storehouse of microbicidal defensin proteins (e.g., neutrophil granules). These destructive forces can be released into the
area around the cell to degrade microbes externally. Neutrophils, especially, can be quite efficient at this secondary antimicrobial
mechanism.
Once degradation is complete, leftover waste products are excreted from the cell in an exocytic vesicle. However, it is important to
note that not all remains of the pathogen are excreted as waste. Macrophages and dendritic cells are also antigen-presenting cells
involved in the specific adaptive immune response. These cells further process the remains of the degraded pathogen and present
key antigens (specific pathogen proteins) on their cellular surface. This is an important step for stimulation of some adaptive
immune responses, as will be discussed in more detail in the next chapter.

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 Figure 13.3.3 : The stages of phagocytosis include the engulfment of a pathogen, the formation of a phagosome, the digestion of the
pathogenic particle in the phagolysosome, and the expulsion of undigested materials from the cell.
Visit this link to view a phagocyte chasing and engulfing a pathogen.

Exercise 13.3.3

What is the difference between a phagosome and a lysosome?

When Phagocytosis Fails

Although phagocytosis successfully destroys many pathogens, some are able to survive and even exploit this defense
mechanism to multiply in the body and cause widespread infection. Protozoans of the genus Leishmania are one example.
These obligate intracellular parasites are flagellates transmitted to humans by the bite of a sand fly. Infections cause serious and
sometimes disfiguring sores and ulcers in the skin and other tissues (Figure 13.3.4). Worldwide, an estimated 1.3 million
people are newly infected with leishmaniasis annually. 1
Salivary peptides from the sand fly activate host macrophages at the site of their bite. The classic or alternate pathway for
complement activation ensues with C3b opsonization of the parasite. Leishmania cells are phagocytosed, lose their flagella,
and multiply in a form known as an amastigote (Leishman-Donovan body) within the phagolysosome. Although many other
pathogens are destroyed in the phagolysosome, survival of the Leishmania amastigotes is maintained by the presence of
surface lipophosphoglycan and acid phosphatase. These substances inhibit the macrophage respiratory burst and lysosomal
enzymes. The parasite then multiplies inside the cell and lyses the infected macrophage, releasing the amastigotes to infect
other macrophages within the same host. Should another sand fly bite an infected person, it might ingest amastigotes and then
transmit them to another individual through another bite.
There are several different forms of leishmaniasis. The most common is a localized cutaneous form of the illness caused by L.
tropica, which typically resolves spontaneously over time but with some significant lymphocyte infiltration and permanent
scarring. A mucocutaneous form of the disease, caused by L. viannia brasilienfsis, produces lesions in the tissue of the nose
and mouth and can be life threatening. A visceral form of the illness can be caused by several of the different Leishmania
species. It affects various organ systems and causes abnormal enlargement of the liver and spleen. Irregular fevers, anemia,
liver dysfunction, and weight loss are all signs and symptoms of visceral leishmaniasis. If left untreated, it is typically fatal.

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 Figure 13.3.4 : (a) Cutaneous leishmaniasis is a disfiguring disease caused by the intracellular flagellate Leishmania tropica,
transmitted by the bite of a sand fly. (b) This light micrograph of a sample taken from a skin lesion shows a large cell, which is
a macrophage infected with L. tropica amastigotes (arrows). The amastigotes have lost their flagella but their nuclei are visible.
Soon the amastigotes will lyse the macrophage and be engulfed by other phagocytes, spreading the infection. (credit a:
modification of work by Otis Historical Archives of “National Museum of Health & Medicine”; credit b: modification of work
by Centers for Disease Control and Prevention)

Key Concepts and Summary

Phagocytes are cells that recognize pathogens and destroy them through phagocytosis.
Recognition often takes place by the use of phagocyte receptors that bind molecules commonly found on pathogens, known as
pathogen-associated molecular patterns (PAMPs).
The receptors that bind PAMPs are called pattern recognition receptors, or PRRs. Toll-like receptors (TLRs) are one type of
PRR found on phagocytes.
Extravasation of white blood cells from the bloodstream into infected tissue occurs through the process of transendothelial
migration.
Phagocytes degrade pathogens through phagocytosis, which involves engulfing the pathogen, killing and digesting it within a
phagolysosome, and then excreting undigested matter.

Footnotes
1. World Health Organization. “Leishmaniasis.” 2016. http://www.who.int/mediacentre/factsheets/fs375/en/.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 13.3: Pathogen Recognition and Phagocytosis is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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13.4: Inflammation and Fever
Learning Objectives
Identify the signs of inflammation and fever and explain why they occur
Explain the advantages and risks posed by inflammatory responses

The inflammatory response, or inflammation, is triggered by a cascade of chemical mediators and cellular responses that may occur
when cells are damaged and stressed or when pathogens successfully breach the physical barriers of the innate immune system.
Although inflammation is typically associated with negative consequences of injury or disease, it is a necessary process insofar as it
allows for recruitment of the cellular defenses needed to eliminate pathogens, remove damaged and dead cells, and initiate repair
mechanisms. Excessive inflammation, however, can result in local tissue damage and, in severe cases, may even become deadly.
Acute Inflammation
An early, if not immediate, response to tissue injury is acute inflammation. Immediately following an injury, vasoconstriction of
blood vessels will occur to minimize blood loss. The amount of vasoconstriction is related to the amount of vascular injury, but it is
usually brief. Vasoconstriction is followed by vasodilation and increased vascular permeability, as a direct result of the release of
histamine from resident mast cells. Increased blood flow and vascular permeability can dilute toxins and bacterial products at the
site of injury or infection. They also contribute to the five observable signs associated with the inflammatory response: erythema
(redness), edema (swelling), heat, pain, and altered function. Vasodilation and increased vascular permeability are also associated
with an influx of phagocytes at the site of injury and/or infection. This can enhance the inflammatory response because phagocytes
may release proinflammatory chemicals when they are activated by cellular distress signals released from damaged cells, by
PAMPs, or by opsonins on the surface of pathogens. Activation of the complement system can further enhance the inflammatory
response through the production of the anaphylatoxin C5a. Figure 13.4.1 illustrates a typical case of acute inflammation at the site
of a skin wound.

Figure 13.4.1 : (a) Mast cells detect injury to nearby cells and release histamine, initiating an inflammatory response. (b) Histamine
increases blood flow to the wound site, and increased vascular permeability allows fluid, proteins, phagocytes, and other immune
cells to enter infected tissue. These events result in the swelling and reddening of the injured site, and the increased blood flow to
the injured site causes it to feel warm. Inflammation is also associated with pain due to these events stimulating nerve pain
receptors in the tissue. The interaction of phagocyte PRRs with cellular distress signals and PAMPs and opsonins on the surface of
pathogens leads to the release of more proinflammatory chemicals, enhancing the inflammatory response.
During the period of inflammation, the release of bradykinin causes capillaries to remain dilated, flooding tissues with fluids and
leading to edema. Increasing numbers of neutrophils are recruited to the area to fight pathogens. As the fight rages on, pus forms
from the accumulation of neutrophils, dead cells, tissue fluids, and lymph. Typically, after a few days, macrophages will help to
clear out this pus. Eventually, tissue repair can begin in the wounded area.

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Chronic Inflammation
When acute inflammation is unable to clear an infectious pathogen, chronic inflammation may occur. This often results in an
ongoing (and sometimes futile) lower-level battle between the host organism and the pathogen. The wounded area may heal at a
superficial level, but pathogens may still be present in deeper tissues, stimulating ongoing inflammation. Additionally, chronic
inflammation may be involved in the progression of degenerative neurological diseases such as Alzheimer’s and Parkinson’s, heart
disease, and metastatic cancer.
Chronic inflammation may lead to the formation of granulomas, pockets of infected tissue walled off and surrounded by WBCs.
Macrophages and other phagocytes wage an unsuccessful battle to eliminate the pathogens and dead cellular materials within a
granuloma. One example of a disease that produces chronic inflammation is tuberculosis, which results in the formation of
granulomas in lung tissues. A tubercular granuloma is called a tubercle (Figure 13.4.2).
Chronic inflammation is not just associated with bacterial infections. Chronic inflammation can be an important cause of tissue
damage from viral infections. The extensive scarring observed with hepatitis C infections and liver cirrhosis is the result of chronic
inflammation.

Figure 13.4.2 : A tubercle is a granuloma in the lung tissue of a patient with tuberculosis. In this micrograph, white blood cells
(stained purple) have walled off a pocket of tissue infected with Mycobacterium tuberculosis. Granulomas also occur in many other
forms of disease. (credit: modification of work by Piotrowski WJ, Górski P, Duda-Szymańska J, Kwiatkowska S)

Exercise 13.4.1

1. Name the five signs of inflammation.

2. Is a granuloma an acute or chronic form of inflammation? Explain.

Chronic Edema
In addition to granulomas, chronic inflammation can also result in long-term edema. A condition known as lymphatic filariasis
(also known as elephantiasis) provides an extreme example. Lymphatic filariasis is caused by microscopic nematodes (parasitic
worms) whose larvae are transmitted between human hosts by mosquitoes. Adult worms live in the lymphatic vessels, where
their presence stimulates infiltration by lymphocytes, plasma cells, eosinophils, and thrombocytes (a condition known as
lymphangitis). Because of the chronic nature of the illness, granulomas, fibrosis, and blocking of the lymphatic system may
eventually occur. Over time, these blockages may worsen with repeated infections over decades, leading to skin thickened with
edema and fibrosis. Lymph (extracellular tissue fluid) may spill out of the lymphatic areas and back into tissues, causing
extreme swelling (Figure 13.4.3). Secondary bacterial infections commonly follow. Because it is a disease caused by a
parasite, eosinophilia (a dramatic rise in the number of eosinophils in the blood) is characteristic of acute infection. However,
this increase in antiparasite granulocytes is not sufficient to clear the infection in many cases.
Lymphatic filariasis affects an estimated 120 million people worldwide, mostly concentrated in Africa and Asia. 1 Improved
sanitation and mosquito control can reduce transmission rates.

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 Figure 13.4.3 : Elephantiasis (chronic edema) of the legs due to filariasis. (credit: modification of work by Centers for Disease
Control and Prevention)

Fever
A fever is an inflammatory response that extends beyond the site of infection and affects the entire body, resulting in an overall
increase in body temperature. Body temperature is normally regulated and maintained by the hypothalamus, an anatomical section
of the brain that functions to maintain homeostasis in the body. However, certain bacterial or viral infections can result in the
production of pyrogens, chemicals that effectively alter the “thermostat setting” of the hypothalamus to elevate body temperature
and cause fever. Pyrogens may be exogenous or endogenous. For example, the endotoxin lipopolysaccharide (LPS), produced by
gram-negative bacteria, is an exogenous pyrogen that may induce the leukocytes to release endogenous pyrogens such as
interleukin-1 (IL-1), IL-6, interferon-γ (IFN-γ), and tumor necrosis factor (TNF). In a cascading effect, these molecules can then
lead to the release of prostaglandin E2 (PGE2) from other cells, resetting the hypothalamus to initiate fever (Figure 13.4.4).

Figure 13.4.4 : The role of the hypothalamus in the inflammatory response. Macrophages recognize pathogens in an area and
release cytokines that trigger inflammation. The cytokines also send a signal up the vagus nerve to the hypothalamus
Like other forms of inflammation, a fever enhances the innate immune defenses by stimulating leukocytes to kill pathogens. The
rise in body temperature also may inhibit the growth of many pathogens since human pathogens are mesophiles with optimum
growth occurring around 35 °C (95 °F). In addition, some studies suggest that fever may also stimulate release of iron-sequestering
compounds from the liver, thereby starving out microbes that rely on iron for growth.2
During fever, the skin may appear pale due to vasoconstriction of the blood vessels in the skin, which is mediated by the
hypothalamus to divert blood flow away from extremities, minimizing the loss of heat and raising the core temperature. The
hypothalamus will also stimulate shivering of muscles, another effective mechanism of generating heat and raising the core
temperature.
The crisis phase occurs when the fever breaks. The hypothalamus stimulates vasodilation, resulting in a return of blood flow to the
skin and a subsequent release of heat from the body. The hypothalamus also stimulates sweating, which cools the skin as the sweat
evaporates.

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Although a low-level fever may help an individual overcome an illness, in some instances, this immune response can be too strong,
causing tissue and organ damage and, in severe cases, even death. The inflammatory response to bacterial superantigens is one
scenario in which a life-threatening fever may develop. Superantigens are bacterial or viral proteins that can cause an excessive
activation of T cells from the specific adaptive immune defense, as well as an excessive release of cytokines that overstimulates the
inflammatory response. For example, Staphylococcus aureus and Streptococcus pyogenes are capable of producing superantigens
that cause toxic shock syndrome and scarlet fever, respectively. Both of these conditions can be associated with very high, life-
threatening fevers in excess of 42 °C (108 °F).

Exercise 13.4.2

1. Explain the difference between exogenous and endogenous pyrogens.

2. How does a fever inhibit pathogens?

Clinical Focus: part 3

Angela’s tests come back negative for all common allergens, and her sputum samples contain no abnormal presence of
pathogenic microbes or elevated levels of members of the normal respiratory microbiota. She does, however, have elevated
levels of inflammatory cytokines in her blood.
The swelling of her airway has still not responded to treatment with antihistamines or corticosteroids. Additional blood work
shows that Angela has a mildly elevated white blood cell count but normal antibody levels. Also, she has a lower-than-normal
level of the complement protein C4.

Exercise 13.4.2
1. What does this new information reveal about the cause of Angela’s constricted airways?
2. What are some possible conditions that could lead to low levels of complement proteins?

Key Concepts and Summary

Inflammation results from the collective response of chemical mediators and cellular defenses to an injury or infection.
Acute inflammation is short lived and localized to the site of injury or infection. Chronic inflammation occurs when the
inflammatory response is unsuccessful, and may result in the formation of granulomas (e.g., with tuberculosis) and scarring
(e.g., with hepatitis C viral infections and liver cirrhosis).
The five cardinal signs of inflammation are erythema, edema, heat, pain, and altered function. These largely result from innate
responses that draw increased blood flow to the injured or infected tissue.
Fever is a system-wide sign of inflammation that raises the body temperature and stimulates the immune response.
Both inflammation and fever can be harmful if the inflammatory response is too severe.

Footnotes
1. Centers for Disease Control and Prevention. “Parasites–Lymphatic Filiariasis.” 2016.
http://www.cdc.gov/parasites/lymphat...info/faqs.html.
2. N. Parrow et al. “Sequestration and Scavenging of Iron in Infection.” Infection and Immunity 81 no. 10 (2013):3503–3514

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 13.4: Inflammation and Fever is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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Chapter 13 Exercises
Review Questions for Chapter 13
Multiple Choice
1) Which of the following best describes the innate nonspecific immune system?
A. a targeted and highly specific response to a single pathogen or molecule
B. a generalized and nonspecific set of defenses against a class or group of pathogens
C. a set of barrier mechanisms that adapts to specific pathogens after repeated exposure
D. the production of antibody molecules against pathogens

2) Which of the following constantly sheds dead cells along with any microbes that may be attached to those cells?
A. epidermis
B. dermis
C. hypodermis
D. mucous membrane

3) Which of the following uses a particularly dense suite of tight junctions to prevent microbes from entering the underlying tissue?
A. the mucociliary escalator
B. the epidermis
C. the blood-brain barrier
D. the urethra

4) Which of the following serve as chemical signals between cells and stimulate a wide range of nonspecific defenses?
A. cytokines
B. antimicrobial peptides
C. complement proteins
D. antibodies

5) Bacteriocins and defensins are types of which of the following?

A. leukotrienes
B. cytokines
C. inflammation-eliciting mediators
D. antimicrobial peptides

6) Which of the following chemical mediators is secreted onto the surface of the skin?
A. cerumen
B. sebum
C. gastric acid
D. prostaglandin

7) Identify the complement activation pathway that is triggered by the binding of an acute-phase protein to a pathogen.
A. classical
B. alternate
C. lectin

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D. cathelicidin

8) Histamine, leukotrienes, prostaglandins, and bradykinin are examples of which of the following?
A. chemical mediators primarily found in the digestive system
B. chemical mediators that promote inflammation
C. antimicrobial peptides found on the skin
D. complement proteins that form MACs

9) White blood cells are also referred to as which of the following?

A. platelets
B. erythrocytes
C. leukocytes
D. megakaryocytes

10) Hematopoiesis occurs in which of the following?

A. liver
B. bone marrow
C. kidneys
D. central nervous system

11) Granulocytes are which type of cell?

A. lymphocyte
B. erythrocyte
C. megakaryocyte
D. leukocyte

12) PAMPs would be found on the surface of which of the following?

A. pathogen
B. phagocyte
C. skin cell
D. blood vessel wall

13) ________ on phagocytes bind to PAMPs on bacteria, which triggers the uptake and destruction of the bacterial pathogens?
A. PRRs
B. AMPs
C. PAMPs
D. PMNs

14) Which of the following best characterizes the mode of pathogen recognition for opsonin-dependent phagocytosis?
A. Opsonins produced by a pathogen attract phagocytes through chemotaxis.
B. A PAMP on the pathogen’s surface is recognized by a phagocyte’s toll-like receptors.
C. A pathogen is first coated with a molecule such as a complement protein, which allows it to be recognized by phagocytes.
D. A pathogen is coated with a molecule such as a complement protein that immediately lyses the cell.

15) Which refers to swelling as a result of inflammation?

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A. erythema
B. edema
C. granuloma
D. vasodilation

16) Which type of inflammation occurs at the site of an injury or infection?

A. acute
B. chronic
C. endogenous
D. exogenous

Fill-in-the-Blanks
17) The muscular contraction of the intestines that results in movement of material through the digestive tract is called ________.

18) ______ are the hair-like appendages of cells lining parts of the respiratory tract that sweep debris away from the lungs.

19) Secretions that bathe and moisten the interior of the intestines are produced by _______ cells.

20) ________ are antimicrobial peptides produced by members of the normal microbiota.

21) ________ is the fluid portion of a blood sample that has been drawn in the presence of an anticoagulant compound.

22) The process by which cells are drawn or attracted to an area by a microbe invader is known as ________.

23) Platelets are also called ________.

24) The cell in the bone marrow that gives rise to all other blood cell types is the ________.

25) PMNs are another name for ________.

26) Kupffer cells residing in the liver are a type of ________.

27) _____________ are similar to basophils, but reside in tissues rather than circulating in the blood.

28) ________, also known as diapedesis, refers to the exit from the bloodstream of neutrophils and other circulating leukocytes.

29) Toll-like receptors are examples of ________.

30) A(n) ________ is a walled-off area of infected tissue that exhibits chronic inflammation.

31) The ________ is the part of the body responsible for regulating body temperature.

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32) Heat and redness, or ________, occur when the small blood vessels in an inflamed area dilate (open up), bringing more blood
much closer to the surface of the skin.

Short Answers
33) Differentiate a physical barrier from a mechanical removal mechanism and give an example of each.

34) Identify some ways that pathogens can breach the physical barriers of the innate immune system.

35) Differentiate the main activation methods of the classic, alternative, and lectin complement cascades.

36) What are the four protective outcomes of complement activation?

37) Explain the difference between plasma and the formed elements of the blood.

38) List three ways that a neutrophil can destroy an infectious bacterium.

39) Briefly summarize the events leading up to and including the process of transendothelial migration.

40) Differentiate exogenous and endogenous pyrogens, and provide an example of each.

Critical Thinking
41) Neutrophils can sometimes kill human cells along with pathogens when they release the toxic contents of their granules into the
surrounding tissue. Likewise, natural killer cells target human cells for destruction. Explain why it is advantageous for the immune
system to have cells that can kill human cells as well as pathogens.

42) In a blood smear taken from a healthy patient, which type of leukocyte would you expect to observe in the highest numbers?

43) If a gram-negative bacterial infection reaches the bloodstream, large quantities of LPS can be released into the blood, resulting
in a syndrome called septic shock. Death due to septic shock is a real danger. The overwhelming immune and inflammatory
responses that occur with septic shock can cause a perilous drop in blood pressure; intravascular blood clotting; development of
thrombi and emboli that block blood vessels, leading to tissue death; failure of multiple organs; and death of the patient. Identify
and characterize two to three therapies that might be useful in stopping the dangerous events and outcomes of septic shock once it
has begun, given what you have learned about inflammation and innate immunity in this chapter.

44) In Lubeck, Germany, in 1930, a group of 251 infants was accidentally administered a tainted vaccine for tuberculosis that
contained live Mycobacterium tuberculosis. This vaccine was administered orally, directly exposing the infants to the deadly
bacterium. Many of these infants contracted tuberculosis, and some died. However, 44 of the infants never contracted tuberculosis.
Based on your knowledge of the innate immune system, what innate defenses might have inhibited M. tuberculosis enough to
prevent these infants from contracting the disease?

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 CHAPTER OVERVIEW

14: Specific Adaptive Host Defenses

14.1: Architecture of the Immune System
14.2: T Lymphocytes and Cellular Immunity
14.3: B Lymphocytes and Humoral Immunity
14.4: Vaccines
14.5: Practical Applications of Monoclonal and Polyclonal Antibodies
Chapter 14 Exercises
Index

Thumbnail: From left to right: erythrocyte ,platelet and lymphocyte. (Public Domain; The National Cancer Institute at Frederick ).

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1
CHAPTER OVERVIEW

Front Matter
TitlePage
InfoPage

1
 OpenStax CNX
14: Specific Adaptive Host Defenses and
Epidemiology

OpenStax
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14.1: Architecture of the Immune System
Learning Objectives
Define memory, primary response, secondary response, and specificity
Distinguish between humoral and cellular immunity
Differentiate between antigens, epitopes, and haptens
Identify cells that express MHC I and/or MHC II molecules and describe the structures and cellular location of MHC I and
MHC II molecules
Identify the cells that are antigen-presenting cells
Describe the process of antigen processing and presentation with MHC I and MHC II
Describe the structure and function of antibodies and distinguish between the different classes of antibodies

Clinical Focus: part 1

Olivia, a one-year old infant, is brought to the emergency room by her parents, who report her symptoms:
excessive crying, irritability, sensitivity to light, unusual lethargy, and vomiting. A physician feels swollen lymph
nodes in Olivia’s throat and armpits. In addition, the area of the abdomen over the spleen is swollen and tender.

Exercise 14.1.1
1. What do these symptoms suggest?
2. What tests might be ordered to try to diagnose the problem?

Specificity and Memory

Adaptive immunity is defined by two important characteristics: specificity and memory. Specificity refers to the adaptive immune
system’s ability to target specific pathogens, and memory refers to its ability to quickly respond to pathogens to which it has
previously been exposed. For example, when an individual recovers from chickenpox, the body develops a memory of the infection
that will specifically protect it from the causative agent, the varicella-zoster virus, if it is exposed to the virus again later.
Specificity and memory are achieved by essentially programming certain cells involved in the immune response to respond rapidly
to subsequent exposures of the pathogen. This programming occurs as a result of the first exposure to a pathogen or vaccine, which
triggers a primary response. Subsequent exposures result in a secondary response that is faster and stronger as a result of the body’s
memory of the first exposure (Figure 14.1.1). This secondary response, however, is specific to the pathogen in question. For
example, exposure to one virus (e.g., varicella-zoster virus) will not provide protection against other viral diseases (e.g., measles,
mumps, or polio).
Adaptive specific immunity involves the actions of two distinct cell types: B lymphocytes (B cells) and T lymphocytes (T cells).
Although B cells and T cells arise from a common hematopoietic stem cell differentiation pathway, their sites of maturation and
their roles in adaptive immunity are very different. B cells mediate humoral immunity in the spaces between other cells and T cells
drive cellular immune it when pathogens are found within our cells. These cells work with antigen presenting cells and with one
another to create even stronger pathways to remembering of the pathogen and quicker response times upon future exposure.
B cells mature in the bone marrow and are responsible for the production of glycoproteins called antibodies, or immunoglobulins.
Antibodies are involved in the body’s defense against pathogens and toxins in the extracellular environment. Mechanisms of
adaptive specific immunity that involve B cells and antibody production are referred to as humoral immunity. The maturation of T
cells occurs in the thymus. T cells function as the central orchestrator of both innate and adaptive immune responses. They are also
responsible for destruction of cells infected with intracellular pathogens. The targeting and destruction of intracellular pathogens by
T cells is called cell-mediated immunity, or cellular immunity.

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 Figure 14.1.1 : This graph illustrates the primary and secondary immune responses related to antibody production after an initial
and secondary exposure to an antigen. Notice that the secondary response is faster and provides a much higher concentration of
antibody.

Exercise 14.1.2

1. List the two defining characteristics of adaptive immunity.

2. Explain the difference between a primary and secondary immune response.
3. How do humoral and cellular immunity differ?

Antigens
Activation of the adaptive immune defenses is triggered by pathogen-specific molecular structures called antigens. Antigens are
similar to the pathogen-associated molecular patterns (PAMPs); however, whereas PAMPs are molecular structures found on
numerous pathogens, antigens are unique to a specific pathogen. The antigens that stimulate adaptive immunity to chickenpox, for
example, are unique to the varicella-zoster virus but significantly different from the antigens associated with other viral pathogens.
The term antigen was initially used to describe molecules that stimulate the production of antibodies; in fact, the term comes from a
combination of the words antibody and generator, and a molecule that stimulates antibody production is said to be antigenic.
However, the role of antigens is not limited to humoral immunity and the production of antibodies; antigens also play an essential
role in stimulating cellular immunity, and for this reason antigens are sometimes more accurately referred to as immunogens. In
this text, however, we will typically refer to them as antigens.
Pathogens possess a variety of structures that may contain antigens. For example, antigens from bacterial cells may be associated
with their capsules, cell walls, fimbriae, flagella, or pili. Bacterial antigens may also be associated with extracellular toxins and
enzymes that they secrete. Viruses possess a variety of antigens associated with their capsids, envelopes, and the spike structures
they use for attachment to cells.
Antigens may belong to any number of molecular classes, including carbohydrates, lipids, nucleic acids, proteins, and
combinations of these molecules. Antigens of different classes vary in their ability to stimulate adaptive immune defenses as well
as in the type of response they stimulate (humoral or cellular). The structural complexity of an antigenic molecule is an important
factor in its antigenic potential. In general, more complex molecules are more effective as antigens. For example, the three-
dimensional complex structure of proteins make them the most effective and potent antigens, capable of stimulating both humoral
and cellular immunity. In comparison, carbohydrates are less complex in structure and therefore less effective as antigens; they can
only stimulate humoral immune defenses. Lipids and nucleic acids are the least antigenic molecules, and in some cases may only
become antigenic when combined with proteins or carbohydrates to form glycolipids, lipoproteins, or nucleoproteins.
One reason the three-dimensional complexity of antigens is so important is that antibodies and T cells do not recognize and interact
with an entire antigen but with smaller exposed regions on the surface of antigens called epitopes. A single antigen may possess
several different epitopes (Figure 14.1.2), and different antibodies may bind to different epitopes on the same antigen (Figure

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14.1.3). For example, the bacterial flagellum is a large, complex protein structure that can possess hundreds or even thousands of
epitopes with unique three-dimensional structures. Moreover, flagella from different bacterial species (or even strains of the same
species) contain unique epitopes that can only be bound by specific antibodies.
An antigen’s size is another important factor in its antigenic potential. Whereas large antigenic structures like flagella possess
multiple epitopes, some molecules are too small to be antigenic by themselves. Such molecules, called haptens, are essentially free
epitopes that are not part of the complex three-dimensional structure of a larger antigen. For a hapten to become antigenic, it must
first attach to a larger carrier molecule (usually a protein) to produce a conjugate antigen. The hapten-specific antibodies produced
in response to the conjugate antigen are then able to interact with unconjugated free hapten molecules. Haptens are not known to be
associated with any specific pathogens, but they are responsible for some allergic responses. For example, the hapten urushiol, a
molecule found in the oil of plants that cause poison ivy, causes an immune response that can result in a severe rash (called contact
dermatitis). Similarly, the hapten penicillin can cause allergic reactions to drugs in the penicillin class.

Figure 14.1.2 : An antigen is a macromolecule that reacts with components of the immune system. A given antigen may contain
several motifs that are recognized by immune cells.

Figure 14.1.3 : A typical protein antigen has multiple epitopes, shown by the ability of three different antibodies to bind to different
epitopes of the same antigen.

Exercise 14.1.3

1. What is the difference between an antigen and an epitope?

2. What factors affect an antigen’s antigenic potential?
3. Why are haptens typically not antigenic, and how do they become antigenic?

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Major Histocompatibility Complex Molecules
The major histocompatibility complex (MHC) molecules are expressed on the surface of healthy cells, identifying them as normal
and “self” to natural killer (NK) cells. MHC molecules also play an important role in the presentation of foreign antigens, which is
a critical step in the activation of T cells and thus an important mechanism of the adaptive immune system. The major
histocompatibility complex (MHC) is a collection of genes coding for MHC molecules found on the surface of all nucleated cells
of the body. In humans, the MHC genes are also referred to as human leukocyte antigen (HLA) genes. Mature red blood cells,
which lack a nucleus, are the only cells that do not express MHC molecules on their surface.
There are two classes of MHC molecules involved in adaptive immunity, MHC I and MHC II (Figure 14.1.4). MHC I molecules
are found on all nucleated cells; they present normal self-antigens as well as abnormal or nonself pathogens to the effector T cells
involved in cellular immunity. In contrast, MHC II molecules are only found on macrophages, dendritic cells, and B cells; they
present abnormal or nonself pathogen antigens for the initial activation of T cells.
Both types of MHC molecules are transmembrane glycoproteins that assemble as dimers in the cytoplasmic membrane of cells, but
their structures are quite different. MHC I molecules are composed of a longer α protein chain coupled with a smaller β2
microglobulin protein, and only the α chain spans the cytoplasmic membrane. The α chain of the MHC I molecule folds into three
separate domains: α1, α2 and α3. MHC II molecules are composed of two protein chains (an α and a β chain) that are approximately
similar in length. Both chains of the MHC II molecule possess portions that span the plasma membrane, and each chain folds into
two separate domains: α1 and α2, and β1, and β2. In order to present abnormal or non-self-antigens to T cells, MHC molecules have
a cleft that serves as the antigen-binding site near the “top” (or outermost) portion of the MHC-I or MHC-II dimer. For MHC I, the
antigen-binding cleft is formed by the α1 and α2 domains, whereas for MHC II, the cleft is formed by the α1 and β1 domains (Figure
14.1.1).

Figure 14.1.4 : MHC I are found on all nucleated body cells, and MHC II are found on macrophages, dendritic cells, and B cells
(along with MHC I). The antigen-binding cleft of MHC I is formed by domains α1 and α2. The antigen-binding cleft of MHC II is
formed by domains α1 and β1.

Exercise 14.1.4

Compare the structures of the MHC I and MHC II molecules.

Antigen-Presenting Cells (APCs)

All nucleated cells in the body have mechanisms for processing and presenting antigens in association with MHC molecules. This
signals the immune system, indicating whether the cell is normal and healthy or infected with an intracellular pathogen. However,
only macrophages, dendritic cells, and B cells have the ability to present antigens specifically for the purpose of activating T cells;
for this reason, these types of cells are sometimes referred to as antigen-presenting cells (APCs).

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While all APCs play a similar role in adaptive immunity, there are some important differences to consider. Macrophages and
dendritic cells are phagocytes that ingest and kill pathogens that penetrate the first-line barriers (i.e., skin and mucous membranes).
B cells, on the other hand, do not function as phagocytes but play a primary role in the production and secretion of antibodies. In
addition, whereas macrophages and dendritic cells recognize pathogens through nonspecific receptor interactions (e.g., PAMPs,
toll-like receptors, and receptors for opsonizing complement or antibody), B cells interact with foreign pathogens or their free
antigens using antigen-specific immunoglobulin as receptors (monomeric IgD and IgM). When the immunoglobulin receptors bind
to an antigen, the B cell internalizes the antigen by endocytosis before processing and presenting the antigen to T cells.

Antigen Presentation with MHC II Molecules

MHC II molecules are only found on the surface of APCs. Macrophages and dendritic cells use similar mechanisms for processing
and presentation of antigens and their epitopes in association with MHC II; B cells use somewhat different mechanisms that will be
described later. For now, we will focus on the steps of the process as they pertain to dendritic cells.
After a dendritic cell recognizes and attaches to a pathogen cell, the pathogen is internalized by phagocytosis and is initially
contained within a phagosome. Lysosomes containing antimicrobial enzymes and chemicals fuse with the phagosome to create a
phagolysosome, where degradation of the pathogen for antigen processing begins. Proteases (protein-degrading) are especially
important in antigen processing because only protein antigen epitopes are presented to T cells by MHC II (Figure 14.1.5).

Figure 14.1.5 : A dendritic cell phagocytoses a bacterial cell and brings it into a phagosome. Lysosomes fuse with the phagosome to
create a phagolysosome, where antimicrobial chemicals and enzymes degrade the bacterial cell. Proteases process bacterial
antigens, and the most antigenic epitopes are selected and presented on the cell’s surface in conjunction with MHC II molecules. T
cells recognize the presented antigens and are thus activated.
APCs do not present all possible epitopes to T cells; only a selection of the most antigenic or immunodominant epitopes are
presented. The mechanism by which epitopes are selected for processing and presentation by an APC is complicated and not well
understood; however, once the most antigenic, immunodominant epitopes have been processed, they associate within the antigen-
binding cleft of MHC II molecules and are translocated to the cell surface of the dendritic cell for presentation to T cells

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 Exercise 14.1.5

1. What are the three kinds of APCs?

2. What role to MHC II molecules play in antigen presentation?
3. What is the role of antigen presentation in adaptive immunity?

Antigen Presentation with MHC I Molecules

MHC I molecules, found on all normal, healthy, nucleated cells, signal to the immune system that the cell is a normal “self” cell. In
a healthy cell, proteins normally found in the cytoplasm are degraded by proteasomes (enzyme complexes responsible for
degradation and processing of proteins) and processed into self-antigen epitopes; these self-antigen epitopes bind within the MHC I
antigen-binding cleft and are then presented on the cell surface. Immune cells, such as NK cells, recognize these self-antigens and
do not target the cell for destruction. However, if a cell becomes infected with an intracellular pathogen (e.g., a virus), protein
antigens specific to the pathogen are processed in the proteasomes and bind with MHC I molecules for presentation on the cell
surface. This presentation of pathogen-specific antigens with MHC I signals that the infected cell must be targeted for destruction
along with the pathogen.
Before elimination of infected cells can begin, APCs must first activate the T cells involved in cellular immunity. If an intracellular
pathogen directly infects the cytoplasm of an APC, then the processing and presentation of antigens can occur as described (in
proteasomes and on the cell surface with MHC I). However, if the intracellular pathogen does not directly infect APCs, an
alternative strategy called cross-presentation is utilized. In cross-presentation, antigens are brought into the APC by mechanisms
normally leading to presentation with MHC II (i.e., through phagocytosis), but the antigen is presented on an MHC I molecule for
CD8 T cells. The exact mechanisms by which cross-presentation occur are not yet well understood, but it appears that cross-
presentation is primarily a function of dendritic cells and not macrophages or B cells.

Exercise 14.1.6

1. Compare and contrast antigen processing and presentation associated with MHC I and MHC II molecules.
2. What is cross-presentation, and when is it likely to occur?

Antibodies
Antibodies (also called immunoglobulins) are glycoproteins that are present in both the blood and tissue fluids. The basic structure
of an antibody monomer consists of four protein chains held together by disulfide bonds (Figure 14.1.6). A disulfide bond is a
covalent bond between the sulfhydryl R groups found on two cysteine amino acids. The two largest chains are identical to each
other and are called the heavy chains. The two smaller chains are also identical to each other and are called the light chains. Joined
together, the heavy and light chains form a basic Y-shaped structure.
The two ‘arms’ of the Y-shaped antibody molecule are known as the Fab region, for “fragment of antigen binding.” The far end of
the Fab region is the variable region, which serves as the site of antigen binding. The amino acid sequence in the variable region
dictates the three-dimensional structure, and thus the specific three-dimensional epitope to which the Fab region is capable of
binding. Although the epitope specificity of the Fab regions is identical for each arm of a single antibody molecule, this region
displays a high degree of variability between antibodies with different epitope specificities. Binding to the Fab region is necessary
for neutralization of pathogens, agglutination or aggregation of pathogens, and antibody-dependent cell-mediated cytotoxicity.
The constant region of the antibody molecule includes the trunk of the Y and lower portion of each arm of the Y. The trunk of the
Y is also called the Fc region, for “fragment of crystallization,” and is the site of complement factor binding and binding to
phagocytic cells during antibody-mediated opsonization.

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 Figure 14.1.6 : (a) The typical four-chain structure of a generic antibody monomer. (b) The corresponding three-dimensional
structure of the antibody IgG. (credit b: modification of work by Tim Vickers)

Exercise 14.1.7

Describe the different functions of the Fab region and the Fc region.

Antibody Classes
The constant region of an antibody molecule determines its class, or isotype. The five classes of antibodies are IgG, IgM, IgA, IgD,
and IgE. Each class possesses unique heavy chains designated by Greek letters γ, μ, α, δ, and ε, respectively. Antibody classes also
exhibit important differences in abundance in serum, arrangement, body sites of action, functional roles, and size (Figure 14.1.7).
IgG is a monomer that is by far the most abundant antibody in human blood, accounting for about 80% of total serum antibody.
IgG penetrates efficiently into tissue spaces, and is the only antibody class with the ability to cross the placental barrier, providing
passive immunity to the developing fetus during pregnancy. IgG is also the most versatile antibody class in terms of its role in the
body’s defense against pathogens.
IgM is initially produced in a monomeric membrane-bound form that serves as an antigen-binding receptor on B cells. The secreted
form of IgM assembles into a pentamer with five monomers of IgM bound together by a protein structure called the J chain.
Although the location of the J chain relative to the Fc regions of the five monomers prevents IgM from performing some of the
functions of IgG, the ten available Fab sites associated with a pentameric IgM make it an important antibody in the body’s arsenal
of defenses. IgM is the first antibody produced and secreted by B cells during the primary and secondary immune responses,
making pathogen-specific IgM a valuable diagnostic marker during active or recent infections.
IgA accounts for about 13% of total serum antibody, and secretory IgA is the most common and abundant antibody class found in
the mucus secretions that protect the mucous membranes. IgA can also be found in other secretions such as breast milk, tears, and
saliva. Secretory IgA is assembled into a dimeric form with two monomers joined by a protein structure called the secretory
component. One of the important functions of secretory IgA is to trap pathogens in mucus so that they can later be eliminated from
the body.
Similar to IgM, IgD is a membrane-bound monomer found on the surface of B cells, where it serves as an antigen-binding receptor.
However, IgD is not secreted by B cells, and only trace amounts are detected in serum. These trace amounts most likely come from
the degradation of old B cells and the release of IgD molecules from their cytoplasmic membranes.
IgE is the least abundant antibody class in serum. Like IgG, it is secreted as a monomer, but its role in adaptive immunity is
restricted to anti-parasitic defenses. The Fc region of IgE binds to basophils and mast cells. The Fab region of the bound IgE then
interacts with specific antigen epitopes, causing the cells to release potent pro-inflammatory mediators. The inflammatory reaction
resulting from the activation of mast cells and basophils aids in the defense against parasites, but this reaction is also central to
allergic reactions.

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 Figure 14.1.7 : The five Immunoglobulin classes

Exercise 14.1.8
1. What part of an antibody molecule determines its class?
2. What class of antibody is involved in protection against parasites?
3. Describe the difference in structure between IgM and IgG.

Antigen-Antibody Interactions
Different classes of antibody play important roles in the body’s defense against pathogens. These functions include neutralization
of pathogens, opsonization for phagocytosis, agglutination, complement activation, and antibody-dependent cell-mediated
cytotoxicity. For most of these functions, antibodies also provide an important link between adaptive specific immunity and innate
nonspecific immunity.
Neutralization involves the binding of certain antibodies (IgG, IgM, or IgA) to epitopes on the surface of pathogens or toxins,
preventing their attachment to cells. For example, Secretory IgA can bind to specific pathogens and block initial attachment to
intestinal mucosal cells. Similarly, specific antibodies can bind to certain toxins, blocking them from attaching to target cells and

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thus neutralizing their toxic effects. Viruses can be neutralized and prevented from infecting a cell by the same mechanism (Figure
14.1.8).

Figure 14.1.8 : Neutralization involves the binding of specific antibodies to antigens found on bacteria, viruses, and toxins,
preventing them from attaching to target cells.
Opsonization is the coating of a pathogen with molecules, such as complement factors, C-reactive protein, and serum amyloid A, to
assist in phagocyte binding to facilitate phagocytosis. IgG antibodies also serve as excellent opsonins, binding their Fab sites to
specific epitopes on the surface of pathogens. Phagocytic cells such as macrophages, dendritic cells, and neutrophils have receptors
on their surfaces that recognize and bind to the Fc portion of the IgG molecules; thus, IgG helps such phagocytes attach to and
engulf the pathogens they have bound (Figure 14.1.9).

Figure 14.1.9 : Antibodies serve as opsonins and inhibit infection by tagging pathogens for destruction by macrophages, dendritic
cells, and neutrophils. These phagocytic cells use Fc receptors to bind to IgG-opsonized pathogens and initiate the first step of
attachment before phagocytosis.
Agglutination or aggregation involves the cross-linking of pathogens by antibodies to create large aggregates (Figure 14.1.10). IgG
has two Fab antigen-binding sites, which can bind to two separate pathogen cells, clumping them together. When multiple IgG
antibodies are involved, large aggregates can develop; these aggregates are easier for the kidneys and spleen to filter from the blood
and easier for phagocytes to ingest for destruction. The pentameric structure of IgM provides ten Fab binding sites per molecule,
making it the most efficient antibody for agglutination.
Another important function of antibodies is activation of the complement cascade. As discussed in the previous chapter, the
complement system is an important component of the innate defenses, promoting the inflammatory response, recruiting phagocytes
to site of infection, enhancing phagocytosis by opsonization, and killing gram-negative bacterial pathogens with the membrane
attack complex (MAC). Complement activation can occur through three different pathways, but the most efficient is the classical
pathway, which requires the initial binding of IgG or IgM antibodies to the surface of a pathogen cell, allowing for recruitment and
activation of the C1 complex.

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 Figure 14.1.10: Antibodies, especially IgM antibodies, agglutinate bacteria by binding to epitopes on two or more bacteria
simultaneously. When multiple pathogens and antibodies are present, aggregates form when the binding sites of antibodies bind
with separate pathogens.
Yet another important function of antibodies is antibody-dependent cell-mediated cytotoxicity (ADCC), which enhances killing of
pathogens that are too large to be phagocytosed. This process is best characterized for natural killer cells (NK cells), as shown in
Figure 14.1.11, but it can also involve macrophages and eosinophils. ADCC occurs when the Fab region of an IgG antibody binds
to a large pathogen; Fc receptors on effector cells (e.g., NK cells) then bind to the Fc region of the antibody, bringing them into
close proximity with the target pathogen. The effector cell then secretes powerful cytotoxins (e.g., perforin and granzymes) that kill
the pathogen.

Figure 14.1.11: In this example of ADCC, antibodies bind to a large pathogenic cell that is too big for phagocytosis and then bind
to Fc receptors on the membrane of a natural killer cell. This interaction brings the NK cell into close proximity, where it can kill
the pathogen through release of lethal extracellular cytotoxins.

Exercise 14.1.9
1. Where is IgA normally found?
2. Which class of antibody crosses the placenta, providing protection to the fetus?
3. Compare the mechanisms of opsonization and antibody-dependent cell-mediated cytotoxicity.

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Key Concepts and Summary
Adaptive immunity is an acquired defense against foreign pathogens that is characterized by specificity and memory. The first
exposure to an antigen stimulates a primary response, and subsequent exposures stimulate a faster and strong secondary
response.
Adaptive immunity is a dual system involving humoral immunity (antibodies produced by B cells) and cellular immunity (T
cells directed against intracellular pathogens).
Antigens, also called immunogens, are molecules that activate adaptive immunity. A single antigen possesses smaller
epitopes, each capable of inducing a specific adaptive immune response.
An antigen’s ability to stimulate an immune response depends on several factors, including its molecular class, molecular
complexity, and size.
Major histocompatibility complex (MHC) is a collection of genes coding for glycoprotein molecules expressed on the surface
of all nucleated cells.
MHC I molecules are expressed on all nucleated cells and are essential for presentation of normal “self” antigens. Cells that
become infected by intracellular pathogens can present foreign antigens on MHC I as well, marking the infected cell for
destruction.
MHC II molecules are expressed only on the surface of antigen-presenting cells (macrophages, dendritic cells, and B cells).
Antigen presentation with MHC II is essential for the activation of T cells.
Antigen-presenting cells (APCs) primarily ingest pathogens by phagocytosis, destroy them in the phagolysosomes, process the
protein antigens, and select the most antigenic/immunodominant epitopes with MHC II for presentation to T cells.
Cross-presentation is a mechanism of antigen presentation and T-cell activation used by dendritic cells not directly infected by
the pathogen; it involves phagocytosis of the pathogen but presentation on MHC I rather than MHC II.
Antibodies (immunoglobulins) are Y-shaped glycoproteins with two Fab sites for binding antigens and an Fc portion involved
in complement activation and opsonization.
The five classes of antibody are IgM, IgG, IgA, IgE, and IgD, each differing in size, arrangement, location within the body,
and function. The five primary functions of antibodies are neutralization, opsonization, agglutination, complement activation,
and antibody-dependent cell-mediated cytotoxicity (ADCC).

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 14.1: Architecture of the Immune System is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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14.2: T Lymphocytes and Cellular Immunity
Learning Objectives
Describe the process of T-cell maturation and thymic selection
Explain the genetic events that lead to diversity of T-cell receptors
Compare and contrast the various classes and subtypes of T cells in terms of activation and function
Explain the mechanism by which superantigens effect unregulated T-cell activation

As explained in previously, the antibodies involved in humoral immunity often bind pathogens and toxins before they can attach to
and invade host cells. Thus, humoral immunity is primarily concerned with fighting pathogens in extracellular spaces. However,
pathogens that have already gained entry to host cells are largely protected from the humoral antibody-mediated defenses. Cellular
immunity, on the other hand, targets and eliminates intracellular pathogens through the actions of T lymphocytes, or T cells (Figure
14.2.1). T cells also play a more central role in orchestrating the overall adaptive immune response (humoral as well as cellular)

along with the cellular defenses of innate immunity.

Figure 14.2.1 : This scanning electron micrograph shows a T lymphocyte, which is responsible for the cell-mediated immune
response. The spike-like membrane structures increase surface area, allowing for greater interaction with other cell types and their
signals. (credit: modification of work by NCI)

T Cell Production and Maturation

T cells, like all other white blood cells involved in innate and adaptive immunity, are formed from multipotent hematopoietic stem
cells (HSCs) in the bone marrow. However, unlike the white blood cells of innate immunity, eventual T cells differentiate first into
lymphoid stem cells that then become small, immature lymphocytes, sometimes called lymphoblasts. The first steps of
differentiation occur in the red marrow of bones (Figure 14.2.2), after which immature T lymphocytes enter the bloodstream and
travel to the thymus for the final steps of maturation (Figure 14.2.3). Once in the thymus, the immature T lymphocytes are referred
to as thymocytes.
The maturation of thymocytes within the thymus can be divided into tree critical steps of positive and negative selection,
collectively referred to as thymic selection. The first step of thymic selection occurs in the cortex of the thymus and involves the
development of a functional T-cell receptor (TCR) that is required for activation by APCs. Thymocytes with defective TCRs are
removed by negative selection through the induction of apoptosis (programmed controlled cell death). The second step of thymic
selection also occurs in the cortex and involves the positive selection of thymocytes that will interact appropriately with MHC
molecules. Thymocytes that can interact appropriately with MHC molecules receive a positive stimulation that moves them further
through the process of maturation, whereas thymocytes that do not interact appropriately are not stimulated and are eliminated by
apoptosis. The third and final step of thymic selection occurs in both the cortex and medulla and involves negative selection to
remove self-reacting thymocytes, those that react to self-antigens, by apoptosis. This final step is sometimes referred to as central
tolerance because it prevents self-reacting T cells from reaching the bloodstream and potentially causing autoimmune disease,
which occurs when the immune system attacks healthy “self” cells.

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 Figure 14.2.2 : (a) Red bone marrow can be found in the head of the femur (thighbone) and is also present in the flat bones of the
body, such as the ilium and the scapula. (b) Red bone marrow is the site of production and differentiation of many formed elements
of blood, including erythrocytes, leukocytes, and platelets. The yellow bone marrow is populated primarily with adipose cells.
Despite central tolerance, some self-reactive T cells generally escape the thymus and enter the peripheral bloodstream. Therefore, a
second line of defense called peripheral tolerance is needed to protect against autoimmune disease. Peripheral tolerance involves
mechanisms of anergy and inhibition of self-reactive T cells by regulatory T cells. Anergy refers to a state of nonresponsiveness to
antigen stimulation. In the case of self-reactive T cells that escape the thymus, lack of an essential co-stimulatory signal required
for activation causes anergy and prevents autoimmune activation. Regulatory T cells participate in peripheral tolerance by
inhibiting the activation and function of self-reactive T cells and by secreting anti-inflammatory cytokines.
It is not completely understood what events specifically direct maturation of thymocytes into regulatory T cells. Current theories
suggest the critical events may occur during the third step of thymic selection, when most self-reactive T cells are eliminated.
Regulatory T cells may receive a unique signal that is below the threshold required to target them for negative selection and
apoptosis. Consequently, these cells continue to mature and then exit the thymus, armed to inhibit the activation of self-reactive T
cells. It has been estimated that the three steps of thymic selection eliminate 98% of thymocytes. The remaining 2% that exit the
thymus migrate through the bloodstream and lymphatic system to sites of secondary lymphoid organs/tissues, such as the lymph
nodes, spleen, and tonsils (Figure 14.2.3), where they await activation through the presentation of specific antigens by APCs. Until
they are activated, they are known as mature naïve T cells.

Figure 14.2.3 : The thymus is a bi-lobed, H-shaped glandular organ that is located just above the heart. It is surrounded by a fibrous
capsule of connective tissue. The darkly staining cortex and the lighter staining medulla of individual lobules are clearly visible in
the light micrograph of the thymus of a newborn (top right, LM × 100). (credit micrograph: modification of micrograph provided
by the Regents of University of Michigan Medical School © 2012)

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 Exercise 14.2.1

1. What anatomical sites are involved in T cell production and maturation?

2. What are the three steps involved in thymic selection?
3. Why are central tolerance and peripheral tolerance important? What do they prevent?

T-Cell Receptors
For both helper T cells and cytotoxic T cells, activation is a complex process that requires the interactions of multiple molecules
and exposure to cytokines. The T-cell receptor (TCR) is involved in the first step of pathogen epitope recognition during the
activation process. This process is highly similar to the recognition of an epitope by anitbodies, so it is unsurprising that the TCR
comes from the same receptor family as the antibodies IgD and IgM, the antigen receptors on the B cell membrane surface, and
shares common structural elements.
Similar to antibodies, the TCR has a variable region and a constant region, and the variable region provides the antigen-binding site
(Figure 14.2.4). However, the structure of TCR is smaller and less complex than the immunoglobulin molecules. Whereas
immunoglobulins have four peptide chains and Y-shaped structures, the TCR consists of just two peptide chains (α and β chains),
both of which span the cytoplasmic membrane of the T cell.
TCRs are epitope-specific, and it has been estimated that 25 million T cells with unique epitope-binding TCRs are required to
protect an individual against a wide range of microbial pathogens. Because the human genome only contains about 25,000 genes,
we know that each specific TCR cannot be encoded by its own set of genes. This raises the question of how such a vast population
of T cells with millions of specific TCRs can be achieved. The answer is a process called genetic rearrangement, which occurs in
the thymus during the first step of thymic selection.
The genes that code for the variable regions of the TCR are divided into distinct gene segments called variable (V), diversity (D),
and joining (J) segments. The genes segments associated with the α chain of the TCR consist 70 or more different Vα segments and
61 different Jα segments. The gene segments associated with the β chain of the TCR consist of 52 different Vβ segments, two
different Dβ segments, and 13 different Jβ segments. During the development of the functional TCR in the thymus, genetic
rearrangement in a T cell brings together one Vα segment and one Jα segment to code for the variable region of the α chain.
Similarly, genetic rearrangement brings one of the Vβ segments together with one of the Dβ segments and one of the Jβ segments to
code for the variable region of the β chain. All the possible combinations of rearrangements between different segments of V, D,
and J provide the genetic diversity required to produce millions of TCRs with unique epitope-specific variable regions.

Figure 14.2.4 : A T-cell receptor spans the cytoplasmic membrane and projects variable binding regions into the extracellular space
to bind processed antigens associated with MHC I or MHC II molecules.

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 Exercise 14.2.2
1. What are the similarities and differences between TCRs and immunoglobulins?
2. What process is used to provide millions of unique TCR binding sites?

Classes of T Cells
T cells can be categorized into three distinct classes: helper T cells, regulatory T cells, and cytotoxic T cells. These classes are
differentiated based on their expression of certain surface molecules, their mode of activation, and their functional roles in adaptive
immunity (Table 14.2.1).
All T cells produce cluster of differentiation (CD) molecules, cell surface glycoproteins that can be used to identify and distinguish
between the various types of white blood cells. Although T cells can produce a variety of CD molecules, CD4 and CD8 are the two
most important used for differentiation of the classes. Helper T cells and regulatory T cells are characterized by the expression of
CD4 on their surface, whereas cytotoxic T cells are characterized by the expression of CD8.
Classes of T cells can also be distinguished by the specific MHC molecules and APCs with which they interact for activation.
Helper T cells and regulatory T cells can only be activated by APCs presenting antigens associated with MHC II. In contrast,
cytotoxic T cells recognize antigens presented in association with MHC I, either by APCs or by nucleated cells infected with an
intracellular pathogen.
The different classes of T cells also play different functional roles in the immune system. Helper T cells serve as the central
orchestrators that help activate and direct functions of humoral and cellular immunity. In addition, helper T cells enhance the
pathogen-killing functions of macrophages and NK cells of innate immunity. In contrast, the primary role of regulatory T cells is to
prevent undesirable and potentially damaging immune responses. Their role in peripheral tolerance, for example, protects against
autoimmune disorders, as discussed earlier. Finally, cytotoxic T cells are the primary effector cells for cellular immunity. They
recognize and target cells that have been infected by intracellular pathogens, destroying infected cells along with the pathogens
inside.
Table 14.2.1 : Classes of T Cells

Class Surface CD Molecules Activation Functions

Orchestrate humoral and cellular

APCs presenting antigens immunity
Helper T cells CD4
associated with MHC II Involved in the activation of
macrophages and NK cells
Involved in peripheral tolerance
APCs presenting antigens
Regulatory T cells CD4 and prevention of autoimmune
associated with MHC II
responses

APCs or infected nucleated cells

Destroy cells infected with
Cytotoxic T cells CD8 presenting antigens associated
intracellular pathogens
with MHC I

Exercise 14.2.3

1. What are the unique functions of the three classes of T cells?

2. Which T cells can be activated by antigens presented by cells other than APCs?

Activation and Differentiation of Helper T Cells

At this point the T cells are both “mature” because they have passed all three thymic selection events and “naïve” because their
TCR has not yet been in contact with the epitope it recognizes. Once it’s TCR binds to its recognized epitope the T cell will be
considered activated. Helper T cells can only be activated by APCs presenting processed foreign epitopes in association with MHC
II. The first step in the activation process is TCR recognition of the specific foreign epitope presented within the MHC II antigen-
binding cleft. The second step involves the interaction of CD4 on the helper T cell with a region of the MHC II molecule separate
from the antigen-binding cleft. This second interaction anchors the MHC II-TCR complex and ensures that the helper T cell is
recognizing both the foreign (“nonself”) epitope and “self” antigen of the APC; both recognitions are required for activation of the

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cell. In the third step, the APC and T cell secrete cytokines that activate the helper T cell. The activated helper T cell then
proliferates, dividing by mitosis, to produce a clonal line of helper T cells that differentiate into subtypes with different functions
(Figure 14.2.5).

Figure 14.2.5 : This illustration depicts the activation of a naïve (unactivated) helper T cell by an antigen-presenting cell and the
subsequent proliferation and differentiation of the activated T cell into different subtypes.
Activated helper T cells can differentiate into one of four distinct subtypes, summarized in Table 14.2.2. The differentiation
process is directed by APC-secreted cytokines. Depending on which APC-secreted cytokines interact with an activated helper T
cell, the cell may differentiate into a T helper 1 (TH1) cell, a T helper 2 (TH2) cell, or a memory helper T cell and more rarely TH17
cells. The two types of helper T cells are relatively short-lived effector cells, meaning that they perform various functions of the
immediate immune response. In contrast, memory helper T cells are relatively long lived; they are programmed to “remember” a
specific antigen or epitope in order to mount a rapid, strong, secondary response to subsequent exposures.
TH1 cells secrete their own cytokines that are involved in stimulating and orchestrating other cells involved in adaptive and innate
immunity. For example, they stimulate cytotoxic T cells, enhancing their killing of infected cells and promoting differentiation into
memory cytotoxic T cells. TH1 cells also stimulate macrophages and neutrophils to become more effective in their killing of
intracellular bacteria. They can also stimulate NK cells to become more effective at killing target cells.
TH2 cells play an important role in orchestrating the humoral immune response through their secretion of cytokines that activate B
cells and direct B cell differentiation and antibody production. Various cytokines produced by TH2 cells orchestrate antibody class
switching, which allows B cells to switch between the production of IgM, IgG, IgA, and IgE as needed to carry out specific
antibody functions and to provide pathogen-specific humoral immune responses.
A third subtype of helper T cells called TH17 cells was discovered through observations that immunity to some infections is not
associated with TH1 or TH2 cells. TH17 cells and the cytokines they produce appear to be specifically responsible for the body’s

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defense against chronic mucocutaneous infections. Patients who lack sufficient TH17 cells in the mucosa (e.g., HIV patients) may
be more susceptible to bacteremia and gastrointestinal infections.1
Table 14.2.2 : Subtypes of Helper T Cells
Subtype Functions

Stimulate cytotoxic T cells and produce memory cytotoxic T cells

Stimulate macrophages and neutrophils (PMNs) for more effective
TH1 cells
intracellular killing of pathogens
Stimulate NK cells to kill more effectively
Stimulate B cell activation and differentiation into plasma cells and
TH2 cells memory B cells
Direct antibody class switching in B cells
Stimulate immunity to specific infections such as chronic
TH17 cells
mucocutaneous infections
“Remember” a specific pathogen and mount a strong, rapid secondary
Memory helper T cells
response upon re-exposure

Activation and Differentiation of Cytotoxic T Cells

As with helper T cells, cytotoxic T cells (also referred to as cytotoxic T lymphocytes, or CTLs) are both mature and naive until
contact with the epitope recognized by its TCR. Cytotoxic T cell have a process of activation by APCs in a similar three-step
process to that of helper T cells. The key difference is that the activation of cytotoxic T cells involves recognition of an antigen
presented with MHC I (as opposed to MHC II) and interaction of CD8 (as opposed to CD4) with the receptor complex. After the
successful co-recognition of foreign epitope and self-antigen, the production of cytokines by the APC and the cytotoxic T cell
activate clonal proliferation and differentiation. Activated cytotoxic T cells can differentiate into effector cytotoxic T cells that
target pathogens for destruction or memory cells that are ready to respond to subsequent exposures.
As noted, proliferation and differentiation of cytotoxic T cells is also stimulated by cytokines secreted from TH1 cells activated by
the same foreign epitope. The co-stimulation that comes from these TH1 cells is provided by secreted cytokines. Although it is
possible for activation of cytotoxic T cells to occur without stimulation from TH1 cells, the activation is not as effective or long-
lasting.
Once activated, cytotoxic T cells serve as the effector cells of cellular immunity, recognizing and kill cells infected with
intracellular pathogens through a mechanism very similar to that of NK cells. However, whereas NK cells recognize nonspecific
signals of cell stress or abnormality, cytotoxic T cells recognize infected cells through antigen presentation of pathogen-specific
epitopes associated with MHC I. Once an infected cell is recognized, the TCR of the cytotoxic T cell binds to the epitope and
releases perforin and granzymes that destroy the infected cell (Figure 14.2.6). Perforin is a protein that creates pores in the target
cell, and granzymes are proteases that enter the pores and induce apoptosis. This mechanism of programmed cell death is a
controlled and efficient means of destroying and removing infected cells without releasing the pathogens inside to infect
neighboring cells, as might occur if the infected cells were simply lysed.

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 Figure 14.2.6 : This figure illustrates the activation of a naïve (unactivated) cytotoxic T cell (CTL) by an antigen-presenting MHC I
molecule on an infected body cell. Once activated, the CTL releases perforin and granzymes that invade the infected cell and
induce controlled cell death, or apoptosis.
In this video, you can see a cytotoxic T cell inducing apoptosis in a target cell.

Exercise 14.2.4

1. Compare and contrast the activation of helper T cells and cytotoxic T cells.
2. What are the different functions of helper T cell subtypes?
3. What is the mechanism of CTL-mediated destruction of infected cells?

Superantigens and Unregulated Activation of T Cells

When T cell activation is controlled and regulated, the result is a protective response that is effective in combating infections.
However, if T cell activation is unregulated and excessive, the result can be a life-threatening. Certain bacterial and viral pathogens
produce toxins known as superantigens that can trigger such an unregulated response. Known bacterial superantigens include toxic
shock syndrome toxin (TSST), staphylococcal enterotoxins, streptococcal pyrogenic toxins, streptococcal superantigen, and the
streptococcal mitogenic exotoxin. Viruses known to produce superantigens include Epstein-Barr virus (human herpesvirus 4),
cytomegalovirus (human herpesvirus 5), and others.
The mechanism of T cell activation by superantigens involves their simultaneous binding to MHC II molecules of APCs and the
variable region of the TCR β chain. This binding occurs outside of the antigen-binding cleft of MHC II, so the superantigen will
bridge together and activate MHC II and TCR without specific foreign epitope recognition (Figure 14.2.7). The result is an
excessive, uncontrolled release of cytokines, often called a cytokine storm, which stimulates an excessive inflammatory response.
This can lead to a dangerous decrease in blood pressure, shock, multi-organ failure, and potentially, death.

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 Figure 14.2.7 : (a) The macrophage in this figure is presenting a foreign epitope that does not match the TCR of the T cell. Because
the T cell does not recognize the epitope, it is not activated. (b) The macrophage in this figure is presenting a superantigen that is
not recognized by the TCR of the T cell, yet the superantigen still is able to bridge and bind the MHC II and TCR molecules. This
nonspecific, uncontrolled activation of the T cell results in an excessive release of cytokines that activate other T cells and cause
excessive inflammation. (credit: modification of work by “Microbiotic”/YouTube)

Exercise 14.2.5
1. What are examples of superantigens?
2. How does a superantigen activate a helper T cell?
3. What effect does a superantigen have on a T cell?

Superantigens
Melissa, an otherwise healthy 22-year-old woman, is brought to the emergency room by her concerned boyfriend. She
complains of a sudden onset of high fever, vomiting, diarrhea, and muscle aches. In her initial interview, she tells the attending
physician that she is on hormonal birth control and also is two days into the menstruation portion of her cycle. She is on no
other medications and is not abusing any drugs or alcohol. She is not a smoker. She is not diabetic and does not currently have
an infection of any kind to her knowledge.
While waiting in the emergency room, Melissa’s blood pressure begins to drop dramatically and her mental state deteriorates to
general confusion. The physician believes she is likely suffering from toxic shock syndrome (TSS). TSS is caused by the toxin
TSST-1, a superantigen associated with Staphylococcus aureus, and improper tampon use is a common cause of infections
leading to TSS. The superantigen inappropriately stimulates widespread T cell activation and excessive cytokine release,
resulting in a massive and systemic inflammatory response that can be fatal.
Vaginal or cervical swabs may be taken to confirm the presence of the microbe, but these tests are not critical to perform based
on Melissa’s symptoms and medical history. The physician prescribes rehydration, supportive therapy, and antibiotics to stem
the bacterial infection. She also prescribes drugs to increase Melissa’s blood pressure. Melissa spends three days in the hospital
undergoing treatment; in addition, her kidney function is monitored because of the high risk of kidney failure associated with
TSS. After 72 hours, Melissa is well enough to be discharged to continue her recovery at home.

Exercise 14.2.6

In what way would antibiotic therapy help to combat a superantigen?

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 Clinical Focus: part 2

Olivia’s swollen lymph nodes, abdomen, and spleen suggest a strong immune response to a systemic infection in
progress. In addition, little Olivia is reluctant to turn her head and appears to be experiencing severe neck pain. The
physician orders a complete blood count, blood culture, and lumbar puncture. The cerebrospinal fluid (CSF)
obtained appears cloudy and is further evaluated by Gram stain assessment and culturing for potential bacterial
pathogens. The complete blood count indicates elevated numbers of white blood cells in Olivia’s bloodstream. The
white blood cell increases are recorded at 28.5 K/µL (normal range: 6.0–17.5 K/µL). The neutrophil percentage was
recorded as 60% (normal range: 23–45%). Glucose levels in the CSF were registered at 30 mg/100 mL (normal
range: 50–80 mg/100 mL). The WBC count in the CSF was 1,163/mm3 (normal range: 5–20/mm3).AutoNum"
template (preferably at the end) to the page.

Exercise 14.2.7

1. Based on these results, do you have a preliminary diagnosis?

2. What is a recommended treatment based on this preliminary diagnosis?

Key Concepts and Summary

Immature T lymphocytes are produced in the red bone marrow and travel to the thymus for maturation.
Thymic selection is a three-step process of negative and positive selection that determines which T cells will mature and exit
the thymus into the peripheral bloodstream.
Central tolerance involves negative selection of self-reactive T cells in the thymus, and peripheral toleranceinvolves anergy
and regulatory T cells that prevent self-reactive immune responses and autoimmunity.
The TCR is similar in structure to immunoglobulins, but less complex. Millions of unique epitope-binding TCRs are encoded
through a process of genetic rearrangement of V, D, and J gene segments.
T cells can be divided into three classes—helper T cells, cytotoxic T cells, and regulatory T cells—based on their expression
of CD4 or CD8, the MHC molecules with which they interact for activation, and their respective functions.
Activated helper T cells differentiate into TH1, TH2, TH17, or memory T cell subtypes. Differentiation is directed by the
specific cytokines to which they are exposed. TH1, TH2, and TH17 perform different functions related to stimulation of adaptive
and innate immune defenses. Memory T cells are long-lived cells that can respond quickly to secondary exposures.
Once activated, cytotoxic T cells target and kill cells infected with intracellular pathogens. Killing requires recognition of
specific pathogen epitopes presented on the cell surface using MHC I molecules. Killing is mediated by perforin and
granzymes that induce apoptosis.
Superantigens are bacterial or viral proteins that cause a nonspecific activation of helper T cells, leading to an excessive
release of cytokines (cytokine storm) and a systemic, potentially fatal inflammatory response.

Footnotes
1. Blaschitz C., Raffatellu M. “Th17 cytokines and the gut mucosal barrier.” J Clin Immunol. 2010 Mar; 30(2):196-203. doi:
10.1007/s10875-010-9368-7.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 14.2: T Lymphocytes and Cellular Immunity is shared under a CC BY license and was authored, remixed, and/or curated by
OpenStax.

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14.3: B Lymphocytes and Humoral Immunity
Learning Objectives
Describe the production and maturation of B cells
Compare the structure of B-cell receptors and T-cell receptors
Compare T-dependent and T-independent activation of B cells
Compare the primary and secondary antibody responses

Humoral immunity refers to mechanisms of the adaptive immune defenses that are mediated by antibodies secreted by B
lymphocytes, or B cells. This section will focus on B cells and discuss their production and maturation, receptors, and mechanisms
of activation. As explained previously, the antibodies involved in humoral immunity often bind pathogens and toxins before they
can attach to and invade host cells. Thus, humoral immunity is primarily concerned with fighting pathogens in extracellular spaces.
This also includes mucus secretions.

B Cell Production and Maturation

Humoral immunity refers to mechanisms of the adaptive immune defenses that are mediated by antibodies secreted by B
lymphocytes, or B cells. This section will focus on B cells and discuss their production and maturation, receptors, and mechanisms
of activation. Like T cells, B cells are formed from multipotent hematopoietic stem cells (HSCs) in the bone marrow and follow a
pathway through lymphoid stem cell and lymphoblast. Unlike T cells, however, lymphoblasts destined to become B cells do not
leave the bone marrow and travel to the thymus for maturation. Rather, eventual B cells continue to mature in the bone marrow.
The first step of B cell maturation is an assessment of the functionality of their antigen-binding receptors. This occurs through
positive selection for B cells with normal functional receptors. A mechanism of negative selection is then used to eliminate self-
reacting B cells and minimize the risk of autoimmunity. Negative selection of self-reacting B cells can involve elimination by
apoptosis, editing or modification of the receptors so they are no longer self-reactive, or induction of anergy in the B cell. Immature
B cells that pass the selection in the bone marrow then travel to the spleen for their final stages of maturation. There they become
naïve mature B cells, i.e., mature B cells that have not yet been activated.

Exercise 14.3.1

Compare the maturation of B cells with the maturation of T cells.

B-Cell Receptors
Like T cells, B cells possess antigen-specific receptors with diverse specificities. Although they rely on T cells for optimum
function, B cells can be activated without help from T cells. B-cell receptors (BCRs) for naïve mature B cells are membrane-bound
monomeric forms of IgD and IgM. They have two identical heavy chains and two identical light chains connected by disulfide
bonds into a basic “Y” shape (Figure 14.3.1). The trunk of the Y-shaped molecule, the constant region of the two heavy chains,
spans the B cell membrane. The two antigen-binding sites exposed to the exterior of the B cell are involved in the binding of
specific pathogen epitopes to initiate the activation process. It is estimated that each naïve mature B cell has upwards of 100,000
BCRs on its membrane, and each of these BCRs has an identical epitope-binding specificity.
In order to be prepared to react to a wide range of microbial epitopes, B cells, like T cells, use genetic rearrangement of hundreds
of gene segments to provide the necessary diversity of receptor specificities. The variable region of the BCR heavy chain is made
up of V, D, and J segments, similar to the β chain of the TCR. The variable region of the BCR light chain is made up of V and J
segments, similar to the α chain of the TCR. Genetic rearrangement of all possible combinations of V-J-D (heavy chain) and V-J
(light chain) provides for millions of unique antigen-binding sites for the BCR and for the antibodies secreted after activation.
One important difference between BCRs and TCRs is the way they can interact with antigenic epitopes. Whereas TCRs can only
interact with antigenic epitopes that are presented within the antigen-binding cleft of MHC I or MHC II, BCRs do not require
antigen presentation with MHC; they can interact with epitopes on free antigens or with epitopes displayed on the surface of intact
pathogens. Another important difference is that TCRs only recognize protein epitopes, whereas BCRs can recognize epitopes
associated with different molecular classes (e.g., proteins, polysaccharides, lipopolysaccharides).

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Activation of B cells occurs through different mechanisms depending on the molecular class of the antigen. Activation of a B cell
by a protein antigen requires the B cell to function as an APC, presenting the protein epitopes with MHC II to helper T cells.
Because of their dependence on T cells for activation of B cells, protein antigens are classified as T-dependent antigens. In contrast,
polysaccharides, lipopolysaccharides, and other nonprotein antigens are considered T-independent antigens because they can
activate B cells without antigen processing and presentation to T cells.

Figure 14.3.1 : B-cell receptors are embedded in the membranes of B cells. The variable regions of all of the receptors on a single
cell bind the same specific antigen.

Exercise 14.3.2

1. What types of molecules serve as the BCR?

2. What are the differences between TCRs and BCRs with respect to antigen recognition?
3. Which molecule classes are T-dependent antigens and which are T-independent antigens?

T Cell-Independent Activation of B cells

Activation of B cells without the cooperation of helper T cells is referred to as T cell-independent activation and occurs when
BCRs interact with T-independent antigens. T-independent antigens (e.g., polysaccharide capsules, lipopolysaccharide) have
repetitive epitope units within their structure, and this repetition allows for the cross-linkage of multiple BCRs, providing the first
signal for activation (Figure 14.3.2). Because T cells are not involved, the second signal has to come from other sources, such as
interactions of toll-like receptors with PAMPs or interactions with factors from the complement system.
Once a B cell is activated, it undergoes clonal proliferation and daughter cells differentiate into plasma cells. Plasma cells are
antibody factories that secrete large quantities of antibodies. After differentiation, the surface BCRs disappear and the plasma cell
secretes pentameric IgM molecules that have the same antigen specificity as the BCRs (Figure 14.3.2).
The T cell-independent response is short-lived and does not result in the production of memory B cells. Thus it will not result in a
secondary response to subsequent exposures to T-independent antigens. There is no internalization of the antigen and no interaction
of the MHC II with any other cells.

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 Figure 14.3.2 : T-independent antigens have repeating epitopes that can induce B cell recognition and activation without
involvement from T cells. A second signal, such as interaction of TLRs with PAMPs (not shown), is also required for activation of
the B cell. Once activated, the B cell proliferates and differentiates into antibody-secreting plasma cells.

Exercise 14.3.3

1. What are the two signals required for T cell-independent activation of B cells?
2. What is the function of a plasma cell?

T Cell-Dependent Activation of B cells

T cell-dependent activation of B cells is more complex than T cell-independent activation, but the resulting immune response is
stronger and develops memory. T cell-dependent activation can occur either in response to free protein antigens or to protein
antigens associated with an intact pathogen. Interaction between the BCRs on a naïve mature B cell and a free protein antigen
stimulate internalization of the antigen, (see figure 14.1.15's example with a dendritic cell) whereas interaction with antigens
associated with an intact pathogen initiates the extraction of the antigen from the pathogen before internalization. Once internalized
inside the B cell, the protein antigen is processed and presented with MHC II. The presented antigen is then recognized by helper T
cells specific to the same antigen. The TCR of the helper T cell recognizes the foreign antigen, and the T cell’s CD4 molecule
interacts with MHC II on the B cell. The coordination between B cells and helper T cells that are specific to the same antigen is
referred to as linked recognition.
Once activated by linked recognition, TH2 cells produce and secrete cytokines that activate the B cell and cause proliferation into
clonal daughter cells. After several rounds of proliferation, additional cytokines provided by the TH2 cells stimulate the
differentiation of activated B cell clones into memory B cells, which will quickly respond to subsequent exposures to the same
protein epitope, and plasma cells that lose their membrane BCRs and initially secrete pentameric IgM (Figure 14.3.3).
After initial secretion of IgM, cytokines secreted by TH2 cells stimulate the plasma cells to switch from IgM production to
production of IgG, IgA, or IgE. This process, called class switching or isotype switching, allows plasma cells cloned from the same
activated B cell to produce a variety of antibody classes with the same epitope specificity. Class switching is accomplished by
genetic rearrangement of gene segments encoding the constant region, which determines an antibody’s class. The variable region is
not changed, so the new class of antibody retains the original epitope specificity.

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 Figure 14.3.3 : In T cell-dependent activation of B cells, there are several steps. (A) the B cell recognizes and internalizes an
antigen. (B) The antigen is processed and an epitope presented on the MHC II. The B cell searches for the T cell that recognizes the
same antigen as it is presenting. (C) The helper T cell interacts with the antigen presented by the B cell, which activates the T cell
and stimulates the release of cytokines that then activate the B cell. (D) Activation of the B cell triggers proliferation and
differentiation into memory B cells and plasma cells.

Exercise 14.3.4

1. What steps are required for T cell-dependent activation of B cells?

2. What is antibody class switching and why is it important?

Primary and Secondary Responses

T cell-dependent activation of B cells plays an important role in both the primary and secondary responses associated with adaptive
immunity. With the first exposure to a protein antigen, a T cell-dependent primary antibody response occurs. The initial stage of the
primary response is a lag period, or latent period, of approximately 10 days, during which no antibody can be detected in serum.
This lag period is the time required for all of the steps of the primary response, including naïve mature B cell binding of antigen
with BCRs, antigen processing and presentation, helper T cell activation, B cell activation, and clonal proliferation. The end of the
lag period is characterized by a rise in IgM levels in the serum, as TH2 cells stimulate B cell differentiation into plasma cells. IgM
levels reach their peak around 14 days after primary antigen exposure; at about this same time, TH2 stimulates antibody class
switching, and IgM levels in serum begin to decline. Meanwhile, levels of IgG increase until they reach a peak about three weeks
into the primary response (Figure 14.3.4).
During the primary response, some of the cloned B cells are differentiated into memory B cells programmed to respond to
subsequent exposures. This secondary response occurs more quickly and forcefully than the primary response. The lag period is
decreased to only a few days and the production of IgG is significantly higher than observed for the primary response (Figure
14.3.4). In addition, the antibodies produced during the secondary response are more effective and bind with higher affinity to the

targeted epitopes. Plasma cells produced during secondary responses live longer than those produced during the primary response,
so levels of specific antibody remain elevated for a longer period of time.

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 Figure 14.3.4 : Compared to the primary response, the secondary antibody response occurs more quickly and produces antibody
levels that are higher and more sustained. The secondary response mostly involves IgG.

Exercise 14.3.5
1. What events occur during the lag period of the primary antibody response?
2. Why do antibody levels remain elevated longer during the secondary antibody response?

Key Concepts and Summary

B lymphocytes or B cells produce antibodies involved in humoral immunity. B cells are produced in the bone marrow, where
the initial stages of maturation occur, and travel to the spleen for final steps of maturation into naïve mature B cells.
B-cell receptors (BCRs) are membrane-bound monomeric forms of IgD and IgM that bind specific antigen epitopes with their
Fab antigen-binding regions. Diversity of antigen binding specificity is created by genetic rearrangement of V, D, and J
segments similar to the mechanism used for TCR diversity.
Protein antigens are called T-dependent antigens because they can only activate B cells with the cooperation of helper T cells.
Other molecule classes do not require T cell cooperation and are called T-independent antigens.
T cell-independent activation of B cells involves cross-linkage of BCRs by repetitive nonprotein antigen epitopes. It is
characterized by the production of IgM by plasma cells and does not produce memory B cells.
T cell-dependent activation of B cells involves processing and presentation of protein antigens to helper T cells, activation of
the B cells by cytokines secreted from activated TH2 cells, and plasma cells that produce different classes of antibodies as a
result of class switching. Memory B cells are also produced.
Secondary exposures to T-dependent antigens result in a secondary antibody response initiated by memory B cells. The
secondary response develops more quickly and produces higher and more sustained levels of antibody with higher affinity for
the specific antigen.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

14.3: B Lymphocytes and Humoral Immunity is shared under a CC BY license and was authored, remixed, and/or curated by LibreTexts.

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14.4: Vaccines
Learning Objectives
Compare the various kinds of artificial immunity
Differentiate between variolation and vaccination
Describe different types of vaccines and explain their respective advantages and disadvantages

For many diseases, prevention is the best form of treatment, and few strategies for disease prevention are as effective as
vaccination. Vaccination is a form of artificial immunity. By artificially stimulating the adaptive immune defenses, a vaccine
triggers memory cell production similar to that which would occur during a primary response. In so doing, the patient is able to
mount a strong secondary response upon exposure to the pathogen—but without having to first suffer through an initial infection.
In this section, we will explore several different kinds of artificial immunity along with various types of vaccines and the
mechanisms by which they induce artificial immunity.

Classifications of Adaptive Immunity

All forms of adaptive immunity can be described as either active or passive. Active immunity refers to the activation of an
individual’s own adaptive immune defenses, whereas passive immunity refers to the transfer of adaptive immune defenses from
another individual or animal. Active and passive immunity can be further subdivided based on whether the protection is acquired
naturally or artificially.
Natural active immunity is adaptive immunity that develops after natural exposure to a pathogen (Figure 14.4.1). Examples would
include the lifelong immunity that develops after recovery from a chickenpox or measles infection (although an acute infection is
not always necessary to activate adaptive immunity). The length of time that an individual is protected can vary substantially
depending upon the pathogen and antigens involved. For example, activation of adaptive immunity by protein spike structures
during an intracellular viral infection can activate lifelong immunity, whereas activation by carbohydrate capsule antigens during
an extracellular bacterial infection may activate shorter-term immunity.
Natural passive immunity involves the natural passage of antibodies from a mother to her child before and after birth. IgG is the
only antibody class that can cross the placenta from mother’s blood to the fetal blood supply. Placental transfer of IgG is an
important passive immune defense for the infant, lasting up to six months after birth. Secretory IgA can also be transferred from
mother to infant through breast milk.
Artificial passive immunity refers to the transfer of antibodies produced by a donor (human or animal) to another individual. This
transfer of antibodies may be done as a prophylactic measure (i.e., to prevent disease after exposure to a pathogen) or as a strategy
for treating an active infection. For example, artificial passive immunity is commonly used for post-exposure prophylaxis against
rabies, hepatitis A, hepatitis B, and chickenpox (in high risk individuals). Active infections treated by artificial passive immunity
include cytomegalovirus infections in immunocompromised patients and Ebola virus infections. In 1995, eight patients in the
Democratic Republic of the Congo with active Ebola infections were treated with blood transfusions from patients who were
recovering from Ebola. Only one of the eight patients died (a 12.5% mortality rate), which was much lower than the expected 80%
mortality rate for Ebola in untreated patients.1 Artificial passive immunity is also used for the treatment of diseases caused by
bacterial toxins, including tetanus, botulism, and diphtheria.
Artificial active immunity is the foundation for vaccination. It involves the activation of adaptive immunity through the deliberate
exposure of an individual to weakened or inactivated pathogens, or preparations consisting of key pathogen antigens.

Access for free at OpenStax 14.4.1 https://bio.libretexts.org/@go/page/31868

 Figure 14.4.1 : The four classifications of immunity. (credit top left photo: modification of work by USDA; credit top right photo:
modification of work by “Michaelberry”/Wikimedia; credit bottom left photo: modification of work by Centers for Disease Control
and Prevention; credit bottom right photo: modification of work by Friskila Silitonga, Indonesia, Centers for Disease Control and
Prevention)

Exercise 14.4.1

1. What is the difference between active and passive immunity?

2. What kind of immunity is conferred by a vaccine?

Herd Immunity
The four kinds of immunity just described result from an individual’s adaptive immune system. For any given disease, an
individual may be considered immune or susceptible depending on his or her ability to mount an effective immune response upon
exposure. Thus, any given population is likely to have some individuals who are immune and other individuals who are
susceptible. If a population has very few susceptible individuals, even those susceptible individuals will be protected by a
phenomenon called herd immunity. Herd immunity has nothing to do with an individual’s ability to mount an effective immune
response; rather, it occurs because there are too few susceptible individuals in a population for the disease to spread effectively.
Vaccination programs create herd immunity by greatly reducing the number of susceptible individuals in a population. Even if
some individuals in the population are not vaccinated, as long as a certain percentage is immune (either naturally or artificially), the
few susceptible individuals are unlikely to be exposed to the pathogen. However, because new individuals are constantly entering
populations (for example, through birth or relocation), vaccination programs are necessary to maintain herd immunity.

Vaccination: Obligation or Choice

A growing number of parents are choosing not to vaccinate their children. They are dubbed “antivaxxers,” and the majority of
them believe that vaccines are a cause of autism (or other disease conditions), a link that has now been thoroughly disproven.
Others object to vaccines on religious or moral grounds (e.g., the argument that Gardasil vaccination against HPV may
promote sexual promiscuity), on personal ethical grounds (e.g., a conscientious objection to any medical intervention), or on
political grounds (e.g., the notion that mandatory vaccinations are a violation of individual liberties).2
It is believed that this growing number of unvaccinated individuals has led to new outbreaks of whooping cough and measles.
We would expect that herd immunity would protect those unvaccinated in our population, but herd immunity can only be

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 maintained if enough individuals are being vaccinated.
Vaccination is clearly beneficial for public health. But from the individual parent’s perspective the view can be murkier.
Vaccines, like all medical interventions, have associated risks, and while the risks of vaccination may be extremely low
compared to the risks of infection, parents may not always understand or accept the consensus of the medical community. Do
such parents have a right to withhold vaccination from their children? Should they be allowed to put their children—and
society at large—at risk?
Many governments insist on childhood vaccinations as a condition for entering public school, but it has become easy in most
states to opt out of the requirement or to keep children out of the public system. Since the 1970s, West Virginia and Mississippi
have had in place a stringent requirement for childhood vaccination, without exceptions, and neither state has had a case of
measles since the early 1990s. California lawmakers recently passed a similar law in response to a measles outbreak in 2015,
making it much more difficult for parents to opt out of vaccines if their children are attending public schools. Given this track
record and renewed legislative efforts, should other states adopt similarly strict requirements?
What role should health-care providers play in promoting or enforcing universal vaccination? Studies have shown that many
parents’ minds can be changed in response to information delivered by health-care workers, but is it the place of health-care
workers to try to persuade parents to have their children vaccinated? Some health-care providers are understandably reluctant
to treat unvaccinated patients. Do they have the right to refuse service to patients who decline vaccines? Do insurance
companies have the right to deny coverage to antivaxxers? These are all ethical questions that policymakers may be forced to
address as more parents skirt vaccination norms.

Variolation and Vaccination

Thousands of years ago, it was first recognized that individuals who survived a smallpox infection were immune to subsequent
infections. The practice of inoculating individuals to actively protect them from smallpox appears to have originated in the 10th
century in China, when the practice of variolation was described (Figure 14.4.2). Variolation refers to the deliberate inoculation of
individuals with infectious material from scabs or pustules of smallpox victims. Infectious materials were either injected into the
skin or introduced through the nasal route. The infection that developed was usually milder than naturally acquired smallpox, and
recovery from the milder infection provided protection against the more serious disease.
Although the majority of individuals treated by variolation developed only mild infections, the practice was not without risks. More
serious and sometimes fatal infections did occur, and because smallpox was contagious, infections resulting from variolation could
lead to epidemics. Even so, the practice of variolation for smallpox prevention spread to other regions, including India, Africa, and
Europe.

Figure 14.4.2 : Variolation for smallpox originated in the Far East and the practice later spread to Europe and Africa. This Japanese
relief depicts a patient receiving a smallpox variolation from the physician Ogata Shunsaku (1748–1810).
Although variolation had been practiced for centuries, the English physician Edward Jenner (1749–1823) is generally credited with
developing the modern process of vaccination. Jenner observed that milkmaids who developed cowpox, a disease similar to
smallpox but milder, were immune to the more serious smallpox. This led Jenner to hypothesize that exposure to a less virulent
pathogen could provide immune protection against a more virulent pathogen, providing a safer alternative to variolation. In 1796,

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Jenner tested his hypothesis by obtaining infectious samples from a milkmaid’s active cowpox lesion and injecting the materials
into a young boy (Figure 14.4.3). The boy developed a mild infection that included a low-grade fever, discomfort in his axillae
(armpit) and loss of appetite. When the boy was later infected with infectious samples from smallpox lesions, he did not contract
smallpox.3 This new approach was termed vaccination, a name deriving from the use of cowpox (Latin vacca meaning “cow”) to
protect against smallpox. Today, we know that Jenner’s vaccine worked because the cowpox virus is genetically and antigenically
related to the Variola viruses that caused smallpox. Exposure to cowpox antigens resulted in a primary response and the production
of memory cells that identical or related epitopes of Variola virus upon a later exposure to smallpox.
The success of Jenner’s smallpox vaccination led other scientists to develop vaccines for other diseases. Perhaps the most notable
was Louis Pasteur, who developed vaccines for rabies, cholera, and anthrax. During the 20th and 21stcenturies, effective vaccines
were developed to prevent a wide range of diseases caused by viruses (e.g., chickenpox and shingles, hepatitis, measles, mumps,
polio, and yellow fever) and bacteria (e.g., diphtheria, pneumococcal pneumonia, tetanus, and whooping cough,).

Figure 14.4.3 : (a) A painting of Edward Jenner depicts a cow and a milkmaid in the background. (b) Lesions on a patient infected
with cowpox, a zoonotic disease caused by a virus closely related to the one that causes smallpox. (credit b: modification of work
by the Centers for Disease Control and Prevention)

Exercise 14.4.2

1. What is the difference between variolation and vaccination for smallpox?

2. Explain why vaccination is less risky than variolation.

Classes of Vaccines
For a vaccine to provide protection against a disease, it must expose an individual to pathogen-specific antigens that will stimulate
a protective adaptive immune response. By its very nature, this entails some risk. As with any pharmaceutical drug, vaccines have
the potential to cause adverse effects. However, the ideal vaccine causes no severe adverse effects and poses no risk of contracting
the disease that it is intended to prevent. Various types of vaccines have been developed with these goals in mind. These different
classes of vaccines are described in the next section and summarized in Table 14.4.1.

Live Attenuated Vaccines

Live attenuated vaccines expose an individual to a weakened strain of a pathogen with the goal of establishing a subclinical
infection that will activate the adaptive immune defenses. Pathogens are attenuated to decrease their virulence using methods such
as genetic manipulation (to eliminate key virulence factors) or long-term culturing in an unnatural host or environment (to promote
mutations and decrease virulence).
By establishing an active infection, live attenuated vaccines stimulate a more comprehensive immune response than some other
types of vaccines. Live attenuated vaccines activate both cellular and humoral immunity and stimulate the development of memory
for long-lasting immunity. In some cases, vaccination of one individual with a live attenuated pathogen can even lead to natural
transmission of the attenuated pathogen to other individuals. This can cause the other individuals to also develop an active,
subclinical infection that activates their adaptive immune defenses.
Disadvantages associated with live attenuated vaccines include the challenges associated with long-term storage and transport as
well as the potential for a patient to develop signs and symptoms of disease during the active infection (particularly in
immunocompromised patients). There is also a risk of the attenuated pathogen reverting back to full virulence. Table 14.4.1 lists
examples live attenuated vaccines.

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Inactivated Vaccines
Inactivated vaccines contain whole pathogens that have been killed or inactivated with heat, chemicals, or radiation. For inactivated
vaccines to be effective, the inactivation process must not affect the structure of key antigens on the pathogen.
Because the pathogen is killed or inactive, inactivated vaccines do not produce an active infection, and the resulting immune
response is weaker and less comprehensive than that provoked by a live attenuated vaccine. Typically the response involves only
humoral immunity, and the pathogen cannot be transmitted to other individuals. In addition, inactivated vaccines usually require
higher doses and multiple boosters, possibly causing inflammatory reactions at the site of injection.
Despite these disadvantages, inactivated vaccines do have the advantages of long-term storage stability and ease of transport. Also,
there is no risk of causing severe active infections. However, inactivated vaccines are not without their side effects. Table 14.4.1
lists examples of inactivated vaccines.

Subunit Vaccines
Whereas live attenuated and inactive vaccines expose an individual to a weakened or dead pathogen, subunit vaccinesonly expose
the patient to the key antigens of a pathogen—not whole cells or viruses. Subunit vaccines can be produced either by chemically
degrading a pathogen and isolating its key antigens or by producing the antigens through genetic engineering. Because these
vaccines contain only the essential antigens of a pathogen, the risk of side effects is relatively low. Table 14.4.1 lists examples of
subunit vaccines.

Toxoid Vaccines
Like subunit vaccines, toxoid vaccines do not introduce a whole pathogen to the patient; they contain inactivated bacterial toxins,
called toxoids. Toxoid vaccines are used to prevent diseases in which bacterial toxins play an important role in pathogenesis. These
vaccines activate humoral immunity that neutralizes the toxins. Table 14.4.1lists examples of toxoid vaccines.

Conjugate Vaccines
A conjugate vaccine is a type of subunit vaccine that consists of a protein conjugated to a capsule polysaccharide. Conjugate
vaccines have been developed to enhance the efficacy of subunit vaccines against pathogens that have protective polysaccharide
capsules that help them evade phagocytosis, causing invasive infections that can lead to meningitis and other serious conditions.
The subunit vaccines against these pathogens introduce T-independent capsular polysaccharide antigens that result in the
production of antibodies that can opsonize the capsule and thus combat the infection; however, children under the age of two years
do not respond effectively to these vaccines. Children do respond effectively when vaccinated with the conjugate vaccine, in which
a protein with T-dependent antigens is conjugated to the capsule polysaccharide. The conjugated protein-polysaccharide antigen
stimulates production of antibodies against both the protein and the capsule polysaccharide. Table 14.4.1 lists examples of
conjugate vaccines.
Table 14.4.1 : Classes of Vaccines

Class Description Advantages Disadvantages Examples

Cellular and humoral Difficult to store and

immunity transport
Chickenpox, German
Weakened strain of whole Risk of infection in measles, measles, mumps,
Live attenuated
pathogen Long-lasting immunity immunocompromised tuberculosis, typhoid fever,
patients yellow fever

Transmission to contacts Risk of reversion

Ease of storage and Weaker immunity

Whole pathogen killed or transport (humoral only) Cholera, hepatitis A,
Inactivated inactivated with heat,
No risk of severe active Higher doses and more influenza, plague, rabies
chemicals, or radiation
infection boosters required

Limited longevity Anthrax, hepatitis B,

influenza, meningitis,
Multiple doses required
Subunit Immunogenic antigens Lower risk of side effects papillomavirus,
No protection against pneumococcal pneumonia,
antigenic variation whooping cough

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 Class Description Advantages Disadvantages Examples

Humoral immunity to Botulism, diphtheria,

Toxoid Inactivated bacterial toxin Does not prevent infection
neutralize toxin pertussis, tetanus

Costly to produce
T-dependent response to Meningitis
capsule No protection against
Capsule polysaccharide (Haemophilus influenzae,
Conjugate antigenic variation
conjugated to protein Streptococcus pneumoniae,
Better response in young May interfere with other Neisseria meningitides)
children vaccines

Exercise 14.4.3

1. What is the risk associated with a live attenuated vaccine?

2. Why is a conjugated vaccine necessary in some cases?

DNA Vaccines
DNA vaccines represent a relatively new and promising approach to vaccination. A DNA vaccine is produced by incorporating
genes for antigens into a recombinant plasmid vaccine. Introduction of the DNA vaccine into a patient leads to uptake of the
recombinant plasmid by some of the patient’s cells, followed by transcription and translation of antigens and presentation of
these antigens with MHC I to activate adaptive immunity. This results in the stimulation of both humoral and cellular immunity
without the risk of active disease associated with live attenuated vaccines.
Although most DNA vaccines for humans are still in development, it is likely that they will become more prevalent in the near
future as researchers are working on engineering DNA vaccines that will activate adaptive immunity against several different
pathogens at once. First-generation DNA vaccines tested in the 1990s looked promising in animal models but were
disappointing when tested in human subjects. Poor cellular uptake of the DNA plasmids was one of the major problems
impacting their efficacy. Trials of second-generation DNA vaccines have been more promising thanks to new techniques for
enhancing cellular uptake and optimizing antigens. DNA vaccines for various cancers and viral pathogens such as HIV, HPV,
and hepatitis B and C are currently in development.
Some DNA vaccines are already in use. In 2005, a DNA vaccine against West Nile virus was approved for use in horses in the
United States. Canada has also approved a DNA vaccine to protect fish from infectious hematopoietic necrosis virus.4 A DNA
vaccine against Japanese encephalitis virus was approved for use in humans in 2010 in Australia.

Clinical Focus: Resolution

Based on Olivia’s symptoms, her physician made a preliminary diagnosis of bacterial meningitis without waiting for positive
identification from the blood and CSF samples sent to the lab. Olivia was admitted to the hospital and treated with intravenous
broad-spectrum antibiotics and rehydration therapy. Over the next several days, her condition began to improve, and new blood
samples and lumbar puncture samples showed an absence of microbes in the blood and CSF with levels of white blood cells
returning to normal. During this time, the lab produced a positive identification of Neisseria meningitidis, the causative agent
of meningococcal meningitis, in her original CSF sample.
N. meningitidis produces a polysaccharide capsule that serves as a virulence factor. N. meningitidis tends to affect infants after
they begin to lose the natural passive immunity provided by maternal antibodies. At one year of age, Olivia’s maternal IgG
antibodies would have disappeared, and she would not have developed memory cells capable of recognizing antigens
associated with the polysaccharide capsule of the N. meningitidis. As a result, her adaptive immune system was unable to
produce protective antibodies to combat the infection, and without antibiotics she may not have survived. Olivia’s infection
likely would have been avoided altogether had she been vaccinated. A conjugate vaccine to prevent meningococcal meningitis
is available and approved for infants as young as two months of age. However, current vaccination schedules in the United
States recommend that the vaccine be administered at age 11–12 with a booster at age 16.

In countries with developed public health systems, many vaccines are routinely administered to children and adults. Vaccine
schedules are changed periodically, based on new information and research results gathered by public health agencies. In the

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United States, the CDC publishes schedules and other updated information about vaccines.

Key Concepts and Summary

Adaptive immunity can be divided into four distinct classifications: natural active immunity, natural passive immunity,
artificial passive immunity, and artificial active immunity.
Artificial active immunity is the foundation for vaccination and vaccine development. Vaccination programs not only confer
artificial immunity on individuals, but also foster herd immunity in populations.
Variolation against smallpox originated in the 10th century in China, but the procedure was risky because it could cause the
disease it was intended to prevent. Modern vaccination was developed by Edward Jenner, who developed the practice of
inoculating patients with infectious materials from cowpox lesions to prevent smallpox.
Live attenuated vaccines and inactivated vaccines contain whole pathogens that are weak, killed, or inactivated. Subunit
vaccines, toxoid vaccines, and conjugate vaccines contain acellular components with antigens that stimulate an immune
response.

Footnotes
1. K. Mupapa, M. Massamba, K. Kibadi, K. Kivula, A. Bwaka, M. Kipasa, R. Colebunders, J. J. Muyembe-Tamfum. “Treatment
of Ebola Hemorrhagic Fever with Blood Transfusions from Convalescent Patients.” Journal of Infectious Diseases 179 Suppl.
(1999): S18–S23.
2. Elizabeth Yale. “Why Anti-Vaccination Movements Can Never Be Tamed.” Religion & Politics, July 22, 2014.
religionandpolitics.org/2014/...never-be-tamed.
3. N. J. Willis. “Edward Jenner and the Eradication of Smallpox.” Scottish Medical Journal 42 (1997): 118–121.
4. M. Alonso and J. C. Leong. “Licensed DNA Vaccines Against Infectious Hematopoietic Necrosis Virus (IHNV).” Recent
Patents on DNA & Gene Sequences (Discontinued) 7 no. 1 (2013): 62–65, issn 1872-2156/2212-3431. doi
10.2174/1872215611307010009.
5. S.B. Halstead and S. J. Thomas. “New Japanese Encephalitis Vaccines: Alternatives to Production in Mouse Brain.” Expert
Review of Vaccines 10 no. 3 (2011): 355–64.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 14.4: Vaccines is shared under a CC BY license and was authored, remixed, and/or curated by OpenStax.

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14.5: Practical Applications of Monoclonal and Polyclonal Antibodies
Learning Objectives
Compare the method of development, use, and characteristics of monoclonal and polyclonal antibodies
Explain the nature of antibody cross-reactivity and why this is less of a problem with monoclonal antibodies

In addition to being crucial for our normal immune response, antibodies provide powerful tools for research and diagnostic
purposes. The high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets,
from drugs to serum proteins to microorganisms. With in vitro assays, antibodies can be used to precipitate soluble antigens,
agglutinate (clump) cells, opsonize and kill bacteria with the assistance of complement, and neutralize drugs, toxins, and viruses.

Measuring Specificity
An antibody’s specificity results from the antigen-binding site formed within the variable regions—regions of the antibody that
have unique patterns of amino acids that can only bind to target antigens with a molecular sequence that provides complementary
charges and noncovalent bonds. There are limitations to antibody specificity, however. Some antigens are so chemically similar that
cross-reactivity occurs; in other words, antibodies raised against one antigen bind to a chemically similar but different antigen.
Consider an antigen that consists of a single protein with multiple epitopes (Figure 14.5.1). This single protein may stimulate the
production of many different antibodies, some of which may bind to chemically identical epitopes on other proteins.
Cross-reactivity is more likely to occur between antibodies and antigens that have low affinity or avidity. Affinity, which can be
determined experimentally, is a measure of the binding strength between an antibody's binding site and an epitope, whereas avidity
is the total strength of all the interactions in an antibody-antigen complex (which may have more than one bonding site). Avidity is
influenced by affinity as well as the structural arrangements of the epitope and the variable regions of the antibody. If an antibody
has a high affinity/avidity for a specific antigen, it is less likely to cross-react with an antigen for which it has a lower
affinity/avidity.

Figure 14.5.1 : An antibody binds to a specific region on an antigen called an epitope. A single antigen can have multiple epitopes
for different, specific antibodies.

Exercise 14.5.1
1. What property makes antibodies useful for research and clinical diagnosis?
2. What is cross-reactivity and why does it occur?

Producing Polyclonal Antibodies

Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a
specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen.
These antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an

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antigen. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies
in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune
system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding
to a specific epitope on the antigen (Figure 14.5.2).
Lab animals are usually injected at least twice with antigen when being used to produce antiserum. The second injection will
activate memory cells that make class IgG antibodies against the antigen. The memory cells also undergo affinity maturation,
resulting in a pool of antibodies with higher average affinity. Affinity maturation occurs because of mutations in the
immunoglobulin gene variable regions, resulting in B cells with slightly altered antigen-binding sites. On re-exposure to the
antigen, those B cells capable of producing antibody with higher affinity antigen-binding sites will be stimulated to proliferate and
produce more antibody than their lower-affinity peers. An adjuvant, which is a chemical that provokes a generalized activation of
the immune system that stimulates greater antibody production, is often mixed with the antigen prior to injection.
Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laboratory, but it
will also contain antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, antisera
must first be “purified” to remove other antibodies before using the antibodies for research or diagnostic assays.

Figure 14.5.2 : This diagram illustrates the process for harvesting polyclonal antibodies produced in response to an antigen.
Clinical Uses of Polyclonal Antisera
Polyclonal antisera are used in many clinical tests that are designed to determine whether a patient is producing antibodies in
response to a particular pathogen. While these tests are certainly powerful diagnostic tools, they have their limitations, because they
are an indirect means of determining whether a particular pathogen is present. Tests based on a polyclonal response can sometimes
lead to a false-positive result—in other words, a test that confirms the presence of an antigen that is, in fact, not present. Antibody-
based tests can also result in a false-negative result, which occurs when the test fails to detect an antibody that is, in fact, present.
The accuracy of antibody tests can be described in terms of test sensitivity and test specificity. Test sensitivity is the probability of
getting a positive test result when the patient is indeed infected. If a test has high sensitivity, the probability of a false negative is
low. Test specificity, on the other hand, is the probability of getting a negative test result when the patient is not infected. If a test
has high specificity, the probability of a false positive is low.
False positives often occur due to cross-reactivity, which can occur when epitopes from a different pathogen are similar to those
found on the pathogen being tested for. For this reason, antibody-based tests are often used only as screening tests; if the results are
positive, other confirmatory tests are used to make sure that the results were not a false positive.

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For example, a blood sample from a patient suspected of having hepatitis C can be screened for the virus using antibodies that bind
to antigens on hepatitis C virus. If the patient is indeed infected with hepatitis C virus, the antibodies will bind to the antigens,
yielding a positive test result. If the patient is not infected with hepatitic C virus, the antibodies will generally not bind to anything
and the test should be negative; however, a false positive may occur if the patient has been previously infected by any of a variety
of pathogens that elicit antibodies that cross-react with the hepatitis C virus antigens. Antibody tests for hepatitis C have high
sensitivity (a low probability of a false negative) but low specificity (a high probability of a false positive). Thus, patients who test
positive must have a second, confirmatory test to rule out the possibility of a false positive. The confirmatory test is a more
expensive and time-consuming test that directly tests for the presence of hepatitis C viral RNA in the blood. Only after the
confirmatory test comes back positive can the patient be definitively diagnosed with a hepatitis C infection. Antibody-based tests
can result in a false negative if, for any reason, the patient’s immune system has not produced detectable levels of antibodies. For
some diseases, it may take several weeks following infection before the immune system produces enough antibodies to cross the
detection threshold of the assay. In immunocompromised patients, the immune system may not be capable of producing a
detectable level of antibodies.
Another limitation of using antibody production as an indicator of disease is that antibodies in the blood will persist long after the
infection has been cleared. Depending on the type of infection, antibodies will be present for many months; sometimes, they may
be present for the remainder of the patient’s life. Thus, a positive antibody-based test only means that the patient was infected at
some point in time; it does not prove that the infection is active.
In addition to their role in diagnosis, polyclonal antisera can activate complement, detect the presence of bacteria in clinical and
food industry settings, and perform a wide array of precipitation reactions that can detect and quantify serum proteins, viruses, or
other antigens. However, with the many specificities of antibody present in a polyclonal antiserum, there is a significant likelihood
that the antiserum will cross-react with antigens to which the individual was never exposed. Therefore, we must always account for
the possibility of false-positive results when working with a polyclonal antiserum.

Exercise 14.5.2

1. What is a false positive and what are some reasons that false positives occur?
2. What is a false negative and what are some reasons that false positives occur?
3. If a patient tests negative on a highly sensitive test, what is the likelihood that the person is infected with the pathogen?

Producing Monoclonal Antibodies

Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain
this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by
monoclonal antibodies (mAbs). Table 14.5.1 compares some of the important characteristics of monoclonal and polyclonal
antibodies.
Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture
techniques. mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from the
spleen of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with
immortal, cancerous B cells called myeloma cells, to yield hybridoma cells. All of the cells are then placed in a selective medium
that allows only the hybridomas to grow; unfused myeloma cells cannot grow, and any unfused B cells die off. The hybridomas,
which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those
producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from
the medium. This is a very expensive and time-consuming process. It may take weeks of culturing and many liters of media to
provide enough mAbs for an experiment or to treat a single patient. Making mAbs is expensive (Figure 14.5.3).

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 Figure 14.5.3 : Monoclonal antibodies (mAbs) are produced by introducing an antigen to a mouse and then fusing polyclonal B
cells from the mouse’s spleen to myeloma cells. The resulting hybridoma cells are cultured and continue to produce antibodies to
the antigen. Hybridomas producing the desired mAb are then grown in large numbers on a selective medium that is periodically
harvested to obtain the desired mAbs.
Table 14.5.1 : Characteristics of Polyclonal and Monoclonal Antibodies

Monoclonal Antibodies Polyclonal Antibodies

Expensive production Inexpensive production

Long production time Rapid production

Large quantities of specific antibodies Large quantities of nonspecific antibodies

Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen

Production is continuous and uniform once the hybridoma is made Different batches vary in composition

Clinical Uses of Monoclonal Antibodies

Since the most common methods for producing monoclonal antibodies use mouse cells, it is necessary to create humanized
monoclonal antibodies for human clinical use. Mouse antibodies cannot be injected repeatedly into humans, because the immune
system will recognize them as being foreign and will respond to them with neutralizing antibodies. This problem can be minimized
by genetically engineering the antibody in the mouse B cell. The variable regions of the mouse light and heavy chain genes are
ligated to human constant regions, and the chimeric gene is then transferred into a host cell. This allows production of a mAb that
is mostly “human” with only the antigen-binding site being of mouse origin.

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Humanized mAbs have been successfully used to treat cancer with minimal side effects. For example, the humanized monoclonal
antibody drug Herceptin has been helpful for the treatment of some types of breast cancer. There have also been a few preliminary
trials of humanized mAb for the treatment of infectious diseases, but none of these treatments are currently in use. In some cases,
mAbs have proven too specific to treat infectious diseases, because they recognize some serovars of a pathogen but not others.
Using a cocktail of multiple mAbs that target different strains of the pathogen can address this problem. However, the great cost
associated with mAb production is another challenge that has prevented mAbs from becoming practical for use in treating
microbial infections.1
One promising technology for inexpensive mAbs is the use of genetically engineered plants to produce antibodies (or plantibodies).
This technology transforms plant cells into antibody factories rather than relying on tissue culture cells, which are expensive and
technically demanding. In some cases, it may even be possible to deliver these antibodies by having patients eat the plants rather
than by extracting and injecting the antibodies. For example, in 2013, a research group cloned antibody genes into plants that had
the ability to neutralize an important toxin from bacteria that can cause severe gastrointestinal disease.2 Eating the plants could
potentially deliver the antibodies directly to the toxin.

Exercise 14.5.3

1. How are humanized monoclonal antibodies produced?

2. What does the “monoclonal” of monoclonal antibodies mean?

Using Monoclonal Antibodies to Combat Ebola

During the 2014–2015 Ebola outbreak in West Africa, a few Ebola-infected patients were treated with ZMapp, a drug that had
been shown to be effective in trials done in rhesus macaques only a few months before.3 ZMapp is a combination of three
mAbs produced by incorporating the antibody genes into tobacco plants using a viral vector. By using three mAbs, the drug is
effective across multiple strains of the virus. Unfortunately, there was only enough ZMapp to treat a tiny number of patients.
While the current technology is not adequate for producing large quantities of ZMapp, it does show that plantibodies—plant-
produced mAbs—are feasible for clinical use, potentially cost effective, and worth further development. The last several years
have seen an explosion in the number of new mAb-based drugs for the treatment of cancer and infectious diseases; however,
the widespread use of such drugs is currently inhibited by their exorbitant cost, especially in underdeveloped parts of the
world, where a single dose might cost more than the patient’s lifetime income. Developing methods for cloning antibody genes
into plants could reduce costs dramatically.

Use of Antibodies in Testing and Identification

Enzyme immunoassays (EIAs) use antibodies to detect the presence of antigens. However, the assays are conducted in microtiter
plates or in vivo. There are many different types of EIAs, but they all involve an antibody molecule whose constant region binds an
enzyme, leaving the variable region free to bind its specific antigen. The addition of a substrate for the enzyme allows the antigen
to be visualized or quantified (Figure 14.5.4).
In EIAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product.
The most widely used enzymes are alkaline phosphatase and horseradish peroxidase for which appropriate substrates are readily
available. In some EIAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form.
EIAs that utilize a fluorogen are called fluorescent enzyme immunoassays (FEIAs). Fluorescence can be detected by either a
fluorescence microscope or a spectrophotometer.

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 Figure 14.5.4 : Enzyme immunoassays, such as the direct ELISA shown here, use an enzyme-antibody conjugate to deliver a
detectable substrate to the site of an antigen. The substrate may be a colorless molecule that is converted into a colored end product
or an inactive fluorescent molecule that fluoresces after enzyme activation. (credit: modification of work by “Cavitri”/Wikimedia
Commons)

The MMR Titer

The MMR vaccine is a combination vaccine that provides protection against measles, mumps, and rubella (German measles).
Most people receive the MMR vaccine as children and thus have antibodies against these diseases. However, for various
reasons, even vaccinated individuals may become susceptible to these diseases again later in life. For example, some children
may receive only one round of the MMR vaccine instead of the recommended two. In addition, the titer of protective
antibodies in an individual’s body may begin to decline with age or as the result of some medical conditions.
To determine whether the titer of antibody in an individual’s bloodstream is sufficient to provide protection, an MMR titer test
can be performed. The test is a simple immunoassay that can be done quickly with a blood sample. The results of the test will
indicate whether the individual still has immunity or needs another dose of the MMR vaccine.
Submitting to an MMR titer is often a pre-employment requirement for healthcare workers, especially those who will
frequently be in contact with young children or immunocompromised patients. Were a healthcare worker to become infected
with measles, mumps, or rubella, the individual could easily pass these diseases on to susceptible patients, leading to an
outbreak. Depending on the results of the MMR titer, healthcare workers might need to be revaccinated prior to beginning
work.

Immunostaining
One powerful use of EIA is immunostaining, in which antibody-enzyme conjugates enhance microscopy. Immunohistochemistry
(IHC) is used for examining whole tissues. As seen in Figure 14.5.5, a section of tissue can be stained to visualize the various cell
types. In this example, a mAb against CD8 was used to stain CD8 cells in a section of tonsil tissue. It is now possible to count the
number of CD8 cells, determine their relative numbers versus the other cell types present, and determine the location of these cells
within this tissue. Such data would be useful for studying diseases such as AIDS, in which the normal function of CD8 cells is
crucial for slowing disease progression.
Immunocytochemistry (ICC) is another valuable form of immunostaining. While similar to IHC, in ICC, extracellular matrix
material is stripped away, and the cell membrane is etched with alcohol to make it permeable to antibodies. This allows antibodies
to pass through the cell membrane and bind to specific targets inside the cell. Organelles, cytoskeletal components, and other
intracellular structures can be visualized in this way. While some ICC techniques use EIA, the enzyme can be replaced with a
fluorescent molecule, making it a fluorescent immunoassay.

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 Figure 14.5.5 : Enzyme-linked antibodies against CD8 were used to stain the CD8 cells in this preparation of bone marrow using a
chromogen. (credit: modification of work by Yamashita M, Fujii Y, Ozaki K, Urano Y, Iwasa M, Nakamura S, Fujii S, Abe M, Sato
Y, Yoshino T)

Exercise 14.5.4
1. What is the difference between immunohistochemistry and immunocytochemistry?
2. What must be true of the product of the enzymatic reaction used in immunohistochemistry?

Enzyme-linked Immunosorbent Assays (ELISAs)

The enzyme-linked immunosorbent assays (ELISAs) are widely used EIAs. In the direct ELISA, antigens are immobilized in the
well of a microtiter plate. An antibody that is specific for a particular antigen and is conjugated to an enzyme is added to each well.
If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate
(chromogen) is added. The presence of the enzyme converts the substrate into a colored end product (Figure 14.5.4). While this
technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is
lower (lower sensitivity).
In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from
a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples. The first step of a sandwich ELISA is
to add the primary antibody to all the wells of a microtiter plate (Figure 14.5.6). The antibody sticks to the plastic by hydrophobic
interactions. After an appropriate incubation time, any unbound antibody is washed away. Comparable washes are used between
each of the subsequent steps to ensure that only specifically bound molecules remain attached to the plate. A blocking protein is
then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well. Some of
the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are
added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which
is a polyclonal antibody that is conjugated to an enzyme. After a final wash, a colorless substrate (chromogen) is added, and the
enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a
spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme,
which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in
the nanogram (10–9 g) per mL range.
In an indirect ELISA, we quantify antigen-specific antibody rather than antigen. We can use indirect ELISA to detect antibodies
against many types of pathogens, including Borrelia burgdorferi (Lyme disease) and HIV. There are three important differences
between indirect and direct ELISAs as shown in Figure 14.5.7. Rather than using antibody to capture antigen, the indirect ELISA
starts with attaching known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells. After blocking the
unbound sites on the plate, patient serum is added; if antibodies are present (primary antibody), they will bind the antigen. After
washing away any unbound proteins, the secondary antibody with its conjugated enzyme is directed against the primary antibody
(e.g., antihuman immunoglobulin). The secondary antibody allows us to quantify how much antigen-specific antibody is present in
the patient’s serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction.
As with several other tests for antibodies discussed in this chapter, there is always concern about cross-reactivity with antibodies
directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV
infection (or any other type of infection) based on a single indirect ELISA assay. We must confirm any suspected positive test,
which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or
a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid
antigen test.

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Figure 14.5.6 : (a) In a sandwich ELISA, a primary antibody is used to first capture an antigen with the primary antibody. A
secondary antibody conjugated to an enzyme that also recognizes epitopes on the antigen is added. After the addition of the
chromogen, a spectrophotometer measures the absorbance of end product, which is directly proportional to the amount of captured
antigen. (b) An ELISA plate shows dilutions of antibodies (left) and antigens (bottom). Higher concentrations result in a darker
final color. (credit b: modification of work by U.S. Fish and Wildlife Service Pacific Region)

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Figure 14.5.7 : The indirect ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen
from the suspected disease agent is attached to microtiter plates. The primary antibody comes from the patient’s serum, which is
subsequently bound by the enzyme-conjugated secondary antibody. Measuring the production of end product allows us to detect or
quantify the amount of antigen-specific antibody present in the patient’s serum.

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 Exercise 14.5.5

1. What is the purpose of the secondary antibody in a direct ELISA?

2. What do the direct and indirect ELISAs quantify?

Key Concepts and Summary

Antibodies bind with high specificity to antigens used to challenge the immune system, but they may also show cross-
reactivity by binding to other antigens that share chemical properties with the original antigen.
Injection of an antigen into an animal will result in a polyclonal antibody response in which different antibodies are produced
that react with the various epitopes on the antigen.
Polyclonal antisera are useful for some types of laboratory assays, but other assays require more specificity. Diagnostic tests
that use polyclonal antisera are typically only used for screening because of the possibility of false-positive and false-negative
results.
Monoclonal antibodies provide higher specificity than polyclonal antisera because they bind to a single epitope and usually
have high affinity.
Monoclonal antibodies are typically produced by culturing antibody-secreting hybridomas derived from mice. mAbs are
currently used to treat cancer, but their exorbitant cost has prevented them from being used more widely to treat infectious
diseases. Still, their potential for laboratory and clinical use is driving the development of new, cost-effective solutions such as
plantibodies.
Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to
bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen
or a fluorogen.
Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular
structures (immunocytochemistry).
Direct ELISA is used to quantify an antigen in solution. The primary antibody captures the antigen, and the secondary antibody
delivers an enzyme. Production of end product from the chromogenic substrate is directly proportional to the amount of
captured antigen.
Indirect ELISA is used to detect antibodies in patient serum by attaching antigen to the well of a microtiter plate, allowing the
patient (primary) antibody to bind the antigen and an enzyme-conjugated secondary antibody to detect the primary antibody.

Footnotes
1. Saylor, Carolyn, Ekaterina Dadachova and Arturo Casadevall, “Monoclonal Antibody-Based Therapies for Microbial
Diseases,” Vaccine 27 (2009): G38-G46.
2. Nakanishi, Katsuhiro et al., “Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1,”
PloS One 8, no. 11 (2013): e80712.
3. Qiu, Xiangguo et al., “Reversion of Advanced Ebola Virus Disease in Nonhuman Primates with ZMapp,” Nature 514 (2014):
47–53.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern
State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University)
with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

This page titled 14.5: Practical Applications of Monoclonal and Polyclonal Antibodies is shared under a CC BY license and was authored,
remixed, and/or curated by OpenStax.

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Chapter 14 Exercises
Review Questions for Chapter 14
Multiple Choice

1) Antibodies are produced by ________.

a. plasma cells
b. T cells
c. bone marrow
d. Macrophages

2) Cellular adaptive immunity is carried out by ________.

a. B cells
b. T cells
c. bone marrow
d. neutrophils

3) A single antigen molecule may be composed of many individual ________.

a. T-cell receptors
b. B-cell receptors
c. MHC II
d. epitopes

4) Which class of molecules is the most antigenic?

a. polysaccharides
b. lipids
c. proteins
d. carbohydrates

5) MHC I molecules present

a. processed foreign antigens from proteasomes.
b. processed self-antigens from phagolysosome.
c. antibodies.
d. T cell antigens.

6) MHC II molecules present

a. processed self-antigens from proteasomes.
b. processed foreign antigens from phagolysosomes.
c. antibodies.
d. T cell receptors.

7) Which type of antigen-presenting molecule is found on all nucleated cells?

a. MHC II
b. MHC I
c. antibodies

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 d. B-cell receptors

8) Which type of antigen-presenting molecule is found only on macrophages, dendritic cells, and B cells?
a. MHC I
b. MHC II
c. T-cell receptors
d. B-cell receptors

9) What is a superantigen?
a. a protein that is highly efficient at stimulating a single type of productive and specific T cell response
b. a protein produced by antigen-presenting cells to enhance their presentation capabilities
c. a protein produced by T cells as a way of increasing the antigen activation they receive from antigen-presenting cells
d. a protein that activates T cells in a nonspecific and uncontrolled manner

10) To what does the TCR of a helper T cell bind?

a. antigens presented with MHC I molecules
b. antigens presented with MHC II molecules
c. free antigen in a soluble form
d. haptens only

11) Cytotoxic T cells will bind with their TCR to which of the following?
a. antigens presented with MHC I molecules
b. antigens presented with MHC II molecules
c. free antigen in a soluble form
d. haptens only

12) A ________ molecule is a glycoprotein used to identify and distinguish white blood cells.
a. T-cell receptor
b. B-cell receptor
c. MHC I
d. cluster of differentiation

13) Name the T helper cell subset involved in antibody production.

a. TH1
b. TH2
c. TH17
d. CTL

14) Which of the following would be a T-dependent antigen?

a. lipopolysaccharide
b. glycolipid
c. protein
d. carbohydrate

15) Which of the following would be a BCR?

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 a. CD4
b. MHC II
c. MHC I
d. IgD

16) Which of the following does not occur during the lag period of the primary antibody response?
a. activation of helper T cells
b. class switching to IgG
c. presentation of antigen with MHC II
d. binding of antigen to BCRs

17) A patient is bitten by a dog with confirmed rabies infection. After treating the bite wound, the physician injects the patient with
antibodies that are specific for the rabies virus to prevent the development of an active infection. This is an example of:
a. Natural active immunity
b. Artificial active immunity
c. Natural passive immunity
d. Artificial passive immunity

18) A patient gets a cold, and recovers a few days later. The patient's classmates come down with the same cold roughly a week
later, but the original patient does not get the same cold again. This is an example of:
a. Natural active immunity
b. Artificial active immunity
c. Natural passive immunity
d. Artificial passive immunity

19) For many uses in the laboratory, polyclonal antibodies work well, but for some types of assays, they lack sufficient ________
because they cross-react with inappropriate antigens.
a. specificity
b. sensitivity
c. accuracy
d. reactivity

20) How are monoclonal antibodies produced?

a. Antibody-producing B cells from a mouse are fused with myeloma cells and then the cells are grown in tissue culture.
b. A mouse is injected with an antigen and then antibodies are harvested from its serum.
c. They are produced by the human immune system as a natural response to an infection.
d. They are produced by a mouse’s immune system as a natural response to an infection.

21) When using an EIA to study microtubules or other structures inside a cell, we first chemically fix the cell and then treat the
cells with alcohol. What is the purpose of this alcohol treatment?
a. It makes holes in the cell membrane large enough for antibodies to pass.
b. It makes the membrane sticky so antibodies will bind and be taken up by receptor-mediated endocytosis.
c. It removes negative charges from the membrane, which would otherwise repulse the antibodies.
d. It prevents nonspecific binding of the antibodies to the cell membrane.

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22) In a direct fluorescent antibody test, which of the following would we most likely be looking for using a fluorescently-labeled
mAb?
a. bacteria in a patient sample
b. bacteria isolated from a patient and grown on agar plates
c. antiserum from a patient smeared onto a glass slide
d. antiserum from a patient that had bound to antigen-coated beads

Fill-in-the-Blanks

23) There are two critically important aspects of adaptive immunity. The first is specificity, while the second is ________.

24) ________ immunity involves the production of antibody molecules that bind to specific antigens.

25) The heavy chains of an antibody molecule contain ________ region segments, which help to determine its class or isotype.

26) The variable regions of the heavy and light chains form the ________ sites of an antibody.

27) MHC molecules are used for antigen ________ to T cells.

28) MHC II molecules are made up of two subunits (α and β) of approximately equal size, whereas MHC I molecules consist of a
larger α subunit and a smaller subunit called ________.

29) A ________ T cell will become activated by presentation of foreign antigen associated with an MHC I molecule.

30) A ________ T cell will become activated by presentation of foreign antigen in association with an MHC II molecule.

31) A TCR is a protein dimer embedded in the plasma membrane of a T cell. The ________ region of each of the two protein
chains is what gives it the capability to bind to a presented antigen.

32) Peripheral tolerance mechanisms function on T cells after they mature and exit the ________.

33) Both ________ and effector T cells are produced during differentiation of activated T cells.

34) ________ antigens can stimulate B cells to become activated but require cytokine assistance delivered by helper T cells.

35) T-independent antigens can stimulate B cells to become activated and secrete antibodies without assistance from helper T cells.
These antigens possess ________ antigenic epitopes that cross-link BCRs.

36) A(n) ________ pathogen is in a weakened state; it is still capable of stimulating an immune response but does not cause a
disease.

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37) ________ immunity occurs when antibodies from one individual are harvested and given to another to protect against disease
or treat active disease.

38) In the practice of ________, scabs from smallpox victims were used to immunize susceptible individuals against smallpox.

39) When we inject an animal with the same antigen a second time a few weeks after the first, ________ takes place, which means
the antibodies produced after the second injection will on average bind the antigen more tightly.

40) When using mAbs to treat disease in humans, the mAbs must first be ________ by replacing the mouse constant region DNA
with human constant region DNA.

41) If we used normal mouse mAbs to treat human disease, multiple doses would cause the patient to respond with ________
against the mouse antibodies.

42) A polyclonal response to an infection occurs because most antigens have multiple ________,

43) When slowly adding antigen to an antiserum, the amount of precipitin would gradually increase until reaching the ________;
addition of more antigen after this point would actually decrease the amount of precipitin.

44) To detect antibodies against bacteria in the bloodstream using an EIA, we would run a(n) ________, which we would start by
attaching antigen from the bacteria to the wells of a microtiter plate.

Short Answer

45) What is the difference between humoral and cellular adaptive immunity?

46) What is the difference between an antigen and a hapten?

47) Describe the mechanism of antibody-dependent cell-mediated cytotoxicity.

48) What is the basic difference in effector function between helper and cytotoxic T cells?

49) What necessary interactions are required for activation of helper T cells and activation/effector function of cytotoxic T cells?

50) Briefly compare the pros and cons of inactivated versus live attenuated vaccines.

51) Describe two reasons why polyclonal antibodies are more likely to exhibit cross-reactivity than monoclonal antibodies.

52) Explain why hemolysis in the complement fixation test is a negative test for infection.

53) What is meant by the term “neutralizing antibodies,” and how can we quantify this effect using the viral neutralization assay?

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54) Why is it important in a sandwich ELISA that the antigen has multiple epitopes? And why might it be advantageous to use
polyclonal antisera rather than mAb in this assay?

Critical Thinking

55) Which mechanism of antigen presentation would be used to present antigens from a cell infected with a virus?

56) Which pathway of antigen presentation would be used to present antigens from an extracellular bacterial infection?

57) A patient lacks the ability to make functioning T cells because of a genetic disorder. Would this patient’s B cells be able to
produce antibodies in response to an infection? Explain your answer.

58) Suppose you were screening produce in a grocery store for the presence of E. coli contamination. Would it be better to use a
polyclonal anti-E. coli antiserum or a mAb against an E. coli membrane protein? Explain.

59) Both IgM and IgG antibodies can be used in precipitation reactions. However, one of these immunoglobulin classes will form
precipitates at much lower concentrations than the other. Which class is this, and why is it so much more efficient in this regard?

60) When shortages of donated blood occur, O-negative blood may be given to patients, even if they have a different blood type.
Why is this the case? If O-negative blood supplies were depleted, what would be the next-best choice for a patient with a different
blood type in critical need of a transfusion? Explain your answers.

61) Label the primary and secondary antibodies, and discuss why the production of end product will be proportional to the amount
of antigen.

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CHAPTER OVERVIEW

Back Matter
Index

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Index
A Ames test atom
A site 6.5: Mutations 2.1: Atoms, Isotopes, Ions, and Molecules - The
Amino acids Building Blocks
6.4: Protein Synthesis (Translation)
2.6: Proteins atomic mass
absorbance
aminoglycosides 2.1: Atoms, Isotopes, Ions, and Molecules - The
3.1: How Microscopes Work Building Blocks
acellular 11.5: Drug Targets on Prokaryote Microorganisms
Amphipathic atomic number
1.5: Types of Microorganisms 2.1: Atoms, Isotopes, Ions, and Molecules - The
acid 2.5: Lipids
Building Blocks
2.2: Water amplitude ATP
Acidophiles 3.1: How Microscopes Work
7.1: Energy, Matter, and Enzymes
8.3: The Effects of pH and Temperature on Anabolism attachment
Microbial Growth 7.1: Energy, Matter, and Enzymes
9.2: The Viral Cycles
activator Analytical epidemiology attenuation
6.7: Gene Regulation and Operon Theory 12.6: Tracking Infectious Diseases
6.7: Gene Regulation and Operon Theory
active site anergy autotrophs
7.1: Energy, Matter, and Enzymes 14.2: T Lymphocytes and Cellular Immunity
7.1: Energy, Matter, and Enzymes
acute inflammation anion auxotrophs
13.4: Inflammation and Fever 2.1: Atoms, Isotopes, Ions, and Molecules - The
6.5: Mutations
Adaptive Immunity Building Blocks
anoxygenic photosynthesis
14.1: Architecture of the Immune System
7.4: Photosynthesis and the Importance of Light
B
ADCC bacitracin
14.1: Architecture of the Immune System antibodies
14.1: Architecture of the Immune System 11.5: Drug Targets on Prokaryote Microorganisms
adenine Bacteria
6.2: Structure and Replication of DNA anticodon
6.4: Protein Synthesis (Translation) 1.5: Types of Microorganisms
adenosine triphosphate bactericides
7.1: Energy, Matter, and Enzymes Antifungal drugs
11.6: Drugs for Non-prokaryote Microbes 11.1: Controlling Microbial Growth
adhesion bacteriology
2.2: Water Antigens
14.1: Architecture of the Immune System 1.5: Types of Microorganisms
12.2: Characteristics and Steps of Infectious
Diseases Antihelminthic drugs Bacteriophages
aerotolerant anaerobes 11.6: Drugs for Non-prokaryote Microbes 9.3: Isolation, Culture, and Identification of Viruses
8.2: Oxygen Requirements for Microbial Growth antimetabolites bacteriostatic
affinity 11.5: Drug Targets on Prokaryote Microorganisms 11.1: Controlling Microbial Growth
14.5: Practical Applications of Monoclonal and Antimicrobial drugs balanced chemical equation
Polyclonal Antibodies 2.1: Atoms, Isotopes, Ions, and Molecules - The
11.4: Discovering Antimicrobial Drugs
Agarose gel electrophoresis Antimicrobial resistance
Building Blocks
10.2: Visualizing and Characterizing DNA base
11.7: Mechanisms for Resistance
agglutination antiparallel
2.2: Water
14.1: Architecture of the Immune System basophils
6.2: Structure and Replication of DNA
alarmones Antiprotozoan drugs
13.2: Second Line Defenses: Cells and Fluids
6.7: Gene Regulation and Operon Theory Betaproteobacteria
11.6: Drugs for Non-prokaryote Microbes
Alcohols antiretroviral drugs
4.2: Classifying Prokaryotes and Examples
11.3: Using Chemicals to Control Microorganisms binary fission
11.6: Drugs for Non-prokaryote Microbes
alexidine antisepsis
8.1: How Microbes Grow
11.3: Using Chemicals to Control Microorganisms binomial nomenclature
11.1: Controlling Microbial Growth
algae Antiviral drugs
1.4: A Systematic Approach
1.5: Types of Microorganisms biofilms
11.6: Drugs for Non-prokaryote Microbes
aliphatic hydrocarbon apoenzyme
8.1: How Microbes Grow
2.3: Carbon and Organic Molecules bioinformatics
7.1: Energy, Matter, and Enzymes
Alkaliphiles Archaea
10.3: Whole Genome Methods and Industrial
Applications
8.3: The Effects of pH and Temperature on
1.5: Types of Microorganisms biological
Microbial Growth
Alkylating agents aromatic hydrocarbon 12.4: How Diseases Spread
2.3: Carbon and Organic Molecules biological safety levels
11.3: Using Chemicals to Control Microorganisms
allosteric inhibitor artificial active immunity 11.1: Controlling Microbial Growth
14.4: Vaccines biological Vector transmission
7.1: Energy, Matter, and Enzymes
allosteric inhibitors artificial passive immunity 12.4: How Diseases Spread
14.4: Vaccines biosynthesis
7.1: Energy, Matter, and Enzymes
allosteric sites aseptic technique 9.2: The Viral Cycles
11.1: Controlling Microbial Growth Biotechology
7.1: Energy, Matter, and Enzymes
Alphaproteobacteria 10.1: Microbes and the Tools of Genetic Engineering
4.2: Classifying Prokaryotes and Examples

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Bisbiguanides chemical reactivity cyclic photophosphorylation
11.3: Using Chemicals to Control Microorganisms 2.1: Atoms, Isotopes, Ions, and Molecules - The 7.4: Photosynthesis and the Importance of Light
buffer Building Blocks cytokine storm
2.2: Water Chemically defined media 14.2: T Lymphocytes and Cellular Immunity
1.6: Tools and Media Used for Bacterial Growth cytopathic effects
C Chemotrophs 9.3: Isolation, Culture, and Identification of Viruses
7.1: Energy, Matter, and Enzymes cytosine
CAP
6.7: Gene Regulation and Operon Theory
chloramphenicol 6.2: Structure and Replication of DNA
11.5: Drug Targets on Prokaryote Microorganisms cytoskeleton
capillary action
2.2: Water
Chlorhexidine 5.1: Characteristics of Eukaryotic Cells
11.3: Using Chemicals to Control Microorganisms cytotoxic T cells
capnophile
8.2: Oxygen Requirements for Microbial Growth
Chromosomal Theory of Inheritance 14.2: T Lymphocytes and Cellular Immunity
6.1: Using Microbiology to Discover the Secrets of
capsid Life
9.1: Viruses D
chronic inflammation
capsule 13.4: Inflammation and Fever
dark repair
4.1: Unique Characteristics of Prokaryotic Cells 6.5: Mutations
cilia
Capsule Staining 5.1: Characteristics of Eukaryotic Cells
death
3.2: Staining Microscopic Specimens and 5.2: Classifying Eukaryotic Microbes and Examples 8.1: How Microbes Grow
Descriptions decimal reduction time
Coenzymes
Carbohydrates 7.1: Energy, Matter, and Enzymes 11.1: Controlling Microbial Growth
2.4: Carbohydrates degenerate
cofactor
catabolism 7.1: Energy, Matter, and Enzymes 6.4: Protein Synthesis (Translation)
7.1: Energy, Matter, and Enzymes Deltaproteobacteria
7.3: Alternate Forms of Catabolism: Fermentation,
cognate amino acid
6.4: Protein Synthesis (Translation) 4.2: Classifying Prokaryotes and Examples
Lipids and Proteins
catabolite activator protein cohesion Denaturation
2.2: Water 2.6: Proteins
6.7: Gene Regulation and Operon Theory
catalase commercial sterilization dendritic cells
11.1: Controlling Microbial Growth 13.2: Second Line Defenses: Cells and Fluids
8.2: Oxygen Requirements for Microbial Growth
catalysts Competitive inhibitors deoxyribonucleic acid
7.1: Energy, Matter, and Enzymes 2.7: Nucleic Acids
7.1: Energy, Matter, and Enzymes
cation complement activatio Deoxyribonucleotides
14.1: Architecture of the Immune System 6.2: Structure and Replication of DNA
2.1: Atoms, Isotopes, Ions, and Molecules - The
Building Blocks complex deoxyribose
cell envelope 9.1: Viruses 6.2: Structure and Replication of DNA
6.3: Structure and Transcription of RNA
4.1: Unique Characteristics of Prokaryotic Cells compound
cell morphology 2.1: Atoms, Isotopes, Ions, and Molecules - The
dermis
Building Blocks 12.1: Normal Microbiota of the Body
4.1: Unique Characteristics of Prokaryotic Cells
cell wall conjugate vaccine Descriptive epidemiology
14.4: Vaccines 12.6: Tracking Infectious Diseases
4.1: Unique Characteristics of Prokaryotic Cells
5.1: Characteristics of Eukaryotic Cells conjugate vaccines Desiccation
cellular arrangement 14.4: Vaccines 11.2: Using Physical Methods to Control
Microorganisms
4.1: Unique Characteristics of Prokaryotic Cells Conjugated proteins
cellular immunity 2.6: Proteins
Differential media
1.6: Tools and Media Used for Bacterial Growth
14.1: Architecture of the Immune System conjugation
cellular uptake 6.6: How Asexual Prokaryotes Achieve Genetic
diffraction
Diversity 3.1: How Microscopes Work
11.7: Mechanisms for Resistance
cellulose conjugation pilus Direct Contact transmission
6.6: How Asexual Prokaryotes Achieve Genetic 12.4: How Diseases Spread
2.4: Carbohydrates
Centers for Disease Control and Diversity direct viable cell count
conjunctiva 8.1: How Microbes Grow
Prevention 12.1: Normal Microbiota of the Body disaccharides
12.5: The Language of Epidemiologists
constitutively expressed 2.4: Carbohydrates
central tolerance 6.7: Gene Regulation and Operon Theory Disinfection
14.2: T Lymphocytes and Cellular Immunity
Contact transmission 11.1: Controlling Microbial Growth
Centrosomes 12.4: How Diseases Spread dispersion
5.1: Characteristics of Eukaryotic Cells
contractile vacuoles 3.1: How Microscopes Work
charged tRNA 5.2: Classifying Eukaryotic Microbes and Examples dissociation
6.4: Protein Synthesis (Translation)
contrast 2.2: Water
chemical bond 3.1: How Microscopes Work DNA
2.1: Atoms, Isotopes, Ions, and Molecules - The
Building Blocks
covalent bond 2.7: Nucleic Acids
2.1: Atoms, Isotopes, Ions, and Molecules - The 6.1: Using Microbiology to Discover the Secrets of
chemical reaction Building Blocks Life
2.1: Atoms, Isotopes, Ions, and Molecules - The DNA probe
Building Blocks
critical item
11.1: Controlling Microbial Growth 10.2: Visualizing and Characterizing DNA

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doubling time Enzymes Flagella Staining
8.1: How Microbes Grow 7.1: Energy, Matter, and Enzymes 3.2: Staining Microscopic Specimens and
drug modification eosinophils Descriptions
11.7: Mechanisms for Resistance 13.2: Second Line Defenses: Cells and Fluids Florence Nightingale
Drug resistance Epidemic diseases 12.6: Tracking Infectious Diseases
11.7: Mechanisms for Resistance 12.5: The Language of Epidemiologists fluorescent
Epidemiology 3.1: How Microscopes Work
E 12.5: The Language of Epidemiologists fluoroquinolones
epidermis 11.5: Drug Targets on Prokaryote Microorganisms
E site
6.4: Protein Synthesis (Translation) 12.1: Normal Microbiota of the Body focal
epigenetic regulation 12.2: Characteristics and Steps of Infectious
edema Diseases
13.4: Inflammation and Fever 6.7: Gene Regulation and Operon Theory
epitopes focal infection
electrolyte 12.2: Characteristics and Steps of Infectious
2.1: Atoms, Isotopes, Ions, and Molecules - The 14.1: Architecture of the Immune System
Diseases
Building Blocks Epsilonproteobacteria focal length
electron carriers 4.2: Classifying Prokaryotes and Examples
3.1: How Microscopes Work
7.1: Energy, Matter, and Enzymes erythema focal point
electron configuration 13.4: Inflammation and Fever
3.1: How Microscopes Work
2.1: Atoms, Isotopes, Ions, and Molecules - The erythrocytes fomites
Building Blocks 13.2: Second Line Defenses: Cells and Fluids
11.1: Controlling Microbial Growth
Electron Microscopy Eukarya
3.2: Staining Microscopic Specimens and
Formaldehyde
1.5: Types of Microorganisms
Descriptions 11.3: Using Chemicals to Control Microorganisms
eukaryote formed elements
electron orbital
1.4: A Systematic Approach
2.1: Atoms, Isotopes, Ions, and Molecules - The 13.2: Second Line Defenses: Cells and Fluids
Building Blocks eutrophs frameshift mutation
electron transfer 4.2: Classifying Prokaryotes and Examples
6.5: Mutations
2.1: Atoms, Isotopes, Ions, and Molecules - The evaporation free ribosomes)
Building Blocks 2.2: Water
5.1: Characteristics of Eukaryotic Cells
electronegativity exergonic reaction frequency
2.1: Atoms, Isotopes, Ions, and Molecules - The 7.1: Energy, Matter, and Enzymes
3.1: How Microscopes Work
Building Blocks Exocytosis functional group
electrophoresis 5.1: Characteristics of Eukaryotic Cells
2.3: Carbon and Organic Molecules
10.2: Visualizing and Characterizing DNA exoenzymes Fungal pathogens
element 12.3: Virulence Factors in Infection
12.3: Virulence Factors in Infection
2.1: Atoms, Isotopes, Ions, and Molecules - The Experimental epidemiology Fungi
Building Blocks
12.6: Tracking Infectious Diseases
elongation 1.5: Types of Microorganisms
exposure fungicides
6.4: Protein Synthesis (Translation)
12.2: Characteristics and Steps of Infectious
enantiomers 11.1: Controlling Microbial Growth
Diseases
2.3: Carbon and Organic Molecules Extracellular Matrix fungistatic
Endemic diseases 11.1: Controlling Microbial Growth
5.1: Characteristics of Eukaryotic Cells
12.5: The Language of Epidemiologists extracellular polymeric substance
endergonic reactions 8.1: How Microbes Grow
G
7.1: Energy, Matter, and Enzymes Extravasation Gammaproteobacteria
Endocytosis 13.3: Pathogen Recognition and Phagocytosis 4.2: Classifying Prokaryotes and Examples
5.1: Characteristics of Eukaryotic Cells gene expression
endomembrane F 6.7: Gene Regulation and Operon Theory
5.1: Characteristics of Eukaryotic Cells F plasmid gene therapy
Endospore Staining 6.6: How Asexual Prokaryotes Achieve Genetic
10.4: Genetic Engineering - Risks, Benefits, and
Perceptions
3.2: Staining Microscopic Specimens and Diversity
Descriptions F+ cell generalized
endospores 6.6: How Asexual Prokaryotes Achieve Genetic
9.2: The Viral Cycles
4.1: Unique Characteristics of Prokaryotic Cells Diversity generalized transduction
endosymbiosis Facultative anaerobes 6.6: How Asexual Prokaryotes Achieve Genetic
Diversity
1.3: Foundations of Modern Cell Theory 8.2: Oxygen Requirements for Microbial Growth
endosymbiotic theory feedback inhibition generation time
8.1: How Microbes Grow
1.3: Foundations of Modern Cell Theory 7.1: Energy, Matter, and Enzymes
endothelia Fever genetic code
6.4: Protein Synthesis (Translation)
13.1: First Line defense- Physical, Mechanical and 13.4: Inflammation and Fever
Chemical Defenses fimbriae genetic engineering
Enriched media 4.1: Unique Characteristics of Prokaryotic Cells
10.1: Microbes and the Tools of Genetic Engineering
10.4: Genetic Engineering - Risks, Benefits, and
1.6: Tools and Media Used for Bacterial Growth flagella Perceptions
enzymatic bypass 4.1: Unique Characteristics of Prokaryotic Cells
11.7: Mechanisms for Resistance

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genomics HEPA inactivated vaccines
10.3: Whole Genome Methods and Industrial 11.2: Using Physical Methods to Control 14.4: Vaccines
Applications Microorganisms inactivation, prevention
geometric isomer herd immunity 11.7: Mechanisms for Resistance
2.3: Carbon and Organic Molecules 14.4: Vaccines Incidence
germ theory of disease heritability 12.5: The Language of Epidemiologists
1.3: Foundations of Modern Cell Theory 6.1: Using Microbiology to Discover the Secrets of inclusions
germinate Life
4.1: Unique Characteristics of Prokaryotic Cells
4.1: Unique Characteristics of Prokaryotic Cells heterotrophs indirect Contact transmission
Glutaraldehyde 7.1: Energy, Matter, and Enzymes
12.4: How Diseases Spread
11.3: Using Chemicals to Control Microorganisms Hfr cell induced fit
glycans 6.6: How Asexual Prokaryotes Achieve Genetic
7.1: Energy, Matter, and Enzymes
Diversity
2.4: Carbohydrates induced mutations
Glycocalyx holoenzyme
6.5: Mutations
7.1: Energy, Matter, and Enzymes
4.1: Unique Characteristics of Prokaryotic Cells inducer
Glycogen Horizontal Gene Transfer
6.7: Gene Regulation and Operon Theory
6.6: How Asexual Prokaryotes Achieve Genetic
2.4: Carbohydrates inducible operon
Diversity
glycolysis host ranges 6.7: Gene Regulation and Operon Theory
7.2: Catabolism of Carbohydrates inert gas
9.1: Viruses
glycopeptides Humoral Immunity 2.1: Atoms, Isotopes, Ions, and Molecules - The
11.5: Drug Targets on Prokaryote Microorganisms Building Blocks
14.1: Architecture of the Immune System
glycoprotein hybridomas infection
2.6: Proteins 12.2: Characteristics and Steps of Infectious
14.5: Practical Applications of Monoclonal and
glycosidic bonds Diseases
Polyclonal Antibodies
2.4: Carbohydrates hydrocarbon inflammation
Golgi apparatus 13.4: Inflammation and Fever
2.3: Carbon and Organic Molecules
5.1: Characteristics of Eukaryotic Cells hydrogen bond Initiation
Gram staining 6.4: Protein Synthesis (Translation)
2.1: Atoms, Isotopes, Ions, and Molecules - The
3.2: Staining Microscopic Specimens and Building Blocks initiation factors
Descriptions hydrophilic 6.4: Protein Synthesis (Translation)
Granulocytes 2.2: Water integrase inhibitors
13.2: Second Line Defenses: Cells and Fluids hydrophobic 11.6: Drugs for Non-prokaryote Microbes
granulomas 2.2: Water interference
13.4: Inflammation and Fever 2.5: Lipids 3.1: How Microscopes Work
granzymes hyperthemophiles intermediate filaments
14.2: T Lymphocytes and Cellular Immunity 8.4: Other Environmental Conditions that Affect 5.1: Characteristics of Eukaryotic Cells
guanine Growth invasion
6.2: Structure and Replication of DNA hypodermis 12.2: Characteristics and Steps of Infectious
12.1: Normal Microbiota of the Body Diseases
H iodophor
HAI I 11.3: Using Chemicals to Control Microorganisms
12.4: How Diseases Spread ID50 ionic bond
12.2: Characteristics and Steps of Infectious 2.1: Atoms, Isotopes, Ions, and Molecules - The
Halogens Building Blocks
Diseases
11.3: Using Chemicals to Control Microorganisms
IgA Ionizing radiation
Halophiles 6.5: Mutations
14.1: Architecture of the Immune System
8.5: Microbial Relationships 11.2: Using Physical Methods to Control
IgD
halotolerant Microorganisms
14.1: Architecture of the Immune System
8.5: Microbial Relationships irreversible chemical reaction
IgE
heat of vaporization of water 2.1: Atoms, Isotopes, Ions, and Molecules - The
14.1: Architecture of the Immune System Building Blocks
2.2: Water
IgG isomers
Heavy Metals
14.1: Architecture of the Immune System 2.3: Carbon and Organic Molecules
11.3: Using Chemicals to Control Microorganisms
IgM Isoniazid
helical
14.1: Architecture of the Immune System 11.5: Drug Targets on Prokaryote Microorganisms
9.1: Viruses
image point Isoprenoids
helminth
3.1: How Microscopes Work 2.5: Lipids
1.5: Types of Microorganisms
image point (focus) isotope
Helminthic worms
3.1: How Microscopes Work 2.1: Atoms, Isotopes, Ions, and Molecules - The
12.3: Virulence Factors in Infection
immunogens Building Blocks
helper T cells
14.1: Architecture of the Immune System
14.2: T Lymphocytes and Cellular Immunity
hematopoiesis
immunoglobulins J
14.1: Architecture of the Immune System John Snow
13.2: Second Line Defenses: Cells and Fluids
immunology 12.6: Tracking Infectious Diseases
1.5: Types of Microorganisms

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Joseph Lister M minimum permissive oxygen
12.6: Tracking Infectious Diseases macrolides concentration
11.5: Drug Targets on Prokaryote Microorganisms 8.2: Oxygen Requirements for Microbial Growth
K macrophages missense mutation
Koch’s postulates 13.2: Second Line Defenses: Cells and Fluids 6.5: Mutations
12.2: Characteristics and Steps of Infectious magnification mitochondria
Diseases 5.1: Characteristics of Eukaryotic Cells
3.1: How Microscopes Work
12.5: The Language of Epidemiologists
Mast cells mold
Krebs cycle
13.2: Second Line Defenses: Cells and Fluids 1.5: Types of Microorganisms
7.2: Catabolism of Carbohydrates
matter molecular cloning
2.1: Atoms, Isotopes, Ions, and Molecules - The 10.1: Microbes and the Tools of Genetic Engineering
L Building Blocks Molecular Koch’s postulates
lag maturation 12.2: Characteristics and Steps of Infectious
8.1: How Microbes Grow 9.2: The Viral Cycles Diseases
latency maximum growth pH Monoclonal antibodies
9.2: The Viral Cycles 8.3: The Effects of pH and Temperature on 14.5: Practical Applications of Monoclonal and
lconjunctiva Microbial Growth Polyclonal Antibodies
12.1: Normal Microbiota of the Body maximum permissive oxygen monocytes
LD50 concentration 13.2: Second Line Defenses: Cells and Fluids
12.2: Characteristics and Steps of Infectious 8.2: Oxygen Requirements for Microbial Growth monosaccharides
Diseases 2.4: Carbohydrates
mechanical
leukocytes 12.4: How Diseases Spread Morbidity
13.2: Second Line Defenses: Cells and Fluids 12.5: The Language of Epidemiologists
mechanical Vector transmission
light microscopy 12.4: How Diseases Spread morbidity rates
3.2: Staining Microscopic Specimens and 12.5: The Language of Epidemiologists
Descriptions
Membrane filtration
11.2: Using Physical Methods to Control mortality
lincosamides Microorganisms 12.5: The Language of Epidemiologists
11.5: Drug Targets on Prokaryote Microorganisms
memory mortality rates
Linnaeus 14.1: Architecture of the Immune System 12.5: The Language of Epidemiologists
1.4: A Systematic Approach
memory T cell subtypes MPN
lipases 14.2: T Lymphocytes and Cellular Immunity 8.1: How Microbes Grow
7.3: Alternate Forms of Catabolism: Fermentation,
Lipids and Proteins
Mesophiles mucus
8.4: Other Environmental Conditions that Affect 13.1: First Line defense- Physical, Mechanical and
lipids Growth Chemical Defenses
2.5: Lipids
messenger RNA mutagen
lipoprotein 2.7: Nucleic Acids 6.5: Mutations
2.6: Proteins
metabolism mutation
lithotrophs 7.1: Energy, Matter, and Enzymes 6.5: Mutations
7.1: Energy, Matter, and Enzymes
Metagenomics mycology
litmus paper 10.3: Whole Genome Methods and Industrial 1.5: Types of Microorganisms
2.2: Water Applications
Live attenuated vaccines miasma theory of disease N
14.4: Vaccines 1.3: Foundations of Modern Cell Theory natamycin
local Microaerophiles 11.3: Using Chemicals to Control Microorganisms
12.2: Characteristics and Steps of Infectious 8.2: Oxygen Requirements for Microbial Growth
Diseases native structure
Microarray technology 2.6: Proteins
local infection 10.2: Visualizing and Characterizing DNA
12.2: Characteristics and Steps of Infectious natural active immunity
Diseases
microbe 14.4: Vaccines
1.1: What Our Ancestors Knew natural antibiotic
logarithmic
8.1: How Microbes Grow
Microbial death curves 11.4: Discovering Antimicrobial Drugs
11.1: Controlling Microbial Growth Natural killer
Louis Pasteur
1.2: Spontaneous Generation
Microbial Genetics 13.2: Second Line Defenses: Cells and Fluids
3.3: Cells as Living Things 6: Mechanisms of Microbial Genetics natural passive immunity
Lyophilization microbiology 14.4: Vaccines
11.2: Using Physical Methods to Control 1.5: Types of Microorganisms neutralization
Microorganisms microorganism 14.1: Architecture of the Immune System
lysogenic cycle 1.1: What Our Ancestors Knew neutron
9.2: The Viral Cycles microtubules 2.1: Atoms, Isotopes, Ions, and Molecules - The
lysosomes 5.1: Characteristics of Eukaryotic Cells Building Blocks
5.1: Characteristics of Eukaryotic Cells minimum growth pH neutrophiles
lytic 8.3: The Effects of pH and Temperature on 8.3: The Effects of pH and Temperature on
9.2: The Viral Cycles Microbial Growth Microbial Growth
lytic cycle neutrophils
9.2: The Viral Cycles 13.2: Second Line Defenses: Cells and Fluids

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Nisin obligate anaerobes periodic table
11.3: Using Chemicals to Control Microorganisms 8.2: Oxygen Requirements for Microbial Growth 2.1: Atoms, Isotopes, Ions, and Molecules - The
Nitrites obligate intracellular pathogens Building Blocks
11.3: Using Chemicals to Control Microorganisms 4.2: Classifying Prokaryotes and Examples peripheral tolerance
nitrogenous base oligotrophs 14.2: T Lymphocytes and Cellular Immunity
6.2: Structure and Replication of DNA 4.2: Classifying Prokaryotes and Examples peristalsis
NK opacity 13.1: First Line defense- Physical, Mechanical and
Chemical Defenses
13.2: Second Line Defenses: Cells and Fluids 3.1: How Microscopes Work
noble gas operator Peritrichous
4.1: Unique Characteristics of Prokaryotic Cells
2.1: Atoms, Isotopes, Ions, and Molecules - The 6.7: Gene Regulation and Operon Theory
Building Blocks operons Peroxidase
Noncompetitive inhibitors 8.2: Oxygen Requirements for Microbial Growth
6.7: Gene Regulation and Operon Theory
7.1: Energy, Matter, and Enzymes opportunistic pathogens Peroxygens
noncritical item 11.3: Using Chemicals to Control Microorganisms
12.2: Characteristics and Steps of Infectious
11.1: Controlling Microbial Growth Diseases pH paper
Noncyclic photophosphorylation opsonization 2.2: Water
7.4: Photosynthesis and the Importance of Light 14.1: Architecture of the Immune System pH scale
Nonionizing radiation Optimum oxygen concentration 2.2: Water
6.5: Mutations 8.2: Oxygen Requirements for Microbial Growth Phagocytes
11.2: Using Physical Methods to Control organic molecule 13.3: Pathogen Recognition and Phagocytosis
Microorganisms phagocytosis
2.3: Carbon and Organic Molecules
nonpolar covalent bond Organotrophs 5.1: Characteristics of Eukaryotic Cells
2.1: Atoms, Isotopes, Ions, and Molecules - The 13.3: Pathogen Recognition and Phagocytosis
7.1: Energy, Matter, and Enzymes
Building Blocks phagolysosome
nonsense mutation oxazolidinones
13.3: Pathogen Recognition and Phagocytosis
11.5: Drug Targets on Prokaryote Microorganisms
6.5: Mutations phenol coefficient
Nonspecific innate immunity oxygenic photosynthesis
11.8: Testing the Effectiveness of Antimicrobial
7.4: Photosynthesis and the Importance of Light
13.1: First Line defense- Physical, Mechanical and Chemicals and Drugs
Chemical Defenses Phenolics
northern blot P 11.3: Using Chemicals to Control Microorganisms
10.2: Visualizing and Characterizing DNA P site phosphodiester
nosocomial infections 6.4: Protein Synthesis (Translation) 2.7: Nucleic Acids
12.4: How Diseases Spread PAGE phosphodiester backbones
nuclear envelope 10.2: Visualizing and Characterizing DNA 6.2: Structure and Replication of DNA
5.1: Characteristics of Eukaryotic Cells pamps phospholipases
nuclear membrane 13.3: Pathogen Recognition and Phagocytosis 7.3: Alternate Forms of Catabolism: Fermentation,
5.1: Characteristics of Eukaryotic Cells pandemic diseases Lipids and Proteins
nucleic acid 12.5: The Language of Epidemiologists Phospholipids
2.7: Nucleic Acids parasitic pathogens 2.5: Lipids
6.1: Using Microbiology to Discover the Secrets of 12.3: Virulence Factors in Infection phosphorescence
Life
Pasteurization 3.1: How Microscopes Work
Nucleic acids 11.2: Using Physical Methods to Control photosynthesis
6.2: Structure and Replication of DNA Microorganisms 7.4: Photosynthesis and the Importance of Light
nucleoid pathogen photosynthetic pigments
4.1: Unique Characteristics of Prokaryotic Cells 1.5: Types of Microorganisms 7.4: Photosynthesis and the Importance of Light
nucleolus pathogenic mechanisms Photosystems
5.1: Characteristics of Eukaryotic Cells 12.3: Virulence Factors in Infection 7.4: Photosynthesis and the Importance of Light
nucleotide pattern recognition receptors Phototrophs
2.7: Nucleic Acids 13.3: Pathogen Recognition and Phagocytosis 7.1: Energy, Matter, and Enzymes
nucleotide bases PCR phylogeny
6.1: Using Microbiology to Discover the Secrets of 10.2: Visualizing and Characterizing DNA 1.4: A Systematic Approach
Life
pellicles physical barriers
nucleotide excision repair 5.2: Classifying Eukaryotic Microbes and Examples 13.1: First Line defense- Physical, Mechanical and
6.5: Mutations
penetration Chemical Defenses
nucleotides 9.2: The Viral Cycles pili
6.2: Structure and Replication of DNA
pentose 4.1: Unique Characteristics of Prokaryotic Cells
nucleus 6.3: Structure and Transcription of RNA planktonic
5.1: Characteristics of Eukaryotic Cells
peptide bonds 8.1: How Microbes Grow
numerical aperture 2.6: Proteins plantibodies
3.1: How Microscopes Work
Peptides 14.5: Practical Applications of Monoclonal and
2.6: Proteins Polyclonal Antibodies
O peptidoglycan plaques
obligate aerobes 4.1: Unique Characteristics of Prokaryotic Cells 9.3: Isolation, Culture, and Identification of Viruses
8.2: Oxygen Requirements for Microbial Growth perforin plasma membrane
14.2: T Lymphocytes and Cellular Immunity 4.1: Unique Characteristics of Prokaryotic Cells

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plasmids Proteins Reflection
4.1: Unique Characteristics of Prokaryotic Cells 2.6: Proteins 3.1: How Microscopes Work
platelets Proteobacteria refraction
13.2: Second Line Defenses: Cells and Fluids 4.2: Classifying Prokaryotes and Examples 3.1: How Microscopes Work
PMNs proteomics refractive index
13.2: Second Line Defenses: Cells and Fluids 10.3: Whole Genome Methods and Industrial 3.1: How Microscopes Work
point mutation Applications regulatory T cells
6.5: Mutations protist 14.2: T Lymphocytes and Cellular Immunity
Polyacrylamide gel electrophoresis 1.5: Types of Microorganisms release
10.2: Visualizing and Characterizing DNA Protists 9.2: The Viral Cycles
polyclonal antibody 5.2: Classifying Eukaryotic Microbes and Examples Reporter genes
14.5: Practical Applications of Monoclonal and proton 10.3: Whole Genome Methods and Industrial
Polyclonal Antibodies 2.1: Atoms, Isotopes, Ions, and Molecules - The Applications
Polyclonal antisera Building Blocks repressible operon
14.5: Practical Applications of Monoclonal and protozoan 6.7: Gene Regulation and Operon Theory
Polyclonal Antibodies 1.5: Types of Microorganisms repressor
polyhedral protozoology 6.7: Gene Regulation and Operon Theory
9.1: Viruses 1.5: Types of Microorganisms Reservoirs
polymerase chain reaction (PCR) PRRs 12.4: How Diseases Spread
10.2: Visualizing and Characterizing DNA 13.3: Pathogen Recognition and Phagocytosis resident microbiota
Polymyxins pseudopeptidoglycan 12.2: Characteristics and Steps of Infectious
11.5: Drug Targets on Prokaryote Microorganisms 4.1: Unique Characteristics of Prokaryotic Cells Diseases
polynucleotide pseudopodia resolution
2.7: Nucleic Acids 5.2: Classifying Eukaryotic Microbes and Examples 3.1: How Microscopes Work
polyphyletic Psychrophiles restriction fragment length polymorphism
5.2: Classifying Eukaryotic Microbes and Examples 8.4: Other Environmental Conditions that Affect 10.2: Visualizing and Characterizing DNA
polysaccharides Growth reverse transcriptase inhibitors,
2.4: Carbohydrates psychrotrophs 11.6: Drugs for Non-prokaryote Microbes
portals of entry 8.4: Other Environmental Conditions that Affect reverse transcriptase PCR
Growth
12.2: Characteristics and Steps of Infectious 10.2: Visualizing and Characterizing DNA
Diseases purines RFLP
portals of exit 6.2: Structure and Replication of DNA
10.2: Visualizing and Characterizing DNA
12.2: Characteristics and Steps of Infectious pyrimidine ribonucleic acid
Diseases 2.7: Nucleic Acids
2.7: Nucleic Acids
pour plate pyrimidines Ribonucleotides
8.1: How Microbes Grow 6.2: Structure and Replication of DNA
6.3: Structure and Transcription of RNA
prevalence ribose
12.5: The Language of Epidemiologists Q 6.3: Structure and Transcription of RNA
primary infection Quaternary ammonium compounds ribosomal RNA
12.2: Characteristics and Steps of Infectious 11.3: Using Chemicals to Control Microorganisms 2.7: Nucleic Acids
Diseases
quaternary structure ribosomes
Primary pathogens 2.6: Proteins 4.1: Unique Characteristics of Prokaryotic Cells
12.2: Characteristics and Steps of Infectious
Diseases
quats riboswitches
11.3: Using Chemicals to Control Microorganisms
primary response 6.7: Gene Regulation and Operon Theory
14.1: Architecture of the Immune System
quorum sensing ribozymes
8.1: How Microbes Grow
primary structure 6.3: Structure and Transcription of RNA
2.6: Proteins RNA
prions R 2.7: Nucleic Acids
9.4: Viroids, Virusoids, and Prions R plasmids 6.3: Structure and Transcription of RNA
product 6.6: How Asexual Prokaryotes Achieve Genetic RNA interference
Diversity 10.3: Whole Genome Methods and Industrial
2.1: Atoms, Isotopes, Ions, and Molecules - The
Building Blocks radioisotope Applications
prokaryote 2.1: Atoms, Isotopes, Ions, and Molecules - The rough endoplasmic reticulum
Building Blocks 5.1: Characteristics of Eukaryotic Cells
1.4: A Systematic Approach
reactive oxygen species runs
Prospective epidemiology
8.2: Oxygen Requirements for Microbial Growth 4.1: Unique Characteristics of Prokaryotic Cells
12.6: Tracking Infectious Diseases
recombinant DNA molecules
protease inhibitors
11.6: Drugs for Non-prokaryote Microbes
10.1: Microbes and the Tools of Genetic Engineering S
recombinant DNA pharmaceuticals Salvarsan
proteases
10.3: Whole Genome Methods and Industrial 11.4: Discovering Antimicrobial Drugs
7.3: Alternate Forms of Catabolism: Fermentation, Applications
Lipids and Proteins Sanger DNA sequencing
recombinant DNA technology,
proteinaceous 10.2: Visualizing and Characterizing DNA
10.1: Microbes and the Tools of Genetic Engineering
9.4: Viroids, Virusoids, and Prions saturated
redox reactions
2.5: Lipids
7.1: Energy, Matter, and Enzymes

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secondary infection Sterols Thymic selection
12.2: Characteristics and Steps of Infectious 2.5: Lipids 14.2: T Lymphocytes and Cellular Immunity
Diseases stop codon thymine
secondary response 6.4: Protein Synthesis (Translation) 6.2: Structure and Replication of DNA
14.1: Architecture of the Immune System structural isomers 6.3: Structure and Transcription of RNA
secondary structure 2.3: Carbon and Organic Molecules tincture
2.6: Proteins substituted hydrocarbon 11.3: Using Chemicals to Control Microorganisms
Selective Media 2.3: Carbon and Organic Molecules toxoid vaccines
1.6: Tools and Media Used for Bacterial Growth Substrates 14.4: Vaccines
selective toxicity 7.1: Energy, Matter, and Enzymes transcription
11.5: Drug Targets on Prokaryote Microorganisms Subunit vaccines 2.7: Nucleic Acids
semicritical item 14.4: Vaccines transcription factors
11.1: Controlling Microbial Growth Sulfites 6.7: Gene Regulation and Operon Theory
Sepsis 11.3: Using Chemicals to Control Microorganisms Transcriptomics
11.1: Controlling Microbial Growth Sulfonamides 10.3: Whole Genome Methods and Industrial
serial dilutions Applications
11.5: Drug Targets on Prokaryote Microorganisms
8.1: How Microbes Grow Superantigens transduction
sessile 6.6: How Asexual Prokaryotes Achieve Genetic
14.2: T Lymphocytes and Cellular Immunity
Diversity
8.1: How Microbes Grow supercritical fluid
Sigma Factor transendothelial migration
11.3: Using Chemicals to Control Microorganisms
13.3: Pathogen Recognition and Phagocytosis
6.7: Gene Regulation and Operon Theory surface tension
silent mutation transfer RNA
2.2: Water
2.7: Nucleic Acids
6.5: Mutations Surfactants
slime layers transformation
11.3: Using Chemicals to Control Microorganisms
6.6: How Asexual Prokaryotes Achieve Genetic
4.1: Unique Characteristics of Prokaryotic Cells synthetic antimicrobial Diversity
smooth endoplasmic reticulum 11.4: Discovering Antimicrobial Drugs translation
5.1: Characteristics of Eukaryotic Cells systemic 2.7: Nucleic Acids
solvent 12.2: Characteristics and Steps of Infectious 6.4: Protein Synthesis (Translation)
2.2: Water Diseases transmissible spongiform
Southern blot systemic infection encephalopathies
10.2: Visualizing and Characterizing DNA 12.2: Characteristics and Steps of Infectious
9.4: Viroids, Virusoids, and Prions
specialized transduction Diseases
transmission
6.6: How Asexual Prokaryotes Achieve Genetic
12.2: Characteristics and Steps of Infectious
Diversity T Diseases
9.2: The Viral Cycles
target mimicry transmittance
specific heat capacity 11.7: Mechanisms for Resistance 3.1: How Microscopes Work
2.2: Water
target modification transparency
specificity 11.7: Mechanisms for Resistance 3.1: How Microscopes Work
14.1: Architecture of the Immune System
14.5: Practical Applications of Monoclonal and
target overproduction transposons
Polyclonal Antibodies 11.7: Mechanisms for Resistance 6.6: How Asexual Prokaryotes Achieve Genetic
sphere of hydration taxonomy Diversity
2.2: Water 1.4: A Systematic Approach triclosan
Spontaneous mutations tcr 11.3: Using Chemicals to Control Microorganisms
6.5: Mutations 14.2: T Lymphocytes and Cellular Immunity Triglycerides
Sporadic diseases Termination 2.5: Lipids
12.5: The Language of Epidemiologists 6.4: Protein Synthesis (Translation) trimethoprim
sporulation tertiary structure 11.5: Drug Targets on Prokaryote Microorganisms
4.1: Unique Characteristics of Prokaryotic Cells 2.6: Proteins tumbles
spread plate tetracyclines 4.1: Unique Characteristics of Prokaryotic Cells
8.1: How Microbes Grow 11.5: Drug Targets on Prokaryote Microorganisms
Starch Th1 cells U
2.4: Carbohydrates 14.2: T Lymphocytes and Cellular Immunity uncoating
start codon th17 cells 9.2: The Viral Cycles
6.4: Protein Synthesis (Translation) 14.2: T Lymphocytes and Cellular Immunity unsaturated
stationary Th2 cells 2.5: Lipids
8.1: How Microbes Grow 14.2: T Lymphocytes and Cellular Immunity uracil
sterilant Theory of Spontaneous Generation 6.3: Structure and Transcription of RNA
11.1: Controlling Microbial Growth 1.2: Spontaneous Generation
sterile field 3.3: Cells as Living Things
V
Thermophiles
11.1: Controlling Microbial Growth vaccination
8.4: Other Environmental Conditions that Affect
sterilization Growth 14.4: Vaccines
11.1: Controlling Microbial Growth
thrombocytes van der Waals interaction
Steroids 13.2: Second Line Defenses: Cells and Fluids 2.1: Atoms, Isotopes, Ions, and Molecules - The
2.5: Lipids Building Blocks

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Variolation Virions virusoids
14.4: Vaccines 9.1: Viruses 9.4: Viroids, Virusoids, and Prions
Vector transmission viroids
12.4: How Diseases Spread 9.4: Viroids, Virusoids, and Prions W
Vehicle transmission virology wavelength
12.4: How Diseases Spread 1.5: Types of Microorganisms 3.1: How Microscopes Work
vertical gene transfer virulence
6.2: Structure and Replication of DNA 12.2: Characteristics and Steps of Infectious Y
Viral cultivation Diseases
yeast
9.3: Isolation, Culture, and Identification of Viruses virulence factors
1.5: Types of Microorganisms
Viral filtrate 12.3: Virulence Factors in Infection
virus
9.3: Isolation, Culture, and Identification of Viruses
1.5: Types of Microorganisms
Z
viricides Z ring
11.1: Controlling Microbial Growth Viruses
8.1: How Microbes Grow
9.1: Viruses

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Index
A Ames test atom
A site 6.5: Mutations 2.1: Atoms, Isotopes, Ions, and Molecules - The
Amino acids Building Blocks
6.4: Protein Synthesis (Translation)
2.6: Proteins atomic mass
absorbance
aminoglycosides 2.1: Atoms, Isotopes, Ions, and Molecules - The
3.1: How Microscopes Work Building Blocks
acellular 11.5: Drug Targets on Prokaryote Microorganisms
Amphipathic atomic number
1.5: Types of Microorganisms 2.1: Atoms, Isotopes, Ions, and Molecules - The
acid 2.5: Lipids
Building Blocks
2.2: Water amplitude ATP
Acidophiles 3.1: How Microscopes Work
7.1: Energy, Matter, and Enzymes
8.3: The Effects of pH and Temperature on Anabolism attachment
Microbial Growth 7.1: Energy, Matter, and Enzymes
9.2: The Viral Cycles
activator Analytical epidemiology attenuation
6.7: Gene Regulation and Operon Theory 12.6: Tracking Infectious Diseases
6.7: Gene Regulation and Operon Theory
active site anergy autotrophs
7.1: Energy, Matter, and Enzymes 14.2: T Lymphocytes and Cellular Immunity
7.1: Energy, Matter, and Enzymes
acute inflammation anion auxotrophs
13.4: Inflammation and Fever 2.1: Atoms, Isotopes, Ions, and Molecules - The
6.5: Mutations
Adaptive Immunity Building Blocks
anoxygenic photosynthesis
14.1: Architecture of the Immune System
7.4: Photosynthesis and the Importance of Light
B
ADCC bacitracin
14.1: Architecture of the Immune System antibodies
14.1: Architecture of the Immune System 11.5: Drug Targets on Prokaryote Microorganisms
adenine Bacteria
6.2: Structure and Replication of DNA anticodon
6.4: Protein Synthesis (Translation) 1.5: Types of Microorganisms
adenosine triphosphate bactericides
7.1: Energy, Matter, and Enzymes Antifungal drugs
11.6: Drugs for Non-prokaryote Microbes 11.1: Controlling Microbial Growth
adhesion bacteriology
2.2: Water Antigens
14.1: Architecture of the Immune System 1.5: Types of Microorganisms
12.2: Characteristics and Steps of Infectious
Diseases Antihelminthic drugs Bacteriophages
aerotolerant anaerobes 11.6: Drugs for Non-prokaryote Microbes 9.3: Isolation, Culture, and Identification of Viruses
8.2: Oxygen Requirements for Microbial Growth antimetabolites bacteriostatic
affinity 11.5: Drug Targets on Prokaryote Microorganisms 11.1: Controlling Microbial Growth
14.5: Practical Applications of Monoclonal and Antimicrobial drugs balanced chemical equation
Polyclonal Antibodies 2.1: Atoms, Isotopes, Ions, and Molecules - The
11.4: Discovering Antimicrobial Drugs
Agarose gel electrophoresis Antimicrobial resistance
Building Blocks
10.2: Visualizing and Characterizing DNA base
11.7: Mechanisms for Resistance
agglutination antiparallel
2.2: Water
14.1: Architecture of the Immune System basophils
6.2: Structure and Replication of DNA
alarmones Antiprotozoan drugs
13.2: Second Line Defenses: Cells and Fluids
6.7: Gene Regulation and Operon Theory Betaproteobacteria
11.6: Drugs for Non-prokaryote Microbes
Alcohols antiretroviral drugs
4.2: Classifying Prokaryotes and Examples
11.3: Using Chemicals to Control Microorganisms binary fission
11.6: Drugs for Non-prokaryote Microbes
alexidine antisepsis
8.1: How Microbes Grow
11.3: Using Chemicals to Control Microorganisms binomial nomenclature
11.1: Controlling Microbial Growth
algae Antiviral drugs
1.4: A Systematic Approach
1.5: Types of Microorganisms biofilms
11.6: Drugs for Non-prokaryote Microbes
aliphatic hydrocarbon apoenzyme
8.1: How Microbes Grow
2.3: Carbon and Organic Molecules bioinformatics
7.1: Energy, Matter, and Enzymes
Alkaliphiles Archaea
10.3: Whole Genome Methods and Industrial
Applications
8.3: The Effects of pH and Temperature on
1.5: Types of Microorganisms biological
Microbial Growth
Alkylating agents aromatic hydrocarbon 12.4: How Diseases Spread
2.3: Carbon and Organic Molecules biological safety levels
11.3: Using Chemicals to Control Microorganisms
allosteric inhibitor artificial active immunity 11.1: Controlling Microbial Growth
14.4: Vaccines biological Vector transmission
7.1: Energy, Matter, and Enzymes
allosteric inhibitors artificial passive immunity 12.4: How Diseases Spread
14.4: Vaccines biosynthesis
7.1: Energy, Matter, and Enzymes
allosteric sites aseptic technique 9.2: The Viral Cycles
11.1: Controlling Microbial Growth Biotechology
7.1: Energy, Matter, and Enzymes
Alphaproteobacteria 10.1: Microbes and the Tools of Genetic Engineering
4.2: Classifying Prokaryotes and Examples

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Bisbiguanides chemical reactivity cyclic photophosphorylation
11.3: Using Chemicals to Control Microorganisms 2.1: Atoms, Isotopes, Ions, and Molecules - The 7.4: Photosynthesis and the Importance of Light
buffer Building Blocks cytokine storm
2.2: Water Chemically defined media 14.2: T Lymphocytes and Cellular Immunity
1.6: Tools and Media Used for Bacterial Growth cytopathic effects
C Chemotrophs 9.3: Isolation, Culture, and Identification of Viruses
7.1: Energy, Matter, and Enzymes cytosine
CAP
6.7: Gene Regulation and Operon Theory
chloramphenicol 6.2: Structure and Replication of DNA
11.5: Drug Targets on Prokaryote Microorganisms cytoskeleton
capillary action
2.2: Water
Chlorhexidine 5.1: Characteristics of Eukaryotic Cells
11.3: Using Chemicals to Control Microorganisms cytotoxic T cells
capnophile
8.2: Oxygen Requirements for Microbial Growth
Chromosomal Theory of Inheritance 14.2: T Lymphocytes and Cellular Immunity
6.1: Using Microbiology to Discover the Secrets of
capsid Life
9.1: Viruses D
chronic inflammation
capsule 13.4: Inflammation and Fever
dark repair
4.1: Unique Characteristics of Prokaryotic Cells 6.5: Mutations
cilia
Capsule Staining 5.1: Characteristics of Eukaryotic Cells
death
3.2: Staining Microscopic Specimens and 5.2: Classifying Eukaryotic Microbes and Examples 8.1: How Microbes Grow
Descriptions decimal reduction time
Coenzymes
Carbohydrates 7.1: Energy, Matter, and Enzymes 11.1: Controlling Microbial Growth
2.4: Carbohydrates degenerate
cofactor
catabolism 7.1: Energy, Matter, and Enzymes 6.4: Protein Synthesis (Translation)
7.1: Energy, Matter, and Enzymes Deltaproteobacteria
7.3: Alternate Forms of Catabolism: Fermentation,
cognate amino acid
6.4: Protein Synthesis (Translation) 4.2: Classifying Prokaryotes and Examples
Lipids and Proteins
catabolite activator protein cohesion Denaturation
2.2: Water 2.6: Proteins
6.7: Gene Regulation and Operon Theory
catalase commercial sterilization dendritic cells
11.1: Controlling Microbial Growth 13.2: Second Line Defenses: Cells and Fluids
8.2: Oxygen Requirements for Microbial Growth
catalysts Competitive inhibitors deoxyribonucleic acid
7.1: Energy, Matter, and Enzymes 2.7: Nucleic Acids
7.1: Energy, Matter, and Enzymes
cation complement activatio Deoxyribonucleotides
14.1: Architecture of the Immune System 6.2: Structure and Replication of DNA
2.1: Atoms, Isotopes, Ions, and Molecules - The
Building Blocks complex deoxyribose
cell envelope 9.1: Viruses 6.2: Structure and Replication of DNA
6.3: Structure and Transcription of RNA
4.1: Unique Characteristics of Prokaryotic Cells compound
cell morphology 2.1: Atoms, Isotopes, Ions, and Molecules - The
dermis
Building Blocks 12.1: Normal Microbiota of the Body
4.1: Unique Characteristics of Prokaryotic Cells
cell wall conjugate vaccine Descriptive epidemiology
14.4: Vaccines 12.6: Tracking Infectious Diseases
4.1: Unique Characteristics of Prokaryotic Cells
5.1: Characteristics of Eukaryotic Cells conjugate vaccines Desiccation
cellular arrangement 14.4: Vaccines 11.2: Using Physical Methods to Control
Microorganisms
4.1: Unique Characteristics of Prokaryotic Cells Conjugated proteins
cellular immunity 2.6: Proteins
Differential media
1.6: Tools and Media Used for Bacterial Growth
14.1: Architecture of the Immune System conjugation
cellular uptake 6.6: How Asexual Prokaryotes Achieve Genetic
diffraction
Diversity 3.1: How Microscopes Work
11.7: Mechanisms for Resistance
cellulose conjugation pilus Direct Contact transmission
6.6: How Asexual Prokaryotes Achieve Genetic 12.4: How Diseases Spread
2.4: Carbohydrates
Centers for Disease Control and Diversity direct viable cell count
conjunctiva 8.1: How Microbes Grow
Prevention 12.1: Normal Microbiota of the Body disaccharides
12.5: The Language of Epidemiologists
constitutively expressed 2.4: Carbohydrates
central tolerance 6.7: Gene Regulation and Operon Theory Disinfection
14.2: T Lymphocytes and Cellular Immunity
Contact transmission 11.1: Controlling Microbial Growth
Centrosomes 12.4: How Diseases Spread dispersion
5.1: Characteristics of Eukaryotic Cells
contractile vacuoles 3.1: How Microscopes Work
charged tRNA 5.2: Classifying Eukaryotic Microbes and Examples dissociation
6.4: Protein Synthesis (Translation)
contrast 2.2: Water
chemical bond 3.1: How Microscopes Work DNA
2.1: Atoms, Isotopes, Ions, and Molecules - The
Building Blocks
covalent bond 2.7: Nucleic Acids
2.1: Atoms, Isotopes, Ions, and Molecules - The 6.1: Using Microbiology to Discover the Secrets of
chemical reaction Building Blocks Life
2.1: Atoms, Isotopes, Ions, and Molecules - The DNA probe
Building Blocks
critical item
11.1: Controlling Microbial Growth 10.2: Visualizing and Characterizing DNA

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doubling time Enzymes Flagella Staining
8.1: How Microbes Grow 7.1: Energy, Matter, and Enzymes 3.2: Staining Microscopic Specimens and
drug modification eosinophils Descriptions
11.7: Mechanisms for Resistance 13.2: Second Line Defenses: Cells and Fluids Florence Nightingale
Drug resistance Epidemic diseases 12.6: Tracking Infectious Diseases
11.7: Mechanisms for Resistance 12.5: The Language of Epidemiologists fluorescent
Epidemiology 3.1: How Microscopes Work
E 12.5: The Language of Epidemiologists fluoroquinolones
epidermis 11.5: Drug Targets on Prokaryote Microorganisms
E site
6.4: Protein Synthesis (Translation) 12.1: Normal Microbiota of the Body focal
epigenetic regulation 12.2: Characteristics and Steps of Infectious
edema Diseases
13.4: Inflammation and Fever 6.7: Gene Regulation and Operon Theory
epitopes focal infection
electrolyte 12.2: Characteristics and Steps of Infectious
2.1: Atoms, Isotopes, Ions, and Molecules - The 14.1: Architecture of the Immune System
Diseases
Building Blocks Epsilonproteobacteria focal length
electron carriers 4.2: Classifying Prokaryotes and Examples
3.1: How Microscopes Work
7.1: Energy, Matter, and Enzymes erythema focal point
electron configuration 13.4: Inflammation and Fever
3.1: How Microscopes Work
2.1: Atoms, Isotopes, Ions, and Molecules - The erythrocytes fomites
Building Blocks 13.2: Second Line Defenses: Cells and Fluids
11.1: Controlling Microbial Growth
Electron Microscopy Eukarya
3.2: Staining Microscopic Specimens and
Formaldehyde
1.5: Types of Microorganisms
Descriptions 11.3: Using Chemicals to Control Microorganisms
eukaryote formed elements
electron orbital
1.4: A Systematic Approach
2.1: Atoms, Isotopes, Ions, and Molecules - The 13.2: Second Line Defenses: Cells and Fluids
Building Blocks eutrophs frameshift mutation
electron transfer 4.2: Classifying Prokaryotes and Examples
6.5: Mutations
2.1: Atoms, Isotopes, Ions, and Molecules - The evaporation free ribosomes)
Building Blocks 2.2: Water
5.1: Characteristics of Eukaryotic Cells
electronegativity exergonic reaction frequency
2.1: Atoms, Isotopes, Ions, and Molecules - The 7.1: Energy, Matter, and Enzymes
3.1: How Microscopes Work
Building Blocks Exocytosis functional group
electrophoresis 5.1: Characteristics of Eukaryotic Cells
2.3: Carbon and Organic Molecules
10.2: Visualizing and Characterizing DNA exoenzymes Fungal pathogens
element 12.3: Virulence Factors in Infection
12.3: Virulence Factors in Infection
2.1: Atoms, Isotopes, Ions, and Molecules - The Experimental epidemiology Fungi
Building Blocks
12.6: Tracking Infectious Diseases
elongation 1.5: Types of Microorganisms
exposure fungicides
6.4: Protein Synthesis (Translation)
12.2: Characteristics and Steps of Infectious
enantiomers 11.1: Controlling Microbial Growth
Diseases
2.3: Carbon and Organic Molecules Extracellular Matrix fungistatic
Endemic diseases 11.1: Controlling Microbial Growth
5.1: Characteristics of Eukaryotic Cells
12.5: The Language of Epidemiologists extracellular polymeric substance
endergonic reactions 8.1: How Microbes Grow
G
7.1: Energy, Matter, and Enzymes Extravasation Gammaproteobacteria
Endocytosis 13.3: Pathogen Recognition and Phagocytosis 4.2: Classifying Prokaryotes and Examples
5.1: Characteristics of Eukaryotic Cells gene expression
endomembrane F 6.7: Gene Regulation and Operon Theory
5.1: Characteristics of Eukaryotic Cells F plasmid gene therapy
Endospore Staining 6.6: How Asexual Prokaryotes Achieve Genetic
10.4: Genetic Engineering - Risks, Benefits, and
Perceptions
3.2: Staining Microscopic Specimens and Diversity
Descriptions F+ cell generalized
endospores 6.6: How Asexual Prokaryotes Achieve Genetic
9.2: The Viral Cycles
4.1: Unique Characteristics of Prokaryotic Cells Diversity generalized transduction
endosymbiosis Facultative anaerobes 6.6: How Asexual Prokaryotes Achieve Genetic
Diversity
1.3: Foundations of Modern Cell Theory 8.2: Oxygen Requirements for Microbial Growth
endosymbiotic theory feedback inhibition generation time
8.1: How Microbes Grow
1.3: Foundations of Modern Cell Theory 7.1: Energy, Matter, and Enzymes
endothelia Fever genetic code
6.4: Protein Synthesis (Translation)
13.1: First Line defense- Physical, Mechanical and 13.4: Inflammation and Fever
Chemical Defenses fimbriae genetic engineering
Enriched media 4.1: Unique Characteristics of Prokaryotic Cells
10.1: Microbes and the Tools of Genetic Engineering
10.4: Genetic Engineering - Risks, Benefits, and
1.6: Tools and Media Used for Bacterial Growth flagella Perceptions
enzymatic bypass 4.1: Unique Characteristics of Prokaryotic Cells
11.7: Mechanisms for Resistance

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genomics HEPA inactivated vaccines
10.3: Whole Genome Methods and Industrial 11.2: Using Physical Methods to Control 14.4: Vaccines
Applications Microorganisms inactivation, prevention
geometric isomer herd immunity 11.7: Mechanisms for Resistance
2.3: Carbon and Organic Molecules 14.4: Vaccines Incidence
germ theory of disease heritability 12.5: The Language of Epidemiologists
1.3: Foundations of Modern Cell Theory 6.1: Using Microbiology to Discover the Secrets of inclusions
germinate Life
4.1: Unique Characteristics of Prokaryotic Cells
4.1: Unique Characteristics of Prokaryotic Cells heterotrophs indirect Contact transmission
Glutaraldehyde 7.1: Energy, Matter, and Enzymes
12.4: How Diseases Spread
11.3: Using Chemicals to Control Microorganisms Hfr cell induced fit
glycans 6.6: How Asexual Prokaryotes Achieve Genetic
7.1: Energy, Matter, and Enzymes
Diversity
2.4: Carbohydrates induced mutations
Glycocalyx holoenzyme
6.5: Mutations
7.1: Energy, Matter, and Enzymes
4.1: Unique Characteristics of Prokaryotic Cells inducer
Glycogen Horizontal Gene Transfer
6.7: Gene Regulation and Operon Theory
6.6: How Asexual Prokaryotes Achieve Genetic
2.4: Carbohydrates inducible operon
Diversity
glycolysis host ranges 6.7: Gene Regulation and Operon Theory
7.2: Catabolism of Carbohydrates inert gas
9.1: Viruses
glycopeptides Humoral Immunity 2.1: Atoms, Isotopes, Ions, and Molecules - The
11.5: Drug Targets on Prokaryote Microorganisms Building Blocks
14.1: Architecture of the Immune System
glycoprotein hybridomas infection
2.6: Proteins 12.2: Characteristics and Steps of Infectious
14.5: Practical Applications of Monoclonal and
glycosidic bonds Diseases
Polyclonal Antibodies
2.4: Carbohydrates hydrocarbon inflammation
Golgi apparatus 13.4: Inflammation and Fever
2.3: Carbon and Organic Molecules
5.1: Characteristics of Eukaryotic Cells hydrogen bond Initiation
Gram staining 6.4: Protein Synthesis (Translation)
2.1: Atoms, Isotopes, Ions, and Molecules - The
3.2: Staining Microscopic Specimens and Building Blocks initiation factors
Descriptions hydrophilic 6.4: Protein Synthesis (Translation)
Granulocytes 2.2: Water integrase inhibitors
13.2: Second Line Defenses: Cells and Fluids hydrophobic 11.6: Drugs for Non-prokaryote Microbes
granulomas 2.2: Water interference
13.4: Inflammation and Fever 2.5: Lipids 3.1: How Microscopes Work
granzymes hyperthemophiles intermediate filaments
14.2: T Lymphocytes and Cellular Immunity 8.4: Other Environmental Conditions that Affect 5.1: Characteristics of Eukaryotic Cells
guanine Growth invasion
6.2: Structure and Replication of DNA hypodermis 12.2: Characteristics and Steps of Infectious
12.1: Normal Microbiota of the Body Diseases
H iodophor
HAI I 11.3: Using Chemicals to Control Microorganisms
12.4: How Diseases Spread ID50 ionic bond
12.2: Characteristics and Steps of Infectious 2.1: Atoms, Isotopes, Ions, and Molecules - The
Halogens Building Blocks
Diseases
11.3: Using Chemicals to Control Microorganisms
IgA Ionizing radiation
Halophiles 6.5: Mutations
14.1: Architecture of the Immune System
8.5: Microbial Relationships 11.2: Using Physical Methods to Control
IgD
halotolerant Microorganisms
14.1: Architecture of the Immune System
8.5: Microbial Relationships irreversible chemical reaction
IgE
heat of vaporization of water 2.1: Atoms, Isotopes, Ions, and Molecules - The
14.1: Architecture of the Immune System Building Blocks
2.2: Water
IgG isomers
Heavy Metals
14.1: Architecture of the Immune System 2.3: Carbon and Organic Molecules
11.3: Using Chemicals to Control Microorganisms
IgM Isoniazid
helical
14.1: Architecture of the Immune System 11.5: Drug Targets on Prokaryote Microorganisms
9.1: Viruses
image point Isoprenoids
helminth
3.1: How Microscopes Work 2.5: Lipids
1.5: Types of Microorganisms
image point (focus) isotope
Helminthic worms
3.1: How Microscopes Work 2.1: Atoms, Isotopes, Ions, and Molecules - The
12.3: Virulence Factors in Infection
immunogens Building Blocks
helper T cells
14.1: Architecture of the Immune System
14.2: T Lymphocytes and Cellular Immunity
hematopoiesis
immunoglobulins J
14.1: Architecture of the Immune System John Snow
13.2: Second Line Defenses: Cells and Fluids
immunology 12.6: Tracking Infectious Diseases
1.5: Types of Microorganisms

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Joseph Lister M minimum permissive oxygen
12.6: Tracking Infectious Diseases macrolides concentration
11.5: Drug Targets on Prokaryote Microorganisms 8.2: Oxygen Requirements for Microbial Growth
K macrophages missense mutation
Koch’s postulates 13.2: Second Line Defenses: Cells and Fluids 6.5: Mutations
12.2: Characteristics and Steps of Infectious magnification mitochondria
Diseases 5.1: Characteristics of Eukaryotic Cells
3.1: How Microscopes Work
12.5: The Language of Epidemiologists
Mast cells mold
Krebs cycle
13.2: Second Line Defenses: Cells and Fluids 1.5: Types of Microorganisms
7.2: Catabolism of Carbohydrates
matter molecular cloning
2.1: Atoms, Isotopes, Ions, and Molecules - The 10.1: Microbes and the Tools of Genetic Engineering
L Building Blocks Molecular Koch’s postulates
lag maturation 12.2: Characteristics and Steps of Infectious
8.1: How Microbes Grow 9.2: The Viral Cycles Diseases
latency maximum growth pH Monoclonal antibodies
9.2: The Viral Cycles 8.3: The Effects of pH and Temperature on 14.5: Practical Applications of Monoclonal and
lconjunctiva Microbial Growth Polyclonal Antibodies
12.1: Normal Microbiota of the Body maximum permissive oxygen monocytes
LD50 concentration 13.2: Second Line Defenses: Cells and Fluids
12.2: Characteristics and Steps of Infectious 8.2: Oxygen Requirements for Microbial Growth monosaccharides
Diseases 2.4: Carbohydrates
mechanical
leukocytes 12.4: How Diseases Spread Morbidity
13.2: Second Line Defenses: Cells and Fluids 12.5: The Language of Epidemiologists
mechanical Vector transmission
light microscopy 12.4: How Diseases Spread morbidity rates
3.2: Staining Microscopic Specimens and 12.5: The Language of Epidemiologists
Descriptions
Membrane filtration
11.2: Using Physical Methods to Control mortality
lincosamides Microorganisms 12.5: The Language of Epidemiologists
11.5: Drug Targets on Prokaryote Microorganisms
memory mortality rates
Linnaeus 14.1: Architecture of the Immune System 12.5: The Language of Epidemiologists
1.4: A Systematic Approach
memory T cell subtypes MPN
lipases 14.2: T Lymphocytes and Cellular Immunity 8.1: How Microbes Grow
7.3: Alternate Forms of Catabolism: Fermentation,
Lipids and Proteins
Mesophiles mucus
8.4: Other Environmental Conditions that Affect 13.1: First Line defense- Physical, Mechanical and
lipids Growth Chemical Defenses
2.5: Lipids
messenger RNA mutagen
lipoprotein 2.7: Nucleic Acids 6.5: Mutations
2.6: Proteins
metabolism mutation
lithotrophs 7.1: Energy, Matter, and Enzymes 6.5: Mutations
7.1: Energy, Matter, and Enzymes
Metagenomics mycology
litmus paper 10.3: Whole Genome Methods and Industrial 1.5: Types of Microorganisms
2.2: Water Applications
Live attenuated vaccines miasma theory of disease N
14.4: Vaccines 1.3: Foundations of Modern Cell Theory natamycin
local Microaerophiles 11.3: Using Chemicals to Control Microorganisms
12.2: Characteristics and Steps of Infectious 8.2: Oxygen Requirements for Microbial Growth
Diseases native structure
Microarray technology 2.6: Proteins
local infection 10.2: Visualizing and Characterizing DNA
12.2: Characteristics and Steps of Infectious natural active immunity
Diseases
microbe 14.4: Vaccines
1.1: What Our Ancestors Knew natural antibiotic
logarithmic
8.1: How Microbes Grow
Microbial death curves 11.4: Discovering Antimicrobial Drugs
11.1: Controlling Microbial Growth Natural killer
Louis Pasteur
1.2: Spontaneous Generation
Microbial Genetics 13.2: Second Line Defenses: Cells and Fluids
3.3: Cells as Living Things 6: Mechanisms of Microbial Genetics natural passive immunity
Lyophilization microbiology 14.4: Vaccines
11.2: Using Physical Methods to Control 1.5: Types of Microorganisms neutralization
Microorganisms microorganism 14.1: Architecture of the Immune System
lysogenic cycle 1.1: What Our Ancestors Knew neutron
9.2: The Viral Cycles microtubules 2.1: Atoms, Isotopes, Ions, and Molecules - The
lysosomes 5.1: Characteristics of Eukaryotic Cells Building Blocks
5.1: Characteristics of Eukaryotic Cells minimum growth pH neutrophiles
lytic 8.3: The Effects of pH and Temperature on 8.3: The Effects of pH and Temperature on
9.2: The Viral Cycles Microbial Growth Microbial Growth
lytic cycle neutrophils
9.2: The Viral Cycles 13.2: Second Line Defenses: Cells and Fluids

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Nisin obligate anaerobes periodic table
11.3: Using Chemicals to Control Microorganisms 8.2: Oxygen Requirements for Microbial Growth 2.1: Atoms, Isotopes, Ions, and Molecules - The
Nitrites obligate intracellular pathogens Building Blocks
11.3: Using Chemicals to Control Microorganisms 4.2: Classifying Prokaryotes and Examples peripheral tolerance
nitrogenous base oligotrophs 14.2: T Lymphocytes and Cellular Immunity
6.2: Structure and Replication of DNA 4.2: Classifying Prokaryotes and Examples peristalsis
NK opacity 13.1: First Line defense- Physical, Mechanical and
Chemical Defenses
13.2: Second Line Defenses: Cells and Fluids 3.1: How Microscopes Work
noble gas operator Peritrichous
4.1: Unique Characteristics of Prokaryotic Cells
2.1: Atoms, Isotopes, Ions, and Molecules - The 6.7: Gene Regulation and Operon Theory
Building Blocks operons Peroxidase
Noncompetitive inhibitors 8.2: Oxygen Requirements for Microbial Growth
6.7: Gene Regulation and Operon Theory
7.1: Energy, Matter, and Enzymes opportunistic pathogens Peroxygens
noncritical item 11.3: Using Chemicals to Control Microorganisms
12.2: Characteristics and Steps of Infectious
11.1: Controlling Microbial Growth Diseases pH paper
Noncyclic photophosphorylation opsonization 2.2: Water
7.4: Photosynthesis and the Importance of Light 14.1: Architecture of the Immune System pH scale
Nonionizing radiation Optimum oxygen concentration 2.2: Water
6.5: Mutations 8.2: Oxygen Requirements for Microbial Growth Phagocytes
11.2: Using Physical Methods to Control organic molecule 13.3: Pathogen Recognition and Phagocytosis
Microorganisms phagocytosis
2.3: Carbon and Organic Molecules
nonpolar covalent bond Organotrophs 5.1: Characteristics of Eukaryotic Cells
2.1: Atoms, Isotopes, Ions, and Molecules - The 13.3: Pathogen Recognition and Phagocytosis
7.1: Energy, Matter, and Enzymes
Building Blocks phagolysosome
nonsense mutation oxazolidinones
13.3: Pathogen Recognition and Phagocytosis
11.5: Drug Targets on Prokaryote Microorganisms
6.5: Mutations phenol coefficient
Nonspecific innate immunity oxygenic photosynthesis
11.8: Testing the Effectiveness of Antimicrobial
7.4: Photosynthesis and the Importance of Light
13.1: First Line defense- Physical, Mechanical and Chemicals and Drugs
Chemical Defenses Phenolics
northern blot P 11.3: Using Chemicals to Control Microorganisms
10.2: Visualizing and Characterizing DNA P site phosphodiester
nosocomial infections 6.4: Protein Synthesis (Translation) 2.7: Nucleic Acids
12.4: How Diseases Spread PAGE phosphodiester backbones
nuclear envelope 10.2: Visualizing and Characterizing DNA 6.2: Structure and Replication of DNA
5.1: Characteristics of Eukaryotic Cells pamps phospholipases
nuclear membrane 13.3: Pathogen Recognition and Phagocytosis 7.3: Alternate Forms of Catabolism: Fermentation,
5.1: Characteristics of Eukaryotic Cells pandemic diseases Lipids and Proteins
nucleic acid 12.5: The Language of Epidemiologists Phospholipids
2.7: Nucleic Acids parasitic pathogens 2.5: Lipids
6.1: Using Microbiology to Discover the Secrets of 12.3: Virulence Factors in Infection phosphorescence
Life
Pasteurization 3.1: How Microscopes Work
Nucleic acids 11.2: Using Physical Methods to Control photosynthesis
6.2: Structure and Replication of DNA Microorganisms 7.4: Photosynthesis and the Importance of Light
nucleoid pathogen photosynthetic pigments
4.1: Unique Characteristics of Prokaryotic Cells 1.5: Types of Microorganisms 7.4: Photosynthesis and the Importance of Light
nucleolus pathogenic mechanisms Photosystems
5.1: Characteristics of Eukaryotic Cells 12.3: Virulence Factors in Infection 7.4: Photosynthesis and the Importance of Light
nucleotide pattern recognition receptors Phototrophs
2.7: Nucleic Acids 13.3: Pathogen Recognition and Phagocytosis 7.1: Energy, Matter, and Enzymes
nucleotide bases PCR phylogeny
6.1: Using Microbiology to Discover the Secrets of 10.2: Visualizing and Characterizing DNA 1.4: A Systematic Approach
Life
pellicles physical barriers
nucleotide excision repair 5.2: Classifying Eukaryotic Microbes and Examples 13.1: First Line defense- Physical, Mechanical and
6.5: Mutations
penetration Chemical Defenses
nucleotides 9.2: The Viral Cycles pili
6.2: Structure and Replication of DNA
pentose 4.1: Unique Characteristics of Prokaryotic Cells
nucleus 6.3: Structure and Transcription of RNA planktonic
5.1: Characteristics of Eukaryotic Cells
peptide bonds 8.1: How Microbes Grow
numerical aperture 2.6: Proteins plantibodies
3.1: How Microscopes Work
Peptides 14.5: Practical Applications of Monoclonal and
2.6: Proteins Polyclonal Antibodies
O peptidoglycan plaques
obligate aerobes 4.1: Unique Characteristics of Prokaryotic Cells 9.3: Isolation, Culture, and Identification of Viruses
8.2: Oxygen Requirements for Microbial Growth perforin plasma membrane
14.2: T Lymphocytes and Cellular Immunity 4.1: Unique Characteristics of Prokaryotic Cells

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plasmids Proteins Reflection
4.1: Unique Characteristics of Prokaryotic Cells 2.6: Proteins 3.1: How Microscopes Work
platelets Proteobacteria refraction
13.2: Second Line Defenses: Cells and Fluids 4.2: Classifying Prokaryotes and Examples 3.1: How Microscopes Work
PMNs proteomics refractive index
13.2: Second Line Defenses: Cells and Fluids 10.3: Whole Genome Methods and Industrial 3.1: How Microscopes Work
point mutation Applications regulatory T cells
6.5: Mutations protist 14.2: T Lymphocytes and Cellular Immunity
Polyacrylamide gel electrophoresis 1.5: Types of Microorganisms release
10.2: Visualizing and Characterizing DNA Protists 9.2: The Viral Cycles
polyclonal antibody 5.2: Classifying Eukaryotic Microbes and Examples Reporter genes
14.5: Practical Applications of Monoclonal and proton 10.3: Whole Genome Methods and Industrial
Polyclonal Antibodies 2.1: Atoms, Isotopes, Ions, and Molecules - The Applications
Polyclonal antisera Building Blocks repressible operon
14.5: Practical Applications of Monoclonal and protozoan 6.7: Gene Regulation and Operon Theory
Polyclonal Antibodies 1.5: Types of Microorganisms repressor
polyhedral protozoology 6.7: Gene Regulation and Operon Theory
9.1: Viruses 1.5: Types of Microorganisms Reservoirs
polymerase chain reaction (PCR) PRRs 12.4: How Diseases Spread
10.2: Visualizing and Characterizing DNA 13.3: Pathogen Recognition and Phagocytosis resident microbiota
Polymyxins pseudopeptidoglycan 12.2: Characteristics and Steps of Infectious
11.5: Drug Targets on Prokaryote Microorganisms 4.1: Unique Characteristics of Prokaryotic Cells Diseases
polynucleotide pseudopodia resolution
2.7: Nucleic Acids 5.2: Classifying Eukaryotic Microbes and Examples 3.1: How Microscopes Work
polyphyletic Psychrophiles restriction fragment length polymorphism
5.2: Classifying Eukaryotic Microbes and Examples 8.4: Other Environmental Conditions that Affect 10.2: Visualizing and Characterizing DNA
polysaccharides Growth reverse transcriptase inhibitors,
2.4: Carbohydrates psychrotrophs 11.6: Drugs for Non-prokaryote Microbes
portals of entry 8.4: Other Environmental Conditions that Affect reverse transcriptase PCR
Growth
12.2: Characteristics and Steps of Infectious 10.2: Visualizing and Characterizing DNA
Diseases purines RFLP
portals of exit 6.2: Structure and Replication of DNA
10.2: Visualizing and Characterizing DNA
12.2: Characteristics and Steps of Infectious pyrimidine ribonucleic acid
Diseases 2.7: Nucleic Acids
2.7: Nucleic Acids
pour plate pyrimidines Ribonucleotides
8.1: How Microbes Grow 6.2: Structure and Replication of DNA
6.3: Structure and Transcription of RNA
prevalence ribose
12.5: The Language of Epidemiologists Q 6.3: Structure and Transcription of RNA
primary infection Quaternary ammonium compounds ribosomal RNA
12.2: Characteristics and Steps of Infectious 11.3: Using Chemicals to Control Microorganisms 2.7: Nucleic Acids
Diseases
quaternary structure ribosomes
Primary pathogens 2.6: Proteins 4.1: Unique Characteristics of Prokaryotic Cells
12.2: Characteristics and Steps of Infectious
Diseases
quats riboswitches
11.3: Using Chemicals to Control Microorganisms
primary response 6.7: Gene Regulation and Operon Theory
14.1: Architecture of the Immune System
quorum sensing ribozymes
8.1: How Microbes Grow
primary structure 6.3: Structure and Transcription of RNA
2.6: Proteins RNA
prions R 2.7: Nucleic Acids
9.4: Viroids, Virusoids, and Prions R plasmids 6.3: Structure and Transcription of RNA
product 6.6: How Asexual Prokaryotes Achieve Genetic RNA interference
Diversity 10.3: Whole Genome Methods and Industrial
2.1: Atoms, Isotopes, Ions, and Molecules - The
Building Blocks radioisotope Applications
prokaryote 2.1: Atoms, Isotopes, Ions, and Molecules - The rough endoplasmic reticulum
Building Blocks 5.1: Characteristics of Eukaryotic Cells
1.4: A Systematic Approach
reactive oxygen species runs
Prospective epidemiology
8.2: Oxygen Requirements for Microbial Growth 4.1: Unique Characteristics of Prokaryotic Cells
12.6: Tracking Infectious Diseases
recombinant DNA molecules
protease inhibitors
11.6: Drugs for Non-prokaryote Microbes
10.1: Microbes and the Tools of Genetic Engineering S
recombinant DNA pharmaceuticals Salvarsan
proteases
10.3: Whole Genome Methods and Industrial 11.4: Discovering Antimicrobial Drugs
7.3: Alternate Forms of Catabolism: Fermentation, Applications
Lipids and Proteins Sanger DNA sequencing
recombinant DNA technology,
proteinaceous 10.2: Visualizing and Characterizing DNA
10.1: Microbes and the Tools of Genetic Engineering
9.4: Viroids, Virusoids, and Prions saturated
redox reactions
2.5: Lipids
7.1: Energy, Matter, and Enzymes

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secondary infection Sterols Thymic selection
12.2: Characteristics and Steps of Infectious 2.5: Lipids 14.2: T Lymphocytes and Cellular Immunity
Diseases stop codon thymine
secondary response 6.4: Protein Synthesis (Translation) 6.2: Structure and Replication of DNA
14.1: Architecture of the Immune System structural isomers 6.3: Structure and Transcription of RNA
secondary structure 2.3: Carbon and Organic Molecules tincture
2.6: Proteins substituted hydrocarbon 11.3: Using Chemicals to Control Microorganisms
Selective Media 2.3: Carbon and Organic Molecules toxoid vaccines
1.6: Tools and Media Used for Bacterial Growth Substrates 14.4: Vaccines
selective toxicity 7.1: Energy, Matter, and Enzymes transcription
11.5: Drug Targets on Prokaryote Microorganisms Subunit vaccines 2.7: Nucleic Acids
semicritical item 14.4: Vaccines transcription factors
11.1: Controlling Microbial Growth Sulfites 6.7: Gene Regulation and Operon Theory
Sepsis 11.3: Using Chemicals to Control Microorganisms Transcriptomics
11.1: Controlling Microbial Growth Sulfonamides 10.3: Whole Genome Methods and Industrial
serial dilutions Applications
11.5: Drug Targets on Prokaryote Microorganisms
8.1: How Microbes Grow Superantigens transduction
sessile 6.6: How Asexual Prokaryotes Achieve Genetic
14.2: T Lymphocytes and Cellular Immunity
Diversity
8.1: How Microbes Grow supercritical fluid
Sigma Factor transendothelial migration
11.3: Using Chemicals to Control Microorganisms
13.3: Pathogen Recognition and Phagocytosis
6.7: Gene Regulation and Operon Theory surface tension
silent mutation transfer RNA
2.2: Water
2.7: Nucleic Acids
6.5: Mutations Surfactants
slime layers transformation
11.3: Using Chemicals to Control Microorganisms
6.6: How Asexual Prokaryotes Achieve Genetic
4.1: Unique Characteristics of Prokaryotic Cells synthetic antimicrobial Diversity
smooth endoplasmic reticulum 11.4: Discovering Antimicrobial Drugs translation
5.1: Characteristics of Eukaryotic Cells systemic 2.7: Nucleic Acids
solvent 12.2: Characteristics and Steps of Infectious 6.4: Protein Synthesis (Translation)
2.2: Water Diseases transmissible spongiform
Southern blot systemic infection encephalopathies
10.2: Visualizing and Characterizing DNA 12.2: Characteristics and Steps of Infectious
9.4: Viroids, Virusoids, and Prions
specialized transduction Diseases
transmission
6.6: How Asexual Prokaryotes Achieve Genetic
12.2: Characteristics and Steps of Infectious
Diversity T Diseases
9.2: The Viral Cycles
target mimicry transmittance
specific heat capacity 11.7: Mechanisms for Resistance 3.1: How Microscopes Work
2.2: Water
target modification transparency
specificity 11.7: Mechanisms for Resistance 3.1: How Microscopes Work
14.1: Architecture of the Immune System
14.5: Practical Applications of Monoclonal and
target overproduction transposons
Polyclonal Antibodies 11.7: Mechanisms for Resistance 6.6: How Asexual Prokaryotes Achieve Genetic
sphere of hydration taxonomy Diversity
2.2: Water 1.4: A Systematic Approach triclosan
Spontaneous mutations tcr 11.3: Using Chemicals to Control Microorganisms
6.5: Mutations 14.2: T Lymphocytes and Cellular Immunity Triglycerides
Sporadic diseases Termination 2.5: Lipids
12.5: The Language of Epidemiologists 6.4: Protein Synthesis (Translation) trimethoprim
sporulation tertiary structure 11.5: Drug Targets on Prokaryote Microorganisms
4.1: Unique Characteristics of Prokaryotic Cells 2.6: Proteins tumbles
spread plate tetracyclines 4.1: Unique Characteristics of Prokaryotic Cells
8.1: How Microbes Grow 11.5: Drug Targets on Prokaryote Microorganisms
Starch Th1 cells U
2.4: Carbohydrates 14.2: T Lymphocytes and Cellular Immunity uncoating
start codon th17 cells 9.2: The Viral Cycles
6.4: Protein Synthesis (Translation) 14.2: T Lymphocytes and Cellular Immunity unsaturated
stationary Th2 cells 2.5: Lipids
8.1: How Microbes Grow 14.2: T Lymphocytes and Cellular Immunity uracil
sterilant Theory of Spontaneous Generation 6.3: Structure and Transcription of RNA
11.1: Controlling Microbial Growth 1.2: Spontaneous Generation
sterile field 3.3: Cells as Living Things
V
Thermophiles
11.1: Controlling Microbial Growth vaccination
8.4: Other Environmental Conditions that Affect
sterilization Growth 14.4: Vaccines
11.1: Controlling Microbial Growth
thrombocytes van der Waals interaction
Steroids 13.2: Second Line Defenses: Cells and Fluids 2.1: Atoms, Isotopes, Ions, and Molecules - The
2.5: Lipids Building Blocks

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Variolation Virions virusoids
14.4: Vaccines 9.1: Viruses 9.4: Viroids, Virusoids, and Prions
Vector transmission viroids
12.4: How Diseases Spread 9.4: Viroids, Virusoids, and Prions W
Vehicle transmission virology wavelength
12.4: How Diseases Spread 1.5: Types of Microorganisms 3.1: How Microscopes Work
vertical gene transfer virulence
6.2: Structure and Replication of DNA 12.2: Characteristics and Steps of Infectious Y
Viral cultivation Diseases
yeast
9.3: Isolation, Culture, and Identification of Viruses virulence factors
1.5: Types of Microorganisms
Viral filtrate 12.3: Virulence Factors in Infection
virus
9.3: Isolation, Culture, and Identification of Viruses
1.5: Types of Microorganisms
Z
viricides Z ring
11.1: Controlling Microbial Growth Viruses
8.1: How Microbes Grow
9.1: Viruses

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