BACT 221

CLINICAL BACTERIOLOGY
BS Medical Laboratory Science | 2ND Semester 2024-2025

LESSON 1: BACTERIOLOGY WHAT IS THE 1ST IMPORTANT THAT WAS DISCOVERED?

(The structure of a bacteria)
BACTERIOLOGY GENERAL CHARACTERISTIC
• They are prokaryotes
• Prevent diseases through identification of
❖ “Pro” meaning before and “karyon”
habitats and risk factors
meaning nucleus, nut or kernel.
• Treat diseases through identification of specific
organism leading to specific medicine • do not contain a true nucleus surrounded by
• New tools to treat diseases nuclear membrane
HISTORY ON DISCOVERY • do not contain organelles
❖ all functions take place in the cytoplasm
1. Lucretius and Girolamo Fracastoro - Disease
was caused by “invisible living creatures” or cytoplasmic membrane

2. Francesco Stelluti - made the earliest

microscopic observations on bees and weevils

3. Anton van Leeuwenhoek – first true

microbiologist as first person to observe and
describe microorganisms accurately using self-
made microscope
HISTORY ON GENERATION
1. Aristotle – simpler vertebrates can arise from THE OUTSIDE OF A BACTERIA
spontaneous generation CELL ENVELOPE
• Outermost structure
2. Francesco Redi - demonstrated that maggots • Composed of: outer membrane and periplasm
do not arise spontaneously from decaying meat
(gram negative only), cell wall and plasma
membrane
3. John Needham – boiled mutton broth (tightly • Extreme environment
sealed) eventually became cloudy with
❖ Halophiles
microorganisms.
❖ Thermophile

4. Lazzaro Spallanzi- sealed boiled water and

seeds
HISTORY ON BIOGENESIS
1. Rudolf Virchow – he challenged spontaneous
generation with the concept of biogenesis.

2. Theodore Schwann – no growth occurred in a

flask containing nutrient solution after allowing 1. OUTER MEMBRANE
air to pass through a red-hot tube • Found only on gram (-) bacteria
• Cell’s initial barrier to environment
3. Louis Pasteur – resolved the issue of • Composed of lipopolysaccharide
spontaneous generation ❖ Has porins
• stated that microorganisms are indeed o Water-filled structure that
present in the air and can contaminate controls passage of water and
seemingly sterile solutions nutrients
• started that microbial life can be 2. CELL WALL
destroyed by heat • Also called murein layer
• Function:
4. John Tyndall – He showed that dust carry ❖ Maintains the shape of the cell and
germs which contaminates sterile broth. gives bacteria strength
• “Tyndallization” - form of sterilization ❖ Prevents bursting of the cell from the
for three consecutive days. high osmotic pressure inside it.
OTHER NOTABLE PERSON ❖ Protective peptidoglycan (murein)
1. Joseph Lister - developed the antiseptic system layer
of surgery. He demonstrated the used of phenol ❖ Protection against mechanical
for treating surgical wounds and also sprayed disruption
phenol over the surgical area. • Determines gram-positive and gram-negative
• Has modified: acid fast
2. Robert Koch - discovered Bacillus anthracis ❖ has a strong hydrophobic structure;
causative agent of anthrax. Discovered Mycobacterium and Nocardia
Mycobacterium tuberculosis.

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 1

 Properties Gram Positive Gram Negative MOTILE BACTERIA VS NON – MOTILE BACTERIA
Bacteria Bacteria
Cell Wall The thick cell wall is The outer
composed of membrane is
peptidoglycan; composed of lipids,
teichoic acids may proteins, LPS, and
also be present thin inner
peptidoglycan layer
Shape Spherical, rod- Spherical, oval,
shaped or straight or curved
filamentous rods, helical or
filamentous

Teichoic Acid Present Absent

Flagellar Structure 2 rings in the basal 4 rings in the basal
body body
Resistance to High Low
PILI OR FIMBRIAE
physical • Nonflagellar, proteinaceous, hairlike
disruption appendages that adhere some bacterial cells to
Resistance to Low High one another and to host cells.
lysozyme
Inhibition by basic High Low
• Common/somatic pili - for attachment
dye • Sex pili - transfer of information / mutation
Reproduction Binary fission Binary fission SLIME LAYER
• Unorganized material that is loosely attached to
• Target of antibiotics the cell wall
• No cell wall: Mycoplasma and Ureaplasma • Also made up of polysaccharide
• Other attachments: teichoic acid (anchored to • It can either inhibit phagocytosis or aid in the
the peptidoglycan) and lipoteichoic acid adherence of the bacteria to the host tissue
(anchored to the plasma membrane). or synthetic implants
• It also serves as a point of anchorage for SURFACE POLYMER
flagella
• Also called capsule
• Its synthesis and structure have been the
primary target of antimicrobial agents. • made of polysaccharide polymers or
polypeptides
3. PERIPLASMIC STRUCTURE
• act as virulence factors in helping the pathogen
• Found only on g(-) bacteria
evade phagocytosis
• Helps in securing nutrients from environment
• capsules sometimes must be removed to detect
4. CYTOPLASMIC MEMBRANE the somatic (cell wall) antigens present
• Deepest layer of cell underneath them.
• Function: ❖ boiling a suspension of the
❖ Osmotic barrier; microorganism
❖ Transport of solutes;
❖ Generation of chemical energy;
❖ Cell motility;
❖ Mediation of chromosomal segregation
during replication; and
❖ Housing of molecular sensors that
monitors chemical and physical changes • Bacteria with capsules
on the environment ❖ Bacillus Anthracis
CELL APPENDAGES ❖ Klebsiella Pneumonia
FLAGELLA ❖ Streptococcus Pneumoniae
• Exterior protein filaments that rotate and cause
bacteria to be motile Remember: Not all has CAPSULE.
• The arrangement of the flagella is as follows:
Atrichous without flagellum THE INSIDE OF A BACTERIA
Monotrichous single flagellum on one
end CYTOPLASMIC STRUCTURE
Amphitrichous single flagellum on both 1. RIBOSOME (NON-MEMBRANOUS STRUCTURE)
ends • Site of protein biosynthesis and gives the
Lophotrichous tuff/group of flagella on cytoplasm a granular structure
one end or both ends
Peritrichous spread over the whole
surface 2. GENOME
• Consist of single, circular chromosome
• Appears diffused nucleoid or chromatin body
that is attached to a mesosome.

3. PLASMID
• Extrachromosomal, double stranded element of
DNA that is associated with virulence.
• Located in the cytoplasm and serves as a site
for the gene to code for antibiotic resistance and
toxin production
• Not essential for growth

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 2

 • 2 Types of plasmids: RECOMBINATION
• Process by which genes are transferred or
Large Plasmid production of B lactamases exchanged between homologous regions on two
that provide resistance to B- DNA molecules.
lactam antibiotics like
penicillin and oxacillin • Provides a way for organisms to acquire and
Small Plasmid is resistant to tetracyclines copy new combinations of biochemical pathways
and chloramphenicol from the changes in their environment.

4. INCLUSION BODIES
• Serves as the energy source or food reserve
of the bacteria
• Composed mainly of polysaccharide; they
lessen osmotic pressure
• Examples:
❖ Glycogen
❖ Cyanophysin granules
❖ poly-B-hydroxybutyrate granules EXCHANGE OF INFO HAPPENS IN 3 WAYS
❖ Carboxysomes 1. TRANSFORMATION
❖ Gas vacuoles • Involves the recipient cell uptaking free DNA that
is released into the environment when bacterial
cell (donor) dies and undergoes lysis or cell
disintegration caused by a rupture in the cell
wall.

2. TRANSDUCTION
• It is the transfer of bacterial genes by a
bacteriophage from one cell to another

5. Endospores/Asexual spores (Resistant structures) 3. CONJUGATION

• small, dormant structures located inside the • The transfer of genetic material from a donor cell
bacterial cell to a recipient cell; Occurs between two living
• aid in survival of bacteria against external and cells; uses sex pili
extreme conditions
• produced within vegetative cells of some
Gram-positive bacteria.
• composed of dipicolinic acid and calcium ions
(calcium dipicolinate).
• Examples:
❖ Bacillus and Clostridium
• Types of spores according to location:
1. Terminal spore - Clostridium tetani
2. Subterminal spore - Clostridium
botulinum
3. Central spore - Bacillus anthracis

HOW DO WE NAME A BACTERIA?

CARL LINNAEUS
• Swedish botanist, zoologist, taxonomist, and
physician who formalized binomial
nomenclature
• Father of modern taxonomy
TAXONOMY OF BACTERIA
What is the 2nd important that was discovered?
HOW BACTERIA MULTIPLIES?
1. Gene
2. Genome
3. Chromosome – double stranded, closed circular,
naked macromolecule; contains genome for viability and
other important functions
4. Plasmid – contains genome for MDR
5. Transponder element – contains genome for
movement
WHAT IS THE 3RD IMPORTANT THAT WAS DISCOVERED?
BACTERIA EXCHANGES INFO
MUTATION
• Change in the original nucleotide sequence of a
gene or genes within an original genome
(genotype)
• May be induced by chemical or physical factors
in the environment or by biologic factors such as
introduction of foreign DNA into the cells.

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 3

 HOW DO WE WRITE THE NAME OF A BACTERIA? MICROAEROPHILE
1. Should be capitalized and followed by the species • It is an organism that requires 2% to 10%
epithet, which begins with a lower-case letter. oxygen for growth
• Example: Campylobacter and Treponema
2. Both the genus and species should be italized in pallidum
print, but underlined when written in script
• Example: Escherichia coli or Escherichia coli

3. When bacteria are to as a group, their names are

neither capitalized nor underlined
• Example: staphylococci, streptococci,
salmonellae
terms
ANAEROBE
GENOTYPIC
• These organisms do not require oxygen to grow
• Refers to genetic make up
• Three different types:
PHENOTYPICS 1. Obligate anaerobes
• Features ❖ Absolutely no need for oxygen
• Morphology because they die after
• Staining prolonged exposure to air
• Nutritional requirements ❖ Examples: Clostridium and
• Biochemical Bacteroides
• Susceptibility test
ENERGY PRODUCTION 2. Facultative anaerobes
• Breakdown of chemical substrates (chemical ❖ Clinically significant bacteria;
energy) through the degradative process of grow either in the presence or
catabolism that is coupled with oxidation absence of oxygen
reduction reaction. ❖ Examples: Enterobacteriaceae
• Use of glucose - an essential nutrient for
3. Aerotolerant anaerobes
energy production in organisms
❖ Can survive in the presence
1. RESPIRATION of oxygen but will be unable to
• Embden – Myerhof-Parnas pathway perform metabolic processes
❖ Oxidation of glucose —--> to Pyruvic unless situated in an anaerobic
Acid environment
❖ MAJOR ROUTE of glucose metabolism ❖ Example: Propionibacterium
in most cells. acne
OXYGEN REQUIREMENT
• Kreb cycle (Tricarboxylic acid)
❖ Enzyme system converts PYRUVATE
—--> to CARBON DIOXIDE and ACID
❖ Generate energy in the form of
adenosine triphosphate (ATP).
2. FERMENTATION
• It does not require oxygen (anaerobic), the use
of Krebs cycle or electron transport chain
• Release energy from sugars or other organic
molecules such as amino acids and purines.
• Forms a mixture of end products: lactate, OTHER THAN OXYGEN, THERE ARE STILL SOME WE NEED…
butyrate, ethanol and acetoin).
CARBON DIOXIDE REQUIREMENT
• Carried out by both obligate and facultative
anaerobes
1. CAPNOPHILES
• Require an increased CO2 (5% to 10% CO2)
• Has 4 types
• Example: Haemophilus influenzae, Neisseria
gonorrhoeae and Streptococcus pneumoniae

2. AUTOTROPHS
• Use CO2 as the sole source of carbon

3. HETEROTROPHS
• Use reduced, preformed, organic molecules
AEROBE from other bacteria or organism
• These organisms require oxygen and grow well
with room air 4. PHOTOTROPHS
• Air contains 15% to 21% oxygen and 1% CO2 • Organisms that use light as their energy source
• Example:
5. CHEMOTROPHS
❖ Bordatella, Brucella, Mycobacteria and
Pseudomonas • Organisms that utilize the energy produced by
organic or inorganic compounds oxidation.

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 4

 TEMPERATURE REQUIREMENT 5. Which of the following bacteria is correctly written
1. PSYCHROPHILES / CRYOPHILES
• These organisms grow well at 0C (zero degree) A. Shigella Sonnie
to a maximum of 20 C. B. Francisella tularensis
• Example: Listeria monocytogenes and Yersinia C. Edwardsiella tarda
enterocolitica. D. hemophilus influenzae

2. MESOPHILES 6. Which of the classification system is missing

• These organisms grow 20 C to 45 C Mycoplasma _________
• The most commonly encountered pathogenic
bacteria in the clinical laboratory A. Phylum
B. Specie
3. THERMOPHILES / HYPERTHERMOPHILES C. Genus
• These organisms grow 50 C to 125 C D. Family
• Examples: Bacillus stearothermophilus,
7. Given this picture, the bacteria is identified through its
Sulfolobus, Pyrococcus, Pyrodictium and
Thermus aquaticus.
A. Genotypic characteristics
B. Phenotypic characteristic
4. EXTREMOPHILE
C. Nomenclature
• Prokaryotes that are able to survive in unusual
D. Classification
condition like the absence of oxygen, increased
temperature and living below the earth’s surface
(Bacillus infernus). ADDITIONAL INFO FROM DISCUSSION
Bacteriology
PH REQUIREMENT
• Bacteria - staining (pag color ng bacteria) to
• Optimum pH 6.5 to 7.5
know its shape, if gram + or gram –
Acidophiles grow at pH 0 to 5.5
Neutrophiles grow between pH 5.5 • Parasites - movement
and 8.0 (most clinically • Fungi - check for color, itsura sa microscope
bacteria) • Virus - electron microscope
Alkalophiles grow between pH 8.5
and 11.5 Francesco Stelluti - uses magnifying glass (which are
called mcroscope that time)
SALT REQUIREMENT
• Halophiles - require and grow in increased Common perform in bacteriology: Culture and
concentration of sodium chloride Sensitivity (3-5. days results)
• Example: Staphylococcus aureus and Listeria • cannot perform in parasites, only bacteria
monocytogenes
GAUGE IN A MINUTE Culture
1. Which of these are present both in G(+) and G(-) • To know the bacteria present Sensitivity
bacteria? • To know the antibiotic that is best for the
bacteria and the dosage that needs to be taken
A. Periplasmic, cell wall, outer membrane only
B. Outer membrane, cell wall, peptidoglycan only How to check for infection:
C. Cell wall and cytoplasmic membrane only 1. Get specimen
2. Check specimen physical features
2. This is mainly composed of peptidoglycan and informs 3. Stain organism (gram staining)
the medtech of which gram result the bacteria belongs 4. Biochemical

A. Cell wall Gram Staining - most basic stain used in bacteriology

B. Periplasmic
C. Cell envelope Mycolic Acid
D. Cytoplasmic • mantika, taba, fats
• gram stain can stick but lighter color
3. Which is the intracellular structure that allows
organisms to withstand extreme conditions? Acid Fast Bacteria - uses acid fast stain

A. Fimbriae Gram Positive - Thick peptidoglycan (makapal na

B. Endospores comforter)
C. Murein
D. Intron Gram Negative - Thin Peptidoglycan (floral na kumot)

4. What is considered as a virulence factor and protects Gram Stain - 2 stains (Gram + & Gram – )
bacteria against phagocytosis and dessication?
2nd stain - safranin
A. Cell membrane
B. Capsule Gram Chemical – 4
C. Sex pili
D. Fimbriae G+ G–
Crystal Violet Violet Violet
Gram’s iodine Violet Violet
Alcohol Violet Colorless
Safranin Violet Red / Pink

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 5

Lysozyme - sticks at cell wall and protects cell wall

Gram Positive - mas nilalapitan ng bacteria

Supravital Stain - Multiply through BINARY FISSION

Mycoplasma - no cell wall (anything w plasma)

Mycoplasma - causative agent of walking pneumonia

No cell wall cannot maintain shape they are called

pleomorphic (nagbabago)

Resting - Coccobaccili

Cell Appendages
• bacteria with this are more powerful
• not required for the bacteria to have

Motile - bacteria w flagella

Non-Motile - bacteria w/out flagella

Lophotrichous - fastest

Slime Layer - serve as pandikit or for attachment

Surface Polymer
• jacket
• Used when it is cold or hot
• 35-37.5 degree Celsius
• makes capsule during extreme temperature

Plasmid - bacteria is powerful with these because it

makes bacteria resistant to antibiotics

Bacteria can have both large and small plasmid

Inclusion Bodies - hoard food by the bacteria

Endospore/Asexual Spore
• not used for reproduction, only binary fission
• made in extreme condition
• not all bacteria can produce
• only bacillus and clostridium can make this

Capsule
• made in extreme temperature
• not all bacteria can produce

Ethyl Alcohol Shock Test - pour alcohol to make spore

--------------------- END OF LESSON 1 -------------------

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 6

 •
Lesson 2: the disinfection and sterilization Bleach
❖ HepB – 10 mins
process ❖ HIV – 2 mins

STERILIZATION • Isopropyl > Ethanol – 10 seconds

• Refers to the destruction of all forms of life ❖ Pseudomonas aeruginosa – 30-100%
including spores ❖ E. coli – 40-100%
• There is no degrees – all or nothing ❖ Serratia - 40-100%
❖ S. aureus - 60-95%
DISINFECTION
❖ S. pyogenes - 60-95%
• Refers to the process that eliminate a defined TEMPERATURE
scope of microorganisms including some • Disinfectant are generally used at room
spores temperature of 20-22 °C
• Can be physical or chemical • ____ Temp = _____ killing activity
• Disinfectant pH
• Antiseptic
• Identification of bacteria desired to be killed
DECONTAMINATION • Identification of the surface
• Removal of pathogenic bacteria so items are BIOFILMS
safe to handle or dispose of • May need to increased concentration of
WHY THERE ARE STILL SOME THAT DON’T DIE? disinfectant or contact time
TYPES OF ORGANISM
• Presence of spores – protein, lipid, carbs,
dipicolinic acid and calcium
• Mycolic acid in bacteria
❖ Note: Opposite for virus with lipid-rich
envelope – detergent and wetting agent
• Prions
❖ Abnormal infectious proteins COMPATIBILITY OF DISINFECTANT AND STERILANT
❖ Non-living things • Two disinfectants are NOT BETTER than one
❖ Resistant to heat (121°C), chemicals • Disinfectants may negate other disinfectant
(acid or base), radiation ❖ Bleach and quaternary compound

Sterilization Disinfection Both

Physical Incineration Boiling
Moist heat Pasteurizing
Dry heat Non – ionizing
Filtration radiation
Ionizing radiation
Chemical Ethylene Oxide Alcohol
Hydrogen Peroxide Aldehydes
Halogens
NUMBER OF ORGANISM
• Microbial load – determines the exposure time Peracetic
Acid
that is necessary for 99.9% elimination
CONCENTRATION OF DISINFECTING AGENT Quaternary
• Preparation, dilution and use must be followed Ammonium
Compound
carefully
• 70% alcohol Phenolics
• Povidone-iodine
PRESENCE OF ORGANIC MATERIAL sterilization
• Organic material inactivates the disinfecting PHYSICAL METHOD
agent 1. INCINERATION
• Prevents full contact • Burned to ashes at 870 °C to 980 °C
NATURE OF SURFACE TO BE DISINFECTED • Good for prions
• Nature of the equipment may affect the type of • Safest method
method that can be used • Disadvantage: toxic air emissions and presence
• Is the material need to be sterilized or of heavy metals
disinfected only?

2. MOIST HEAT
• Moist heat or heat under steam pressure
CONTACT TIME • 1 atm or 15 pounds per square inch at
• Identify the organism present first temperature of 121 °C or 132 °C
• Contact time may disinfect only or sterilize ❖ 30 minutes, 121 °C / 4 minutes, 132 °C
• Betadine –1 to 2 minutes culture media

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 7

 ❖ 30-60 minutes, 132 °C – biological BIOSAFETY LEVELN
waste
• Faster than dry heat Biosafety level Remarks
• Moist heat must come in contact with surface 1 - No known potential to hurt healthy people
• Bacillus stearothermophilus (Geobacillus - Used for laboratory teaching exercises
- B. subtilis and Naegleria gruberi
stearothermophilus)
2 - Agents are commonly being sought in clinical
specimens
- HIV, hepatitis virus, Salmonella and prions
- Access limitation during experimentation,
supervision, use of BSC on aerosol producing,
vaccination

3 - Mycobacterium tuberculosis
- Agents are primarily transmitted thru aerosols
- BL2 precautions + engineering controls

• Prions: 4 - Exotic agents such as Congo-Crimean

❖ Brain, spinal cord, eye tissue hemorrhagic fever and Marburg virus
- Agents have no vaccine or therapy
❖ Autoclave for 18 minutes, 134 °C - All personnel and materials need to be
❖ Autoclave for 1 hour, 132 °C decontaminated
❖ Immerse in 1N sodium hydroxide for 1
hour, rinse then autoclave for 1 hour, 5. RADIOATION – IONIZING
121 °C (gravity displacement) or 134 °C • Cold sterilization
(prevacum sterilizer) • Causes mutation in the DNA and produce
❖ Immerse in 1N sodium hydroxide for 1 peroxidase
hour, rinse then autoclave for 1 hour, • Destroy vegetative cells and endospores of both
121 °C (gravity displacement) then clean prokaryotes and eukaryotes
routinely • Gamma rays (1500 to 2500 radiation) & xrays
• Biological Indicator: Bacillus pumilus
3. DRY HEAT • Best for: Plastic syringe, gloves, and catheters
• Longer exposure time
• Usually, 160-180 °C 6. RADIATION – NONIONIZING
• Done for 1.5 – 3 hours • Damage to cellular DNA by producing thymine
• Used for heat stable substance such as dimers
glasswares • Used on exposed surface, operating rooms
• Microorganisms in water are destroyed under
UV lamps
• Ultraviolet rays (10um to 400um) in which
260um is the most lethal

CHEMICAL METHOD
4. FILTRATION 1. ETHYLENE OXIDE
• Most common chemical sterilant
Liquid Filtration Uses cellulose acetate or
cellulose nitrate membrane • Used for sterilizing heat-sensitive objects
with a vacuum • Disadvantage: lengthy cycle time and potential
Air Filtration Uses high – efficiency health hazard
particulate air (HEPA) filter

Removes organisms larger than 2. HYDROGEN PEROXIDE

0.3um • if stabilized with phytate, citrate or malonate
Filtration of Bacteria, Yeast, Uses 0.45um pores of • Vapor form is used to sterilized HEPA filters
and Molds membrane filter
• No toxic by-products
Range: 0.2 – 0.45um DISINFECTION
Remove most bacteria and PHYSICAL METHOD
fungi but not virus 1. BOILING
• 100 °C for 15 minutes
BIOSAFETY CABINET • Kills only vegetative bacteria

Class I allow room air to pass into the 2. PASTEURIZATION

cabinet sterilizing only the air to • 70°C for 30 minutes
be exhausted
• Kills food pathogen without decreasing food
exhausted outside or work area nutrition or flavor
Class II IIA –self-contained, 70% of air
is exhausted into work area 3. TYNDALIZATION
IIB –air is discharged outside • 100°C for 30 minutes for 3 consecutive days
building; for radioisotopes, toxic • Kills endospores
chemicals, carcinogens
Class III has rubber gloves, afford most
protection
4. NON – IONIZING RADIATION
• Example: UV light
Note: must be certified initially, when moved over 18 • Long wavelength and low energy
inches and annually. • Do not penetrate well

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 8

 STERILIZATION AND DISINFECTION (BOTH) 2. MERCURY
CHEMICAL METHOD • Not used anymore
1. ALCOHOLS
3. 1% SILVER NITRATE
• Non-sporicidal and evaporates quickly
• Not used anymore for ophthalmia neonatorum
• Water content – hydrolyzes hydrogen bonds in
bacterial protein • Replaced by erythromycin
• Limited to skin as antiseptic, thermometer or •
injection vial rubber septa

2. HYPOCHLORITE
• Bactericidal, virucidal, fungicidal,
mycobactericidal, sporicidal
• Ration is 1:10
• Inexpensive yet effective
WHAT IF THESE DISINFECTANT, STERILIZATION, AND ANTISEPTIC
• Does not decrease capacity based on quality of TECHNIQUE WERE NOT USED PROPERLY?
water TRUE PATHOGEN
• Disadvantage: may cause oropharyngeal, • Organisms are able to invade the tissue of
esophageal, and ocular irritation; corrodes healthy individual
metal, discolors fabrics and may produce toxic • Normally found outside the host
gas when combined with ammonia or other acid OPPORTUNISTIC PATHOGEN
• Normally do not cause disease in their natural
habitat to a healthy person
• Cause disease if the host is
immunocompromised
• Ex. Neisseria meningitidis and Escherichia coli
INFECTION
A. TYPES OF INFECTION (ACCORDING TO SOURCE)
1. AUTOGENOUS INFECTION
• Infection from the host

3. ALDEHYDE 2. IATROGENIC INFECTION

• Example: glutaraldehyde • From medical treatment or procedure
• Does not corrode lenses, rubber or metals
• Technique is called cold sterilization 3. OPPORTUNISTIC PATHOGEN
• Affects immunocompromised host
4. PERACETIC ACID
• Best if equipment has organic material 4. NOSOCOMIAL PATHOGEN
• Technique is called cold sterilization • Hospital-Acquired infection
• Types of Nosocomial infection:
5. HEAVY METALS 1) Urinary tract infection
2) Lung infection (Pneumonia)
Agent(s) Action(s) Applications & 3) Surgical site infection
Precautions 4) Blood stream infection
Silver nitrate Precipitates proteins Eye drop (1%
(AgNO3) solution) B. TYPES OF INFECTION (ACCORDING TO HOST
Reacts with -SH DISTRIBUTION)
Mercuric chloride groups; lyses cell Disinfectant: toxic 1. LOCAL INFECTION
(HgCl2) membrane at high
concentrations • Confined in one area
• Ex. boils and abscesses
6. PHENOLS
2. FOCAL INFECTION
Agent(s) Action(s) Applications & • From local spreads to other part
Precautions • Ex. Tonsillitis, wound infection by Clostridium
Phenol, carbolic Denature proteins Disinfectant at high tetani, etc.
acid, Lysol, concentration
hexachlorophene Disrupt cell
membrane used in soaps at 3. SYSTEMIC INFECTION
low concentration • Infection spreads from blood and lymph
• Types of Systematic infection:
ANTISEPTIC
1. IODINE Bacteremia Presence of bacteria in the
blood
• Prepared either as tincture with alcohol or Septicemia Active multiplication in the
iodophor coupled with neutral polymer blood
(Povidone-iodine) Pyemia Pus producing organisms
• ALERT: invade blood
Toxemia Presence of toxin in the
❖ If skin is compromised, used in infants blood
(high skin permeability), mucosal
application or excessive skin application,
PUS
then may cause goiter, hypothyroidism,
liver function abnormalities, dermatitis,
and neutropenia
❖ Use charcoal as adsorbing agent
❖ Can diffuse thru breastmilk

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 9

 EXTENT OF INFECTION 4. INDIGENOUS / NORMAL MICROBIAL FLORA OR
MICROBIOTA
Primary Infection Initial Infection Microbiota:
Secondary Infection Caused by Opportunistic Skin Staphylococci, Propionibacterium
Pathogen and Corynebacterium
Latent Infection (Silent Phase) Clinically silent inside the body
Mixed Infection Two or more infection Mouth & Oral cavity Viridans Streptococci
Acute Infection Infection develops and progress
slowly Upper respiratory tract Viridans Streptococci,
Chronic Infection Infection develops slowly but Diphtheroids and S. epidermidis
long lasting
Nasopharynx S. aureus, S. epidermidis and N.
SHINGLES meningitidis

Colon E.coli, Bacteroides and Lactobacilli

Urethra Diphtheroids,
S. epidermidis, alpha and non
hemolytic Streptococci.

5. PHAGOCYTOSIS
HOW THESE COULD BE PASSED?
6. INFLAMMATION
CLASSIFICATION OF INFECTIOUS DISEASE
1. COMMUNICABLE OR CONTAGIOUS DISEASE
7. IMMUNE RESPONSE
• Spread from one host to another (directly or
indirectly)
PHASES OF INFECTION
• Ex. tuberculosis, herpes, flu and chickenpox
1. INCUBATION PERIOD
2. NON-COMMUNICABLE DISEASE • Time between the exposure to pathogen and
• Does not spread from one host to another onset of symptoms
• Ex. tetanus and botulism
2. PRODROMAL PERIOD
ROUTE OF INFECTION
1. DIRECT TRANSMISSION • Appearance of signs and symptoms
• Congenital - S. agalactiae, N. gonorrhoeae and
3. CLINICAL OR ILLNESS PERIOD
syphilis
• Peak of characteristic sign and symptoms of an
• Sexual contact - C. trachomatis, N.
infection
gonorrhoeae and syphilis
• Infectious respiratory droplets - N.
4. DECLINE PERIOD
meningitidis
• Period in which the signs and symptoms begin
• Hand to hand transmission - Rhinovirus
to subside
2. INDIRECT TRANSMISSION
5. CONVALESCENCE OR PERIOD OF RECOVERY
• Fomites
• Period in which the surviving host is
• Water - Salmonella, Shigella and Vibrio recuperating towards
• Arthropod vectors - Borrelia, Francisella and
Yersinia 6. FULL RECOVERY
CLASSIFICATION OF DISEASE (ACCORDING TO
OCCURRENCE) VIRULENCE
1. SPORADIC DISEASE
• Ability of microorganisms to cause disease
• Occasionally
• It is the degree of pathogenicity
2. ENDEMIC DISEASE • Organisms that can establish infection with a
relatively low infection dose. More virulent than
• Constant presence
those that requires high dose for infection.
3. EPIDEMIC DISEASE FACTORS AFFECTING VIRULENCE
• Affects large number of people in a short span of 1. TOXIC FACTORS
time • Toxins are substance produced by pathogenic
microorganisms causing tissue and cellular
4. PANDEMIC DISEASE damage
• Affects across large region around the word • Ex. Diphtheria toxin, tetanospasmin, botulism
toxin
Resistance factors
1. PHYSICAL BARRIER 2. ENZYMATIC FACTORS
• Ex. Stricture of urethral opening, the flushing • Aid in the spread of infection
action of urination and thick mucus plug in the • Ex. Hyaluronidase, coagulase, leukocidin,
cervical opening collagenase
2. CLEANSING MECHANISM
• Nasal hair, cough-sneeze reflex and cell lining of 3. CELLULAR STRUCTURE
trachea • Capsule resist phagocytosis - Encapsulated
3. ANTIMICROBIAL SUBSTANCE bacteria
• Lysozymes - destroy bacterial cell wall EXOTOXIN VS ENDOTOXIN
• Bile salts - disrupt bacterial membranes EXOTOXIN
• On of the most lethal substances
• Gram Positive bacteria

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 10

 • Do not require bacterial death to be release
into circulation
• Example: cytotoxins, neurotoxins and
enterotoxins
• Bacteria: Clostridium botulinum,
Corynebacterium diphtheriae, Staphylococcus
aureus and Streptococcus pyogenes
ENDOTOXIN
• Composed of the lipopolysaccharide
• Present only in gram negative bacteria
• Release when bacteria die and their cell wall
undergo lysis
• Toxicity is due to the LIPID A portion of LPS
❖ LPS may elicit fever, chills, hypotension,
granulocytosis, thrombocytopenia, and
disseminated intravascular coagulation.

ADDITIONAL INFO FROM DISCUSSION

Antiseptic - for body
Disinfectant - non-living things

70% alcohol is stronger than 100%

Critical – involves insertion in the body

Semi-critical - di pinapasok sa katawan

Moist heat - needs pressure, constant temperature, and

contact time

Lead Acetate
• geobacillus
• 18-24 hrs incubation
• Violet if the organism is dead, indication of
complete autoclaving process
• Yellow if still alive

Gray - successful
White - not

Dry heat - Bacillus Niger

Biosafety Class IIB - may host sa taas palabas ng

building

Ethylene oxide
• need ibabad matagal
• not for papers

Pasteurization
• para sa mga bawal kumulo
• di nasisira nutritional content ng food

Tyndalization - prions remains unkilled

1% silver nitrate - for creed's method

Hypochloride/ Bleach/ Zonrox

suffix: -cidal (kills bacteria)
• static (paralyzes bacteria)
• example of this is refrigerator ratio: 1 (zonrox)
:10 (water)

--------------------- END OF LESSON 2 -------------------

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 11

 LESSON 3: MICROBIAL IDENTIFICATION BLOOD / BONE MARROW ASPIRATE

Where do bacteriology tests start?

SPECIMEN REQUISITION
1. Patient’s name
2. Hospital Identification Number
3. Age and Date of Birth
4. Sex
5. Collection date and time
6. Ordering of Physician
7. Exact nature and source of specimen
8. Diagnosis
9. Current antimicrobial therapy
HEPARIN
• Can also be used but may inhibit some gram (+)
and yeast.
OTHER BODY FLUIDS

1. TIMING \
• Specimens should be collected on the acute
phase and before the medicines.
❖ Kasi pag nagamot na, wala na.
• Needle Aspirations > Swabs
❖ Pag nagcollect, dapat diretso sa
laboratory
❖ Within 2 hrs
AMNIOCENTESIS
• Use a holding medium
❖ Stuart’s or Amie’s Medium
❖ Holds only the organism. It does not
support growth

PARACENTESIS
2. SPECIMEN COLLECTION
GENERAL TRANSPORT TIME
2 hours Regular
1 hour Stool and Gastric Biopsy
Bedside Corneal Scrappings
ASAP Bons; Suprapubic Aspirate
Traumatic 15 mins ARTHROCENTESIS

GENERAL STORAGE
Bacteria
- Extracellular RT
- Intracellular 4°C
Fungi
- Yeast RT PERICARDIOCENTESIS
- Mold 37°C
Virus 4°C
Parasite Depends

ABSCESS (LESION, WOUND, PUSTULE, ULCER)

PLEUROCENTESIS / THORACENTESIS

CEREBROSPINAL FLUID (CSF)

SUPERFICIAL AND DEEP (RESPECTIVELY)

0.025% SPS

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 12

 EAR CARY – BLAIR MEDIUM

FEMALE GENITAL TRACT

EYE

MALE GENITAL TRACT

CONJUNCTIVITIS & CORNEAL SCRAPINGS

(RESPECTIVELY)

GI TRACT

HAIR, NAILS, SKIN SCRAPINGS

GASTRIC BIOPSY
HAIR ANATOMY

BED PAN
RESPIRATORY TRACT

2 HOURS TALAGA!
STOOL CONTAINER

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 13

 TISSUE DIRECT MICROSCOPIC
1. Quality of specimen can be assessed
• Ex: Sputum, <25 squamous epithelial cell / LPO
or <10 squamous epithelial cell / HPO
2. Clinical can be given early indication
3. Specimen workup can be compared on the culture
• Ex: 3 bacteria on a smear, 2 on an aerobic
culture
• Ex: 1 bacteria on smear, 4 on an aerobic culture
URINE DIRECT MICROSCOPY
• Not performed on throat, nasopharyngeal and
stool specimens
STAINING
1. STAINS BASED ON PH
1. BASIC DYE
• Commonly used - Cationic dyes with positively
charged groups that adhere to negatively charge
molecules like nucleic acids and proteins.
• Example: Methylene blue, crystal violet,
safranin and malachite green

2. ACIDIC DYE
TYPES OF URINARY CATHETERS
• Anionic dyes with negatively charged groups
that bind to positively charge cell structures.
• Example: Eosin and acid fuchsin
2. STAINS BASED ON COMPLEXITY
1. SIMPLE STAINING
NOTE: if sa lab lang, normal container lang • Single stain is used Directed towards coloring
i-ship to a different facility the forms and shape of the cells
• Example: Methylene blue

2. DIFFERENTIAL STAINING
• Divide bacteria into separate groups
• Directed towards coloring the components of
the elements present.
• Example: Gram staining and Acid-fast bacilli
(AFB) staining

Specimen priority (levels of specimen prioritization) GRAM STAIN

Level Description Specimens

1 Critical / Amniotic fluid
Invasive Blood
Brain
CSF
Heart valves
Pericardial fluid
2 Unpreserved Body fluids (not listed for level 1)
Bone
Drainage from wounds
Feces
Sputum
Tissue
3 Quantitation Catheter tip
required Urine
Tissue for quantitation
4 Preserved Feces in preservative
Urine in preservative
Swabs in holding medium (aerobic &
anaerobic)

RULE
NOTE: Kung paano nilalabel ang blood tube, same din
• All cocci are Gram positive except:
sa bacteriology. Letter Bacteria
GROSS EXAM No Neisseria
• Needed to verify the specimen and add in Boyfriend Branhamella
Muna Moraxella
diagnosis Vilma Veillonella

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 14

 • All bacilli are Gram negative except: cultivation
Letter Bacteria
B Bacillus
• There is a need to process first:
L Lactobacillus
L Listeria
A Actinomyces
C Clostridium

C Corynebacterium
M Mycobacterium • Urine needs to be concentrated by
E Erysiphelothrix centrifugation or filtration
N Nocardia • Bones may need to be homogenized
❖ Homogenization: Grinding or mincing
HEADS UP or squash/crush prep
• Removal of MgRNA • Swabs has many types:
• Aged, dying and autolyzing cells, old cells may ❖ If fiber: vortex in 0.5-1ml saline or broth
lose their ability to retain stains for 10 20 seconds
• Antibiotic-treated bacterial cells have atypical
staining reaction
• Using acidic iodine during staining
• Due to a technical error or the wrong use of
stains
ACID – FAST STAIN

• Loops matter if: urine or tissue from burn

victims plate quantitatively

TYPES OF STAINS
1. Ziehl-Neelsen/Hot staining Method
2. Kinyoun’s/Cold staining Method
3. Pappenheim method – differentiate Mycobacteriuam CULTURE MEDIA
smegmatis from Mycobacterium tuberculosis • Composed of a mixture of nutrients such as
4. Baumgarten method – differentiate Mycobacterium carbon, nitrogen, sulfur, phosphorus, hydrogen,
leprae from Mycobacterium tuberculosis oxygen and buffers
5. Auramine-rhodamine method – selective for the cell • Types:
wall of AFB. ❖ Liquid
CONTINUES OF STAIN BASED ON COMPLEXITY ❖ Semi-solid
3. NEGATIVE STAINING ❖ Solid Medium
• Demonstrate presence of diffuse capsule TYPES OF CULTURE
surrounding some bacteria 1. PURE CULTURE
• Excellent technique for studying bacterial gas • It is composed of only one species
vacuole and viral morphology
• Appearance: bacteria as light-colored bodies 2. MIXED CULTURE
against dark background • It is composed of more than one species
• Example: India Ink or Nigrosin dye
3. STOCK CULTURE
• It is composed several culture species
contained in a separate culture medium (one
species per culture medium). - It is used for
academic and industrial purposes.
CLASSIFICATION OF CULTURE MEDIA
ACCORDING TO CONSISTENCY
1. LIQUID MEDIUM
4. SPECIAL STAINING
• It does not contain any amount of agar
Staining Technique Cellular structure / Bacteria • Also called broth
Dyar Cell wall • Nutrients are dissolved in water
Anthony’s, Hiss, and Gin’s Capsule • Turbidity is measured
Nigrosin Capsule
• It allows the growth of aerobes, anaerobes and
Neisser Metachromatic granules
Albert Metachromatic granules facultative anaerobes
Dorner Endospore • Example: Brain heart infusion, trypticase soy
Schaeffer – Fulton Endospore broth (TSB) and thioglycolate broth.
Gray Flagella
Leifson Flagella
Feulgen DNA
Levaditi’s Sphirochetes
Fontana - Tribondeau Sphirochetes

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 15

2. SEMI – SOLID MEDIUM 2. TUBE MEDIA
• It contains 0.5% to 1% Agar • Prepared as either liquid, slant, butt and slant
• It is used to observe bacterial motility and detect or butt
indole and sulfide production • Examples:
• Ex: Sulfide Indole Motility (SIM) Medium ❖ Triple sugar iron (TSI)
• Anaerobes – bottom part ❖ SIM - Simmon’s citrate agar
• Aerobes – upper layer ❖ Lysine iron agar (LIA)
ACCORDING TO USE
1. NUTRITIVE MEDIA
• Routinely used in the laboratory and without
additional supplement
• Support the growth of most non-fastidious
bacteria
• Non-selective
3. SOLID MEDIUM • Usually composed of meat and soybean extract
• It contains 2% to 3% agarose • Ex: Nutrient agar, Nutrient broth and Tryptone
• Melts at ≥ 95°C soy broth (TSB), Blood Agar Plate (BAP),
• Solidifies below 50°C (now called agar) Chocolate Agar Plate (CAP)
• Ex: Triple sugar Iron (TSI) agar, MacConkey
(MAC) agar, Blood agar plate (BAP) and 2. ENRICHMENT MEDIA (LIQUID – TYPE MEDIA)
Chocolate agar plate (CAP) • Also called back-up broth
• Also called supplemental broth
• Allows detection of:
❖ Small numbers of organism present may
be detected
❖ Organism damaged by the antimicrobial
❖ Detection of anaerobes in aerobic
culture
• Contain specific nutrients and without
additional supplement
ACCORDING TO COMPOSITION • Example: Alkaline peptone water, Selenite F,
1. SYNTHETIC / DEFINED MEDIUM Thioglycollate, Tetrathionate, Gram-negative
• Medium in which all the components are known (GN) broth and Lim broth
• Used for research purposes
• Preferred for isolation of cyanobacterium and 3. DIFFERENTIAL MEDIA
chemoorganotrophs • These media allow the visualization of
• Ex: BG-11 medium metabolic differences between groups of
bacteria
• Example: MAC, BAP, eosin methylene blue
(EMB) and Hektoen Enteric Agar (HEA)

2. NON – SYTHETIC / COMPLEX MEDIUM

• Medium in which some substances are
unknown 4. SELECTIVE MEDIA
• Peptones, Meat and Yeast Extract • These media are incorporated with antibiotics,
• Useful for isolation of medically significant dyes or chemicals to inhibit the growth of other
bacteria organisms.
• Ex: Nutrient broth (NB) broth medium, TSB and • Example: HEA, MAC, Xylose lysine
MAC agar desoxycholate (XLD) agar, Bismuth sulfate agar
(BSA), Mannitol Salt Agar (MSA) and Thayer-
3. TISSUE CULTURE MEDIUM Martin Agar (TMA)
• Used for obligate intracellular bacteria • Other selective media:
(Rickettsia and Chlamydia).
• Example: W138 cells, HeLA 299 Cells and Gentamicin blood agar Streptococcus
Bacitracin chocolate Haemophilus
McCoy cells agar
Blood agar plate with Aeromonas
HeLa 299 cells Human cervical tissue ampicillin
McCoy cells Fibroblast Phenylethyl alcohol Gram positive bacteria
W138 cells Fibroblast Colistin – Nalidixic acid Gram positive bacteria
(CAN) agar
ACCORDING TO DISPENSING / DISTRIBUTION
1. PLATE MEDIA Anong nilalagay para maging selective?
• Distributed into the dish or plate
To prevent growth of Add
Gram Positive Bacteria Crystal / Gentian violet, basic /
carbol fuchsin and bile salt

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 16

 Gram Negative Bacteria Potassium tellurite and sodium 6. Sheep Blood Agar
azide Composition Purpose Remarks
For Swarming Bacteria Alcohol and chloral hydrate Protein source: Supports growth of Detects hemolytic
tryptones all bacteria patters
THE MEDICALLY IMPORTANT CULTURE MEDIA
Soybean protein Also called BAP
1. Brain – Heart Infusion digest
Composition Purpose Remarks Rabbit or horse
Peptone Bacterial Can be broth or Sodium chloride blood may be used,
identification agar but the hemolytic
Phosphate buffer Sheep blood patterns may be
Antimicrobial Can be with blood inconsistent with
Small conc of susceptibility testing or without blood those on sheep
dextrose blood

2. Chocolate Agar 7. Modified Thayer – Martin

Composition Purpose Remarks Composition Purpose Remarks
BAP + hemin/x Neisseria same as blood agar Colistin – inhibit supports growth of has modifications
factor and gonorrheae except that red cells gram (-) N. gonorrheae and such as addition of
nicotinamide are lysed to release N meningitidis trimethoprim to
adenine Haemophilus spp. hemin/x factor and Vancomycin – inhibit Proteus
dinucleotide/NAD/V nicotinamide inhibit gram (+) organisms
Factor adenine
dinucleotide/NAD/V Nystatin - yeast
Factor

3. Columbia CNA With Blood

Composition Purpose Remarks
three peptone Growth of gram (+)
sources bacteria CONTINUES OF ACCORDING TO USE
5. SPECIAL MEDIA
5% defibrinated
blood
• Used to isolate bacteria with specific growth
requirements
colistin and nalidixic • Example: Lowenstein Jensen (LJ) medium and
acid - to inhibit most Thiosulfate citrate–bile salts sucrose (TCBS)
gram (-)
agar.

4. Hektoen – Enteric Agar

Composition Purpose Remarks
bile salts Growth of slows the growth of
Salmonella and non-pathogenic and • Incubation:
pH indicator: Shigella spp. – blue gram (-) enteric Fungi 28 – 30 °C
bromothymol blue green bacteria Bacteria 35 – 37 °C
acid fuchsin dyes also differential non- Virus 35 – 37 °C
SS is orange to
salmon colored
colonies ATMOSPHERIC REQUIREMENT

5. MacConkey Agar --------------------- END OF LESSON 3 -------------------

Composition Purpose Remarks
Crystal violet - For differentiation of lactose fermenter -
inhibit G(+) and gram (-) enteric pink
fungi lactose fermenters
and non-lactose non-lactose
Sugar: lactosepH fermenters fermenter -
indicator: neutral red colorless

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 17

 •
LESSON 4: THE NORMAL FLORA 20% of feces consists of bacteria approximately
1011 organisms/g.

NORMAL FLORA
• Also known as “Human Microbiome or
Microbiota”
• Commensals
• Various bacteria and fungi that are permanent
residents of certain body sites, especially the
skin, oropharynx, colon, and vagina.
THEY CAN CAUSE DISEASES IF (NORMAL FLORA)
1. Increases in numbers beyond what the body can
handle
2. There is a change in habitat
3. Status of Immune System 4. Escherichia coli
ROLES (NORMAL FLORA) • Leading cause of UTI
1. Weight control
2. Inflammatory bowel DSE 5. Bacteroides fragilis
3. Immune response • Important cause of peritonitis associated with
4. Resistance to infections perforation of intestinal wall following trauma or
appendicitis

6. Fusobacterium, Pepto streptococcus,

Enterococcus faecalis
• Also, a cause of UTI and endocarditis

NORMAL FLORA ON THE:

7. Pseudomonas aeruginosa
SKIN • Various infection in hospitalized patient
1. Staphylococcus epidermidis
• Present in 10% of normal stools
• Predominant; superficial
• Non-pathogen of the skin but can infect artificial
• Also present in soil and water
heart valves and prosthetic joints GENITOURINARY TRACT
1. Lactobacillus
2. Candida albicans • Vaginal flora
• Normal flora of the skin but can cause systemic • Produces acid
infections
2. Candida albicans
3. Propionibacterium and Peptococcus • Candida vaginitis
• Anaerobic bacteria
• Only in deeper follicles
3. Escherichia coli and Enterobacter
RESPIRATORY TRACT
1. Streptococci and Staphylococci
• Causes UTI
• Nose
• Most significant: Staphylococcus aureus and
Streptococcus pneumoniae

2. Viridans streptococci and Neisseria

• Streptococcus mutans – dental plaque
• Streptococcus sanguinis – leading cause of
bacterial endocarditis
STOMACH
• None
INTESTINAL TRACT
1. Stomach
• Has low pH

2. Small Intestine
• Small numbers Streptococci, Lactobacilli and
yeasts particularly Candida albicans.
• Mostly in the terminal ileum.

3. Colon
• Major location of bacteria in the body

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 18

 • Commonly associated with outbreaks in
prisons, dorms, athletic environments, and
school

VIRULENCE FACTOR (S.aureus)

• may produce biofilm or slime layer

Protein A inhibits IgG antibody

--------------------- END OF LESSON 4 ------------------- Alpha toxin disrupts smooth muscle and
toxic to RBC, WBC, platelets
and hepatocytes
LESSON 4.1: FAMILY STAPHYLOCOCCACEAE Beta toxin hydrolyzes membrane
phospholipid
Delta toxin cytolytic to RBC and other
STAPHYLOCOCCUS mammalian cells
• There are 45 species and 21 subspecies Gamma toxin wound, skin and deep tissue
infections
• Gram (+) Leucocidin destruction of phagocytes
• Cocci Coagulase & enhances invasion
• Catalase (+) except: S. saccharolyticus Hyaluronidase
and S. aureus subs anaerobius
• Aerobic or facultative anaerobe except: FOLLICULITIS
S. saccharolyticusand S. aureus subs • Infection of hair follicles
anaerobius FURUNCLES
• S. aureus is the most virulent • Hair follicle infection that spreads to tissue
• Non-motile causing boils
• Non-spore forming CARBUNCLES
• Normal flora of skin and mucosal membrane • Furuncles that coalesce
• Causes diseases on sterile site IMPETIGO
• Caused by S. aureus
• Involves epidermis with production of
vesicles that rupture and crust over
SCALDED SKIN SYNDROME
• Also called Ritter disease
• Caused by S. aureus’ exfoliating toxins
• Causes split of intracellular bridges of the
epidermidis resulting to extensive sloughing
• Bacteria remain locally
• Affects neonates
TOXIC SHOCK SYNDROME
• Caused by S. aureus’ toxic shock syndrome
toxin or pyrogenic exotoxin c
• Patient also experiences fever,
desquamation and hypotension leading to
death
FOOD POISONING
• Enterotoxins, most commonly A (78%), D
Novobiocin – Susceptible Novobiocin – Resistant (38%), and B (10%)
S. epidermidis S. cohnii
S. haemolyticus S. kloosii
S. capitis S. saprophyticus
S. homnis subsp hominis S. xylosus
S. saccharolytic
S. lugdunensis

Pathogenesis
S. aureus
• MOT: person-to-person, fomites;
aerosolized
• Microbiota: anterior nares, nasopharynx,
perineal area, skin and mucosa
• Most virulent species

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 19

 Organism Habitat Virulence Factor Diseases
S. epidermidis Skin and Exopolysaccharide Nosocomial bacteria
mucus (slime or biofilm) –
membrane enhances adhesion Iatrogenic infection
and resistance to - Endocarditis
antibiotic - Graft infection
- Prosthetics
Exotoxins: delta toxin - Catheter
S. haemolyticus Same as S. Uncertain Same as S. epidermidis
epidermidis
but fewer in
number
S. lugdunensis Same as S. Uncertain Same as S. epidermidis
epidermidis
but fewer in Resembles S. aureus’
number infection
S. Skin and Uncertain Community-acquired
saprophyticus mucosa of UTI in young sexually
genitourinary active female
tract
Second most common
Micrococcus Skin, Uncertain Rarely implicated for
spp. mucosa and infection
oropharynx

Laboratory Diagnosis
Note: Also review the Culture Media & their purpose!
1. Collection and Transport
• No special consideration
ADDITIONAL INFORMATION FROM DISCUSSION
2. Gram Staining Normal Flora
• Gram (+) • Sterile
• Cells divide longitudinally and horizontally • Good bacteria
forming pairs, tetrads and irregular clusters • Located in certain body parts
• Digestive system has it
3. Cultivation
• Common broth such as BHI, Thio and NB Body
• If selective: Phenylethyl alcohol, colistin- • Sterile = Brain, CSF
nalidixic acid (CNA) agar, MSA, CHROM agar
• Non – sterile = Exposed to environment
• If general: 5% SBA/BAP and CAP
MANNITOL SALT AGAR (MSA)
Opportunistis Infection
• Not regularly used but may purify
Staphylococcus epidermidis • Normal flora that cause disease
• More normal flora, increase cause of
infection

Little Normal Flora

Staphylococcus aureus
• Good bacteria

CHROM AGAR MRSA Intestinal Flora

• Causes poop to be brown
• If wala to, gray yung poop

Skin
• Pinakamadaming normal flora

ADDITIONAL FOR CULTIVATION Candida albicans

• 35°C in ambient air for 24 hours • Common in pregnant women
• May need 48-72 hours if MSA
Nose = non – sterile
Lungs = sterile

Endocarditis
• Inflammation of inner wall of the heart

Stomach
• None because acidic (walang may gusto)

Ileum
• Has normal flora and starts neutralizing pH

E.coli
• Normal flora in intestine (pinakamadami)
• Fecal contamination indicator
• It cause UTI (most common in female)

Pseudomonas aeruginosa
• Common

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 20

 • Bluish – green = with pseudomonas (urine, Antibiotic sensitivity testing
blood, etc.) • other name for novobiocin – susceptible
• Amoy rubber • To identify specie

Note: walang normal flora sa vagina Staphylococcus – Halophiles (gusto maalat)

Lactobacillus Phenylethyl alcohol and colistin – nalidixic acid

• Vaginal flora = neutral pH to Acidic = pampatay ng gram (-)
• Produces lactic acid
S. aureus – yellow halo pigment (after 18 – 24 hrs
Candida vaginitis incubation)
• Infection found in pregnant women with
gestational diabetes MSA – Kulay red before iluto

Culture and Sensitivity test S. epidermidis – Pink kahit gaano katagal i –

• 2 tier process incubate
• Most important test
• Main test Contaminated – tumubong colony na hindi
• Best time = before medicine or kakastart dinaanan ng loop during streaking
lang ng infection
Diameter – 6mm (standard size)
Culture
• 18 – 24 hrs
--------------- END OF LESSON 4. 1 -------------------
• “what bacteria?”
Goodluck, BFFs!! May we get all the
Sensitivity
things that we manifest!
• 18 – 24 hrs (approximately 3 days) Lets slay the Prelim exam, Mwa!!
• “what antibiotic is best?” DL Cutie!! Topnotch Cutie! High Scores Cutie!!
Traumatic (15 mins) - Blood, Amniotic Fluid,
Sinovial Fluid, Bone Marrow, Lamang Loob, Balat
(Skin)

2 hours - saliva, phlegm

ASAP - Bone; Suprapubic Aspirate

Bedside - Corneal Scrappings (Eyes)

Incubate - 18-24 hours

Temp - 35-37 degrees

After having the specimen:

I. Macroscopic - Physical Examination (may
uod ba, etc)
II. Microscopic - Kulayan organism (staining),
gram result, shape, and orientation (ex.
grape like cluster)
III. Culture (18-24 hours) – colony then
biochem (identify specie)
IV. Catalase Testing - Hydrogen Peroxide
(3%) Effervescence (bula) – staphylococcus
V. Sensitivity Testing

Gram positive cocci in grape like cluster

Aspirate is better than swab minimum of 2 (2

separate swab)
• 1 - slide
• 2 - culture media

Hydrogen Peroxide = 3% concentration

Skin swab / wound swab – specimen reaction
Mucus – specimen for mucosal membrane
Rabbit’s plasma – specimen for coagulase test

KEENO STA. MARIA & KYLE ABAD | BS MLS 2-Y2- 2 21