Study Guide for Microbiology (Bio 6) Lab Quiz 2 Denise Lim

The second laboratory exam will have the same format as the first exam with a practical section
and a written section. It will be worth a total of 50 points.

A word of warning about using this study guide: much of the information in this guide is
duplicated in the handout "Diagnostic Tests for Identifying Bacteria". This guide contains ONLY
the information that will be covered by Lab Exam #2 and it also includes questions you may find
helpful in preparing for the exam.

Study tips:
The lab study guides that are provided are intended to help you focus your attention on the major
points covered in lab. They are not suitable replacements for lab attendance, note taking or
completing the required reading. Any material covered in lab may appear on lab quizzes,
including material not covered in this study guide.

Where appropriate draw yourself diagrams or flow charts. Flash cards with just one test per card
are also a useful memorization tool.

When you study, I suggest you use this study guide, together with your notes for each week
(both those downloaded from the web and your own notes) and the lab manual. The textbook is
also useful in some instances. Questions can cover any of the information in the required
reading.

Remember to review the background information, purpose, method, results and interpretation of
results for each experiment. If you need to look up definitions, there is a good glossary at the
back of your lab manual starting on page 619 and a good glossary at the back of your textbook
starting on page 913. Spelling counts.

Knowledge from the first section of the course will be assumed such as good laboratory practice
and safety procedures.

Bacterial Staining Techniques

Know the differences between a simple stain and a differential stain. The theory is explained on
pages 81 and 86 of your lab manual. Know the four steps in a differential stain and the specific
reagents used for each step in the three differential stains we performed (Gram stain, Endospore
stain and Acid-fast stain).

Acid-Fast Stain (Exercise 3-7)

What genus of bacteria is identified using the acid-fast stain? Name two members of this genus
and the diseases they cause.
Know the steps in performing an acid-fast stain, the reagents and the purpose of each reagent.
What component of the acid-fast cell walls gives a high affinity for the primary stain and ability
to resist decolorization?
Know how to interpret results.

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Endospore Stain (Exercise 3-9)
What is an endospore?
Why are endospores resistant to heat and chemicals?
What are the major genera that produce spores?
Know the steps in performing an endospore stain, the reagents and the purpose of each reagent.
Know how to interpret results (visually identify endospores and vegetative cells under a
microscope).

BIOCHEMICAL TESTS FOR IDENTIFYING BACTERIA

In week 7 we began to introduce selective and differential media that enable identification of
individual microbial species in pure cultures (page 107).

You must understand what is meant by the term selective medium and what is meant by the term
differential medium. You must know whether each of the different media we used were
selective, differential or both.

In a selective medium, an agent is included in the medium that inhibits the growth of unwanted
bacteria and encourages the growth of the species you want to examine. For a selective medium
you need to know what selective agent is included in the medium and what is selected for.

Differences between organisms can be determined using differential media that change
appearance if a particular biochemical reaction takes place. Differential media contain a substrate
that can be used by some species but not by others. In order to determine whether this substrate
has been used, the medium also contains an indicator that changes appearance when one or more
of the products are produced. The ability to use a particular substrate is dependent on whether a
species is capable of synthesizing the enzyme that catalyzes that particular reaction.

For a differential medium you need to know what substrate(s) is/are included in the medium,
what product(s) is/are produced if the species can use the substrate and what enzyme catalyzes
the reaction. You also need to know what indicator is used to determine whether the reaction
occurred and how to interpret the change in appearance.

Since many of these media are based on differences in ability to use various substrates for
fermentation it is important that you understand fermentation reactions (refer to your textbook if
necessary).

Be sure to read page 127 and the blue box on page 128 for helpful information on differential
tests.

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1. Mannitol Salts Agar (MSA - bright red agar plates): page 108-109.

This is a selective and differential medium.

High salt (7.5%) NaCl selects for Staphylococcus. Remember that you learned in your osmotic
pressure experiments that Staphylococcal species are halophiles.

MSA can differentiate between the pathogenic species S. aureus and nonpathogenic species S.
epidermidis, based on the ability to use mannitol as a substrate for fermentation.

What enzyme is present in mannitol fermenters?

What indicator is used in this agar?

How would you determine a positive reaction?

2. MacConkey Agar (dark red agar plate, sometimes brownish red if plate is old)
Page122-123.

This is a selective and differential medium.

Crystal violet inhibits gram positive organisms.

Like the EMB agar, this assay differentiates between lactose and non-lactose fermenters.

Bile salts and the pH indicator neutral red react with acid fermentation products to produce a
bright pink color.

E. coli is a lactose fermenting organism that produces greater quantities of acid than other lactose
fermenting bacteria. Because of this, it will produce dark pink, magenta colored colonies and
cause bile salt precipitation to produce a bright pink halo in the agar around the area of growth.
Other lactose fermenting bacteria will not react as dramatically and may produce pale pink or red
colonies, but may not turn the agar pink. Pink or red growth is therefore an indication that the
organism is positive for lactose fermentation. Non-lactose fermenting organisms will produce
white colonies and turn the agar a yellowish color.

3. Phenol Red Broths (red broth in tubes containing an inverted Durham tube) pages 131-
133.

These media are used to test for carbohydrate fermentation. These are differential but not
selective tests. This is one of a series of tests that help identify Gram negative rods, although it
can be used with other organisms as well. Expected results are included in the Table in the week
8 notes.

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If the medium contains glucose, the test will show if an organism is able to use fermentation as a
means of making ATP.

What is the pH indicator? What color does it turn if acid fermentation products are present?

What enzyme is present if lactose is used as a substrate for fermentation?

What enzyme is present if sucrose is used for fermentation?

Some organisms produce CO2 and H2 gas in addition to acids as fermentation end products. This
gas will be trapped in the inverted Durham tube if produced.

4. Catalase Activity (microscope slide), page 141-142.

This test is used to differentiate Streptococcus from Staphylococcus species.

If hydrogen peroxide (H2O2) is produced in bacteria during electron transfer it is highly toxic
and could kill the cell. Catalase enzymes are used to detoxify a cell of hydrogen peroxide.

To determine if bacteria contain catalase, a drop of hydrogen peroxide is placed on a clean

micoscope slide. A loopful of bacterial culture is smeared into the drop. If it bubbles, O2 is being
released indicating the organism is catalase positive. Even the generation of a small amount of
bubble formation is a positive results.

catalase enzyme
2H2O2 (hydrogen peroxide) ---------------------> 2H2O + O2 (free oxygen gas)

5. Oxidase Activity (test strip), page 143-144.

This test is used to differentiate enteric (facultative anaerobic) from non-enteric (aerobic)
gram negative bacteria.

Oxidases are enzymes involved in electron transport chain (ETC). Once the electron acceptors
in the ETC have been reduced by NADH or FADH2, they must be reoxidized to be readied for
the next round of electron transport. Cytochrome oxidase is an example of an enzyme that
catalyzes the oxidation these compounds.

We do not have an indicator to look at the steps of the ETC, so in this test we use an artificial
substrate, tetramethyl-p-phenylenediamine dihydrochloride (TPPD), to look for oxidation by
oxidase enzymes because it turns from colorless to purple when oxidized. This substrate is
embedded in the commercially prepared test strips. Lay the test strip on a piece of clean paper
towel and smear a small loopful of bacterial culture on the test strip. It will turn dark purple

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within 10 to 15 seconds if an oxidase is present. If a purple color appears after a longer period of
time, the result is negative. It is possible to test several culture samples on the same test strip.

cytochrome oxidase
reduced cytochrome + O2 ----------------> oxidized cytochrome + H2O

6. Exoenzyme Tests (starch hydrolysis, caseinase, gelatinase and lipase tests)

The following tests detect the presence of exoenzymes. Exoenzymes are enzymes that are
secreted into the surrounding medium and work on substrates found outside the cell. In general,
these exoenzymes are hydrolytic and break down large biomolecules that are too large to be
easily transported into the cell. These biomolecules must be broken down into their smaller
building blocks before they can be made available as a nutrient source for the cell. Starch must
broken down into glucose, protein into amino acids, and triglycerides into fatty acids and
glycerol.

a. Starch Hydrolysis (Agar Plates) page 161-162.

These plates look like a regular nutrient agar plate like TSA, but it contains high-molecular
weight starch molecules. If an organism produces the exoenzyme, amylase, a zone of clearing
will form around the area of growth when Gram's iodine is added to the plate. Iodine reacts with
starch to produce a dark brown/purplish color.

What is the substrate in this reaction?

What is the product?
What is the enzyme?
What is the indicator?

b. Caseinase Test (milk agar; opaque white agar plates), pages 169-170.

These plates contain casein, the major protein found in milk, which gives the plates the milky
color. If an organism produces the exoenzyme protease that digests the protein, a zone of
clearing will form around the area of growth.

What is the substrate in this reaction?

What is the product?
What is the enzyme?
What is the indicator?

c. Gelatin Hydrolysis (gelatin deeps), pages 171-172.

Gelatin is produced by boiling collagen (the major protein in bone and connective tissues) and
forms a solid matrix when allowed to cool, much like agar. Organisms that produce the enzyme
gelatinase can hydrolyze the protein into amino acids, which causes it to liquefy. After
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incubation of an inoculated tube, test for liquification by cooling the tube in the refrigerator. If
the medium resolidifies, the organism is negative for gelatinase. If the medium remains a liquid,
the organism is positive for gelatinase.

What is the substrate in this reaction?

What is the product?
What is the enzyme?
What is the indicator?

d. Lipase Test (Tributyrin Agar Plates: opaque, white), pages 175-176.

These plates contains the triglyceride tributyrin and test for lipid hydrolysis. The tributyrin
makes the plates an opaque white color. If an organism produces the exoenzyme lipase, a zone of
clearing will form around the area of growth.

What is the substrate in this reaction?

What is the product?
What is the enzyme?
What is the indicator?

7. Methyl Red/Voges-Proskauer Broth (MR-VP, yellow broth) pages 136-138.

One medium is used for two separate, but related tests:

a. The Methyl Red test detects organisms, such as Escherichia coli, capable of performing a
mixed acid fermentation. When glucose is fermented, products remain acid and produce a red
color in the presence of the pH indicator methyl-red (red at acid pH, yellow at neutral pH).

b. The Voges-Proskauer test detects organisms that have the ability to convert acidic
fermentation products into the neutral compound 2,3 butanediol acetylmethylcarbinol.
Enterobacter aerogenes is an example of one of these organisms.

The indicators are Barritt's solutions A & B. Barritt's solution A contains alpha-naphthol;
Barritt's solution B contains KOH; together they react with the acetylmethylcarbinol to produce a
deep rose color.

8. Nitrate Reduction Test (Trypticase nitrate broth): pages 145-148.

This medium is used to assay for the presence of enzymes capable of reducing nitrate to nitrite to
nitrogen gas:

NO3 (nitrate) ----> NO2 (nitrite) ----> N2 (nitrogen gas) or NH3 (ammonia)

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What enzyme must be present for conversion of nitrate to nitrite?

The indicators are Nitrate A and Nitrate B reagents. If medium turns red, then nitrate has been
reduced to nitrite.

However these indicators detect nitrite, so if all nitrite has been further reduced to nitrogen gas
and ammonia, no color would be detected.

To differentiate between a negative reaction (no reduction and no color change) and complete
reduction to nitrogen gas and ammonia (also no color change due to the absence of NO2), a
small amount of zinc dust is added. Zinc will non-enzymatically reduce any unused substrate,
NO3. Any NO2 produced by the zinc dust will react with the previously added indicator and turn
a red color. This means the bacteria had been unable to reduce nitrate and the result is negative.
If, on the other hand, there is still no color change after adding zinc, then the organism was able
to completely reduced nitrate to nitrogen gas and ammonia.

9. Simmons Citrate Agar Slants (green slants) pages 149-150

Simmons citrate agar is a medium used to test for the presence of the enzymes citrate permease,
which transports citrate into the cell, and citrase, which produces oxalacetic acid and acetate.
This test is used to distinguish certain gram negative rods. When an organism metabolizes
citrate during the Krebs cycle (also called the citric acid cycle), carbon dioxide is produced,
which is then converted to sodium carbonate which is alkaline (basic).

The indicator is bromthymol blue which changes from green to blue when the pH rises as a result
of sodium carbonate production.

10. Urease Test (pale orange slants) page 166-167

This medium contains the pH indicator phenol-red, which will turn bright magenta pink/purple
when alkaline in the presence of ammonia.

If the enzyme urease is present urea is broken down to ammonia resulting in a pH rise upon the
production of NH3 during the breakdown of urea by the urease enzyme. If a species cannot
produce the urease enzyme, the medium will remain orange or may have a slight pink tinge.

urease enzyme
CO(NH2)2 (urea) + 2H2O ----------------> CO2 + H2O + 2NH3 (ammonia)

11. SIM (Sulfur Indole Motility) agar deep tubes, pages 177-180.

This medium allows for the testing of three different characteristics: 1) H2S production, 2)
Indole production, and 3) Motility.
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a. H2S production: Cystein, an amino acid found in peptone, and sodium thiosulfate can both
be used as a substrate for H2S production, depending on the enzymes produced by an
organism. H2S gas is colorless and cannot be detected in this state. The H2S will react with
FeSO4 to produce a black precipitate. Black color is positive for H2S production because
FeSO4 is present in the SIM medium as an indicator.

b. Indole production: If an organism is able to use tryptophan as an energy source, then it will
produce the breakdown product indole, which can be detected by the addition of Kovac’s
reagent (p-dimethylamino-benzaldehyde, butanol, and HCl), as an indicator. If indole is
produced a red ring is formed.

c. Motility: Motile bacteria can swim through the low concentration of agar present in a SIM
tube. Turbid growth throughout the agar indicates motility. A line of growth restricted to the
line of the stab indicates no motility.

NOTE: The indole, MR-VP, and citrate tests are sometimes referred to collectively as the
IMViC tests and are used to identify Gram negative coliform species.

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