Study Guide for Microbiology (Bio 6)
Denise Lim
Lab Exam 2 and the Lab Final
The second laboratory exam will consist of approximately 25 practical questions and 25 short
answer/essay questions. It will be worth a total of 50 points. The Lab Final is comprehensive and
will include material from the entire semester. The Lab Final will consist of 50 practical
questions only. There will be no essay questions on the final.
This lab study guide is intended to help you focus your attention on the major points covered in
lab. It is not suitable replacements for lab attendance, note taking or completing the required
reading. Any material covered in lab may appear on the lab exam, including material not covered
in this study guide.
Study tips:
Where appropriate draw yourself diagrams or flow charts. Flash cards with just one test per card
are also a useful memorization tool. Group the biochemical tests by the type of organism it used
to identify.
When you study, I suggest you use this study guide together with the lab notes for each exercise
(both those downloaded from the course website 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, application, 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 and a good glossary at the back of your textbook.
Material for Exam #2
The following is a list of all the lab exercises that will be included in Lab Exam #2.
Ex. 3-8: Acid Fast Staining
Ex. 3-10: Endospore Staining
Ex. 4-4: Mannitol Salts Agar
Ex. 4-5: MacConkey Agar
Ex. 5-3: Phenol Red Broth
Ex. 5-5: Catalase Test
Ex. 5-6: Oxidase Test
Ex. 5-12: Starch Hydrolysis
Ex. 5-14: Casein Hydrolysis Test
Ex. 5-15: Gelatin Hydrolysis Test
Ex. 5-17: Lipid Hydrolysis test
Ex. 5-4: Methyl Red and Voges-Proskauer Tests
Ex. 5-7: Nitrate Reduction Test
Ex. 5-8: Citrate Test
Ex. 5-13: Urea Hydrolysis Test
Ex. 5-20: SIM Medium
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Material for the Final Exam
In addition to the material from Lab Exam #1 and Exam #2, the following exercises will be
included on the comprehensive final exam:
Ex. 4-4: Mannitol Salts Agar
Ex. 5-16: DNA Hydrolysis Test
Ex. 5-27: Coagulase Test
*Ex. 5-24: Novobiocin Test
Ex. 4-3: Bile Esculin Test
*Ex. 5-24: Bacitracin Test
Ex. 5-25: Blood Agar
†NaCl Broth
Staphylococcus & Streptococcus Quick Tests
Ex. 5-30: Enteropluri Tubes
****************************************************************************
Following are helpful study questions or information for each exercise. The questions are
organized by how the tests would be used together if an unknown organism were to be
identified, so you may need to skip around to find all the material you need for the second
exam.
The final lab exam will be cumulative, so you should review everything we have covered
both the first and second exams, as well as any additional exercises
****************************************************************************
I. STAINING
Know the differences between a simple stain and a differential stain. The theory is explained in
your lab manual. Know the specific reagents used for each step and the effect each reagent has
on a specimen for both Acid Fast staining and Endospore staining.
Acid-Fast Stain (Exercise 3-7)
What genus of bacteria is identified using the acid-fast stain? Name two species of this genus
and the diseases they cause. Know the difference between the species name and the disease
name.
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.
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? Be able to name at least two diseases caused by
spore forming organisms.
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Know the steps in performing an endospore stain, the reagents and the purpose of each reagent.
Note that we used the procedure described on page 99 of the lab manual.
Know how to interpret results (visually identify endospores and vegetative cells under a
microscope).
II. SOME GENERAL CONCEPTS FOR BIOCHEMICAL TESTS FOR IDENTIFYING
BACTERIA
Biochemical tests allow us to identify when a particular chemical reaction occurs in a cell. In
order to observe the occurrence of a biochemical reaction, some visible change must occur. An
indicator is anything that changes appearance when a biochemical reaction has taken place.
Indicators include color changes, zones of clearing that may form around an area of growth,
bubbling, or any other changes in the growth medium.
Understand what is meant by the term selective medium and what is meant by the term
differential medium. Know whether each of the different media we used were selective,
differential or both. Don't forget that media is used to grow organisms and that all of these tests
tell us something about some aspect of metabolism. The catalase and oxidase tests are not
growth media because the organisms are grown on TSA before the indicator is applied to the
organism.
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 characteristic is
selected for.
Differential media simply change appearance if a particular biochemical reaction takes place
because of the indicators we have already discussed. Differential media contain a substrate that
can be used by an organism only if it produces the enzymes required to catalyze that particular
biochemical reaction. Sometimes the indicator is already present in the medium, sometimes the
indicator may need to be added, and sometimes the indicator is a change in the media itself after
the reaction occurred.
For each biochemical reaction you need to know:
1) The chemical reaction: substrate ! product.
2) The enzyme that catalyzes the reaction.
3) The indicator that is used to determine whether the reaction occurred.
4) How to interpret the change in appearance.
5) The appropriate use for the test.
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).
NOTE: The Kirby-Bauer Novobiocin and Bacitracin tests used for Staphylococcus and
Streptococcus identification are antibiotic sensitivity tests, NOT biochemical tests.
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III. TESTS GROUPED BY APPLICATION
A.
TESTS TO BE USED ONCE THE GRAM REACTION IS KNOWN
1.
Catalase Activity (Ex. 5-5): Note that this test can be performed on a microscope slide
or directly on an agar medium
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. The culture should not be more than 48 hours old.
catalase enzyme
2H2O2 (hydrogen peroxide) ---------------------> 2H2O + O2 (free oxygen gas)
2.
Oxidase Activity (Ex. 5-6: OxiStrips)
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 called OxiStrips. Put the test strip in an
empty petri dish and smear a small loopful of bacterial culture on the test strip. It will turn dark
purple 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. The culture should not be more that 48 hours old.
cytochrome oxidase
reduced cytochrome + O2 ----------------> oxidized cytochrome + H2O
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B.
1.
SPECIALIZED TESTS
Mannitol Salts Agar (Ex. 4-4: MSA - bright red agar plates)
This medium is both selective and differential.
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.
saprophyticus and S. epidermidis, based on the ability to use mannitol as a substrate for
fermentation.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
2. MacConkey Agar(Ex. 4-5: dark red agar plate, sometimes brownish red if plate is old)
This medium is both selective and differential.
Bile salts and crystal violet inhibit the growth of Gram positive organisms.
This test 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.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
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NOTE: The casease, gelatinase, lipase, Bile Esculin Agar (BEA) and DNase tests all test for
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.
C. TESTS USED TO IDENTIFY GRAM NEGATIVE NON-ENTERICS
1. Casein Hydolysis Test (Ex. 5-15: milk agar; opaque white agar plates)
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?
2. Gelatin Hydrolysis (5-15: gelatin deeps)
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
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?
3. Lipase Test (5-17: Tributyrin Agar Plates: opaque, white)
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?
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4. Nitrate Reduction Test (5-7: Trypticase nitrate broth)
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)
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.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
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D. TESTS FOR GRAM NEGATIVE ENTERICS
1. Phenol Red Broths (Ex. 5-3: red broth in tubes containing an inverted Durham tube)
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.
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.
2.
MacConkey Agar – Ex. 4-5; See SPECIALIZED TESTS above
3.
Methyl Red/Voges-Proskauer Broth (Ex. 5-4: MR-VP, yellow broth).
One medium is used for two separate, but related tests. The two tests distinguish between
different fermentation products. For these tests, it is sufficient to identify the enzymes simply as
fermentation enzymes.
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.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
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4. Simmons Citrate Agar Slants (Ex. 5-8: green slants)
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 begins the breakdown of citrate in a
series of complex reactions that result in an alkaline pH. For this test, it is sufficient to know that
the product is alkaline. The indicator is bromthymol blue which changes from green to blue
when the pH rises.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
5. Urease Test (Ex. 5-13: pale orange slants)
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)
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
6. SIM = Sulfur Indole Motility (Ex. 5-20: agar deep tubes)
This medium allows for the testing of three different characteristics: 1) H2S production, 2)
Indole production, and 3) Motility.
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.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the 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
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reagent (p-dimethylamino-benzaldehyde, butanol, and HCl), as an indicator. If indole is
produced a red ring is formed.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
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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E. TESTS FOR GRAM POSITIVE STAPHYLOCOCCUS
1.
Mannitol Salts Agar – (Ex. 4-4) See under SPECIALIZED TESTS above
2.
DNase test (Ex. 5-16: uncolored agar plate):
The presence of the exoenzyme DNase is also confirmatory for S. aureus. The agar medium
contains DNA. After the culture has been allowed to grow, HCl is added to the plate (similar
to iodine in the starch test). HCl will cause intact DNA to precipitate and produce a cloudy
white color in the agar, unless the DNA has been digested by the DNase enzyme. The
hydrolysis of DNA into single nucleotides will produce a clearing in the agar surrounding
the area of growth.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
3.
Coagulase test (5-27: small plastic tube of rabbit plasma):
The coagulase enzyme converts fibrinogen into fibrin within the tissues of a host organism.
This enhances the pathogencity of a microorganism by walling off the site of infection and
protecting the pathogen from the host immune system. Solidification of the rabbit plasma to
a fibrin clot at the bottom of the tube indicates the presence of the coagulase enzyme.
This test is used to differentiate between Staphylococcus species. A positive test identifies
pathogenic S. aureus. Even though fermentation of mannitol on an MSA plate can also
indicate S. aureus, variants of other species of Staphylococcus have been known to ferment
mannitol as well. Identification of S. aureus should always be confirmed with the coagulase
test.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
4.
Novobiocin Sensitivity Test (Ex. 5-24; Note: This is a Kirby-Bauer antibiotic sensitivity
test, not a biochemical test)
Which organisms are sensitive to Novobiocin? Which are resistant?
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F. TESTS FOR GRAM POSITIVE STREPTOCOCCUS
1.
Bile Esculin Agar - BEA (Ex. 4-3: beige agar plate):
These plates look like a regular nutrient agar plate like TSA, but contain bile, the glycoside
esculin, and iron salts as an indicator. Group D Streptococcus sp. will use the enzyme
esculinase to convert the esculin to 6,7-dihydroxy-coumarin, which reacts with the iron
salts, producing a black color in the agar. Non-Group D Streptococcus sp. do not produce
the black color.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
2.
Hemolysin production (Ex. 5-25: blood agar plates also called BAP):
Important ingredients: red blood cells
This medium is a differential medium used to distinguish species of Streptococcus and
contains 5% sheep's blood. Some species produce proteins called hemolysins. These
proteins can lyse erythrocytes (red blood cells), releasing hemoglobin. Inoculate the plate
with a single streak and incubate at 35°C for 24 to 48 hours.
α-hemolysis (alpha): only the hemoglobin in the RBC is broken down (partial or
incomplete hemolysis). The medium surrounding the colony is cloudy and discolored, turns
green. eg. S. pneumoniae, S. salivarius
β-hemolysis (beta): the RBC is completely broken down and there is a colorless, clear,
sharply defined zone of hemolysis surrounding colonies. eg. S. pyogenes
γ-hemolysis (gamma): no affect on RBC, no lysis. S. faecalis is non-hemolytic after 24
hour incubation, but may become a-hemolytic after a longer incubation.
What is the substrate in each of these reactions?
What is the product for each reaction?
What is the enzyme for each reaction?
What is the indicator for each reaction?
3.
6.5% NaCl broth (Not in the lab manual; purple broth):
The Group D Streptococci can be separated into enterococci (eg. Enterococci faecalis) and
non-enterococci (eg. Streptococcus bovis). Only the enterococci are able to grow in the
presence of high salt. This high salt broth contains a pH indicator that will turn from purple
to yellow if the microorganism can grow.
What is the substrate in this reaction?
What is the product?
What is the enzyme?
What is the indicator?
4.
Bacitracin Sensitivity Test (Ex. 5-24; Note: This is a Kirby-Bauer antibiotic sensitivity
test, not a biochemical test)
Which Streptococcal species is sensitive to Bacitracin? Which are resistant?
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G. RAPID ID TESTS
Know which species of organism is specifically identified by the Staph and Strep tests. Be
able to interpret each test to identify an unknown organism. Be able to explain the
technology or principle that each rapid test is based on.
1.
Staphylococcus Rapid ID Agglutination test – Latex bead agglutination test.
2.
Streptococcus Rapid ID ELISA test – Antibody-antigen-antibody sandwich attached to
the test strip.
3.
Ex. 5-30: Enteropluri Tube (also know as an Enterotube) – A twelve-chambered tube
containing biochemical media used in combination to produce an identification number that
can be looked up in the reference book.
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