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Study Guide for Microbiology (Bio 6)
Denise Lim
Lab Final
The last laboratory exam will be 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. YOU WILL NOT BE ALLOWED A 3X5 CARD FOR THE FINAL.
This lab study guide is intended to help you focus your attention on the major points covered in
lab. It is not a suitable replacement 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.
The following is a list of all the lab exercises that were covered after the first exam. Remember
that the Final Lab Exam is comprehensive.
Ex. 5-4: Methyl Red and Voges-Proskauer Tests
Ex. 5-8: Citrate Test
Ex. 5-13: Urea Hydrolysis Test
Ex. 5-20: SIM Medium
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
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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
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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/application 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.
In this Study Guide, the tests are grouped by application, not chronologically by week.
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TESTS FOR GRAM NEGATIVE ENTERICS
NOTE: The indole, MR-VP, and citrate tests are sometimes referred to collectively as the
IMViC tests and are used to identify Gram negative enteric organisms, otherwise known as
coliforms.
1. Phenol Red Broths (Ex. 5-3: red broth in tubes containing an inverted Durham tube)
See SPECIALIZED TESTS from the 1st Exam
2.
MacConkey Agar – Ex. 4-5; See SPECIALIZED TESTS from the 1st Exam
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.
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.
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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)
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.
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.
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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.
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.
4.
Novobiocin Sensitivity Test (Ex. 5-24; Note: This is a Kirby-Bauer antibiotic sensitivity
test, not a biochemical test)
How is sensitivity or resistance determined?
Which organisms are sensitive to Novobiocin? Which are resistant?
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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.
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.
-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
-hemolysis (beta): the RBC is completely broken down and there is a colorless, clear,
sharply defined zone of hemolysis surrounding colonies. eg. S. pyogenes
-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.
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.
4.
Bacitracin Sensitivity Test (Ex. 5-24; Note: This is a Kirby-Bauer antibiotic sensitivity
test, not a biochemical test)
How does using blood agar change the appearance of this Kirby-Bauer test? Which
Streptococcal species is sensitive to Bacitracin? Which are resistant?
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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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