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General Microbiology Laboratory
Biochemical tests
Tests To Know
Case Study Tests
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Methyl Red/ Voges Proskauer
Indole
Citrate
H2S production in SIM
Urea hydrolysis
Motility
Lactose fermentation
Sucrose fermentation
Glucose fermentation & gas production
Mohammed Laqqan
Introduction
 Microbial metabolic processes are complex, but they permit
the microbiologist to distinguish among microorganisms
grown in culture. Many clinical pathogens can be identified by
inoculating pure cultures into media that contain one or more
specific biochemicals.
 The biochemical reactions that take place can then be
determined by relatively simple indicator reagents, included in
the medium or added to the culture later.
 Some bacteria ferment carbohydrates, producing acidic,
alcoholic, or gaseous end products.
 Many different species are distinguished on the basis of the
carbohydrates they do or do not attack, as well as by the nature
of end products formed during fermentation.
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The nature of products formed in amino acid
metabolism also provides information as to the
identification of bacterial species.
The production of visible pigments distinguishes
certain types of bacteria (pseudomonas, serratia).
Working with pure cultures freshly isolated from
clinical specimens, the microbiologist uses a carefully
selected battery of special media to identify their
outstanding biochemical properties.
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Fermentation of carbohydrates
 Carbohydrates are complex chemical substrates which serve as
energy sources when broken down by bacteria and other cells.
They are composed of carbon, hydrogen, and oxygen (with
hydrogen and oxygen being in the same ratio as water;
[CH2O]) and are usually classed as either sugars or starches.
 Facultative anaerobic and anaerobic bacteria are capable of
fermentation, an anaerobic process during which
carbohydrates are broken down for energy production.
 A wide variety of carbohydrates may be fermented by various
bacteria in order to obtain energy and the types of
carbohydrates which are fermented by a specific organism can
serve as a diagnostic tool for the identification of that
organism.
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 We can detect whether a specific carbohydrate is
fermented by looking for common end products of
fermentation. When carbohydrates are fermented as
a result of bacterial enzymes, the following
fermentation end products may be produced:
1. Acid end products.
2. Acid and gas end products.
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Carbohydrate Utilization
 Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate
utilization tests are designed to detect the change in pH which would occur if fermentation of
the given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH
indicator (phenol red) to turn yellow. If the bacteria do not ferment the carbohydrate then the
media remains red. If gas is produced as a by product of fermentation, then the Durham tube
will have a bubble in it.
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The carbohydrate tests are the:
Glucose (Dextrose) test
Lactose test
Sucrose test
All carbohydrate test media should be inoculated with the transfer loop.
Left tube shows less acid formation than far right
tube, but gas is still made
Center shows no carbohydrate utilization to
produce acid or gas.
Right tube shows acid was produced as
evidenced by the yellow color, and gas was made
(look at the bubble in the Durham tube)
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MR-VP tests
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This test is used to determine two things.
The MR portion (methyl red) is used to determine if glucose can be
converted to acidic products like lactate, acetate, and formate.
The VP portion is used to determine if glucose can be converted to
acetoin.
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These fermentation tests are used to differentiate between certain
intestinal bacteria called coliforms.
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The medium contains dextrose as the carbohydrate source. Some
coliforms will ferment the dextrose to acid products that will cause the
pH to drop below pH 5. This is called mixed acid fermentation. After
incubation the addition of methyl red, a pH indicator which turns red
below pH 4.4, will indicate whether such fermentation has occurred.
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Other coliforms will convert dextrose to less acidic products such as
ethanol or butanediol. These bacteria are negative in the methyl red test.
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 Butanediol fermentation is demonstrated by the VogesProskauer test which measures the presence of acetoin (acetyl
methyl carbinol), a precursor to butanediol.
 This test uses the same medium as the methyl red test and both
tests are usually performed in parallel.
 Barritt's reagents, alpha-naphthol and potassium hydroxide, are
added to a 48 hour culture and the tube is shaken to aerate the
solution. The development of a pink or red color after
agitation is a positive reaction for the production of acetoin.
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 How to Perform Tests: Inoculate 2 MR-VP broths with inoculating
loop. One tube of culture will be used to conduct the MR test, the second
tube serves as the VP test. After 48 hours of incubation, add a few drops
of MR to one tube, and VP reagents to the other tube.
 Properties they test for: Both tests are used to help differentiate
species of the family Enterobacteriaceae.
 MR—tests for acid end products from glucose fermentation.
 VP—tests for acetoin production from glucose fermentation.
 Media and Reagents Used:
 MR-VP Broth
 Methyl Red indicator for acid
 Voges Proskauer reagents—A: 5% Alpha-Naphthol,
B: Potassium Hydroxide,
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Results
 MR (methyl red) test: Methyl red is added to the MR tube. A red color
indicates a positive result (glucose can be converted into acidic end
products (indicating pH below 6) . A yellow color indicates a negative
result, glucose is converted into neutral end products (indicating no
acid production)
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 VP (Vogues Proskauer) test: First alpha-napthol (also called Barritt’s
reagent A) and then potassium hydroxide (also called Barritt’s reagent B)
are added to the VP tube. The culture should be allowed to sit for about 15
minutes for color development to occur. If acetoin was produced then the
culture turns a red color (positive result); if acetoin was not produced then
the culture appears yellowish to copper in color (a negative result).
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End of lecture
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