Download Biochemical Test By: Mahmoud W El

General Microbiology
Laboratory
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
The ability to degrade amino acids to
identifiable end products is often used to
differentiate among bacteria.

Tryptophan, for example, is hydrolyzed to
indole, pyruvic acid and ammonia by
tryptophanase.
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The pyruvic acid can be further metabolized
to produce large amounts of energy.
The ammonia is available for use in synthesis
of new amino acids.
Indole can be detected by reaction with
Kovac's
reagent
(Paradimethylaminobenzaldehyde in alcohol) to
produce a red color.
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POSITIVE CONTROL:
E.coli.
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NEGATIVE CONTROL:
Enterobacter aerogenes
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Trypton Broth 1%.
Kovac’s Reagent.
Test Tube.
Culture from bacteria.
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1. Inoculate tryptone broth (1%) with the
organism.
2. Incubate at 37 oC for 48 hours.
3. Add 10 drops of Kovac's reagent. A red
color in the alcohol (upper) layer is a positive
result.
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
MR-VP medium/broth is two tests in one, the
Methyl Red Test and the Voges-Proskauer test.

It is very useful in separating members of the family
Enterobacteriaceae and some Streptococcus.

Most members of the Enterobacteriaceae give either
a positive MR or a positive VP test.

Several species are positive for both, such as
Proteus mirabilis.
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Principle
 This is to detect the ability of an organism to
produce and maintain stable acid end
products from glucose fermentation.
 Some bacteria produce large amounts of
acids from glucose fermentation that they
overcome the buffering action of the system.
 Methyl Red is a pH indicator, which remains
red in color at a pH of 4.4 or less.
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IMViC test
Methyl Red-Voges Proskauer (MR-VP) Tests
Principle
Glucose
Acidic pathway
Or
Acety methyl carbinol
(ACETOIN)
Mixed acids
 pH less than 4.4
Barrit’s A
Barrit’s B
Methyl Red
indicator
Red color
Neutral pathway
MR positive
E. coli
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VP positive
Klebsiella
Pink color
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
Positive Control: E.Coli
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Negative Control:
Klebsiella pneumoniae
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1. The tubes of MR-VP broth were inoculated
using sterile technique with given bacterial
culture.
2. One tube was served as control.
3. Incubate at 37 0 C for 24-48 hours.
4. Test the presence of mixed acids by the
addition of methyl red indicator.
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Principle
 VP test detects butylene glycol producers.
 Acetyl-methyl carbinol (acetoin) is an
intermediate in the production of butylene
glycol.
 In this test two reagents, 40% KOH and
alpha-naphthol are added to test broth after
incubation and exposed to atmospheric
oxygen.
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
If acetoin is present, it is oxidized in the
presence of air and KOH to diacetyl.

Diacetyl then reacts with guanidine
components of peptone, in the presence of
alpha naphthol to produce red color.

Role of alpha-naphthol is that of a catalyst
and a color intensifier.
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Positive Control:
Klebsiella pneumoniae
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Negative Control:
E.Coli
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1. The tubes of MR-VP broth were inoculated
using sterile technique withgiven bacterial
culture.
2. One tube was served as control.
3. Incubate at 37 0 C for 24-48 hours.
4. Test the presence of acetyl-methyl carbinol
by the addition of Barrit’s reagent.
5. Shacked well and allowed for 15 minutes.
The presence of rose colouration is a positive
result
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 IMVC test
Indole Methyl Red-Voges Proskauer (MR-VP) and
Citrate Tests.
 This Four test used to differentiation between of
Enterobacteriacea member.
Name test
I
M
V
C
E. Coli
+
+
-
-
Enterobacter
-
-
+
+
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GOOD LUCK
GIRLS