IMViC tests:

1. IMViC consists of a series of biochemical tests used to differentiate

isolates of bacterial species and to identify Enterobacteriaceae groups by
analysing biochemical properties and enzymatic reactions in the
presence of specific substrates.
1. I for Indole test,
2. M for methyl red test,
3. Vi for Voges Proskauer test and
4. C stands for citrate utilization test.
Indole production test:
1. Indole is an intracellular component present in both gram positive and
gram-negative bacteria useful in a number of bacterial processes like
spore formation and biofilm formation etc.
2. When bacteria containing the enzyme tryptophanase act upon the
amino acid tryptophan, Indole is obtained.
3. The test used to detect this indole is called the indole production test.
4. Let us take a protein-rich tryptone broth and inoculate with the test
bacteria.
5. If the bacteria contain tryptophanase enzyme, it hydrolyses the
tryptophan present in the broth and forms indole, pyruvic acid and
ammonium ions.
6. Further, these degraded products are treated with Kovac’s reagent
indicator in the presence of heat.
7. The appearance of a red ring on the top of the medium indicates that
the medium contains indole.
8. When formation of a red color ring at top of the tube then the given
organism contains tryptophanase enzyme indicating the Indole positive
test. 
9. Whereas, when formation of a slight yellowish ring at the top of tube
then the given organism lacks tryptophanase enzyme indicating indole
negative test.
Methyl red test:
1. This biochemical test is used to detect the ability of the bacteria to
ferment glucose into stable acids.
2. During fermentation, glucose is oxidized into pyruvate which is further
metabolized into any stable acid by a mixed acid pathway.
3. In both conversions, few bacteria play their metabolic roles and the end
product depends upon the bacterial species that are involved in the
mixed acid pathway.
 4. Now let's go through the test organism lactobacillus that undergoes
both fermentation and mixed acid pathway.
5. The test organism lactobacillus is inoculated into carbohydrate rich -
glucose phosphate broth and fermented at 35 °C for 48–72 hours.
6. In the first step glucose by consuming 2 ADP and 2 NAD is converted
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into pyruvate by releasing 2 ATP and 2 NADH+H molecules.

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7. In the next step, then the lactobacillus releases lactate dehydrogenase.

8. This enzyme helps the pyruvate to react with NADH+H resulting in the
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formation of lactic acid and NAD .+

9. To the resulting lactic acid, a methyl red indicator is added in the

presence of heat to determine the pH.
10.Since the formed lactic acid is acidic, it changes its color from yellow to
red.
11.The development of red color in the test tube indicates positive methyl
red test i.e., the presence of acid producing bacteria in the medium. 
12.If the color of solution in the test tube remains yellow color, indicates
negative methyl red test i.e., the absence of acid producing bacteria in
the medium. 

Voges Proskauer test:

1. This video contemplates how Voges Proskauer test detects enteric
bacteria that convert pyruvic acid to acetoin.
2. Some enterobacteria like Enterobacter aerogenes are inoculated into a
mixture of peptone and carbohydrate rich glucose phosphate broth.
3. This inoculated test tube is incubated at 37 C for 48 hours.
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4. During incubation, the glucose is converted into pyruvic acid, which is

later metabolized to acetoin by test bacteria like Enterobacter
aerogenes.
5. These bacteria contain an enzyme called diacetyl reductase which helps
in conversion of pyruvic acid to acetoin.
6. This produced acetoin is detected by adding VP reagent - 1 and VP
reagent - 2 where VP reagent -1 is 𝛼-naphthol and VP reagent - 2 is 40%
potassium hydroxide.
7. The acetoin in reaction with 𝛼-naphthol and 40% potassium hydroxide
forms diacetyl.
8. This diacetyl is condensed with guanidine of the peptone medium.
9. When methyl red indicator added to this medium results in a pinkish red
coloured complex.
 10.The development of pinkish red colour in the test tube indicates positive
Voges Proskauer test i.e. the broth contains acetoin producing enteric
bacteria.
11.If the colour of solution in the test tube remains yellow, indicates
negative Voges Proskauer test i.e., the absence of acetoin producing
enteric bacteria in the medium. 
 

Citrate utilization test: 

1. Citrate utilization test is used to detect the ability of the bacteria to
utilize the citrate as a source of energy and inorganic ammonium salts as
the source of nitrogen.
2. Let us take the sodium citrate agar medium in a test tube.
3. Add bromothymol blue indicator and inoculate with the test bacteria in
a slant position.
4. Incubate the test tube at 37 C for 18-48 hours.
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5. During incubation, the bacteria with the help of citrase enzyme converts
the citrate present in the medium into pyruvate with release of carbon
dioxide.
6. The excess sodium now reacts with the carbon dioxide and water to
form sodium carbonate which, being alkaline, changes the color of the
solution from green to Prussian blue.
7. The change in colour of the solution indicates the positive test as citrate
gets utilized.
8. No change in colour of the solution indicates the negative test as citrate
is not utilized.