IMVIc

Test
B.K.K.K.Jinad GS/M.Sc./FOOD/36
asa 08/08
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Introduction

The IMVIC tests are used to differentiate the enteric for many years. Identification of enteric
bacilli is of prime importance in controlling intestinal infections by preventing contamination of
food and water supplies. The groups of bacteria that can be found in the intestinal tract of
humans’and lower mammals are classified as member of the familyEnterobacteriaceae.

This family includes

1. Pathogens : e.g. Salmonella and Shigella

2. Occasional pathogens : e.g. Proteus and Klebsiella

3. Normal intestinal flora : e.g. Escherichia and Enterobacter

IMVIC represents the first letter of each individual test within the series:

1. Indole test

2. Methyl Red test

3. Voges-Proskauer Test

4. Citrate test

5.1. Indole Test

Tryptone is an essential amino acid that can undergo oxidation by the enzymes tryptophanase.
Some microorganisms can oxidize tryptophan and this can be used as a biochemical marker.

Tryptophanase

Tryptophan Indole + pyruvic acid + Ammonia

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The medium used for this test is tryptone broth. Indole is a component of the amino acid
tryptophan. When tryptophan is broken down (the presence of indol can be detected through the
use of Kovacs' reagent which is yellow, when reacts with indole and produces a red color on the
surface of the test tube.

Materials

Cultures : Bacterial cultures (24 to 48 hr)

Media : SIM agar or peptone water (Tubes with tryptone/peptone broth)

Reagent : Kovac’s reagent (p-dimethyl amino benzaldehyde)

Equipment : Bunsun burner, inoculating loop, Glassware marking pencil

Procedure

Three tubes of given media were inoculated with culture.

These media were incubated at 37°C for 48 hours.

Then 3 ml of Kovac’s reagent was added to each tube.

Results

Test sample Observation

Given bacterial culture Deep red Colour

Control Yellow
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Discussion

According to the above results it can be seen that E.coli can convert tryptopan in to indole. This
indicates the possible fecal contamination.

5.2. Methyl Red test

This test is used primarily to differentiate E.Coli from E.aerogenes and a qualitative test of acid
produced from oxidation of glucose

iE.Coli - produce large amounts of acid

ii .E.aerogenes - produces neutral or non-acidic end products.

In this test the bacteria that produce stable acid end products by means of mixed acid
fermentation of glucose is identified. All enteric microorganisms ferment glucose with the
production of organic acids, and the formation of acids is detected by the addition of methyl red.
Many gram-negative intestinal bacteria can be differentiated based on the products produced
when they ferment glucose in MR-VP medium Escherichia, Salmonella and Proteus ferment
glucose to produce lactic, acetic, auccinic and formic acids and CO2, H2 and Ethanol. The large
amounts of acids produced lower the pH of the medium. Methyl red (a pH indicator) will turn
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red when added to the medium if the organism was a mixed acid fermenter. Many of these
organisms also produce gas.

Materials

Culture : Bacterial cultures (24 - 48 hrs)

Media : Tubes with MR-VP broth (Methyl red-vogesProskaur)

Reagent : Methyl red indicator

Equipment : Bunsen burner, inoculating loop, glass ware marking pencil

Procedure

Three tubes of given media were inoculated with E.coli culture.

Tubes containing inoculated media were kept at 37°C for 48 hours.

After incubation few drops of methyl red was added each test tube.
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5.3. Voges-Proskaur Test

Vogus - Proskaur test determines the capability of some microorganisms to produce non acidic
or neutral end products such as acetyl methyl carbinol from the organic acid during the
glycolysis. The two tests (Vogus- Proskaur and methyl red) are generally considered together
because they can be performed on the same medium. Organisms those are negative in the methyl

Red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products
do not lower the pH as much as acids do. Enterobacter, Serratia and some species of Bacillus
produce these substances. There is no satisfactory test for determining production of 2, 3
butanediol. The reagent used in this test is Barrit’s reagent.

Materials

Cultures : Bacterial cultures (24 - 48 hr)

Media : Tubes with MR-VP broth.

Reagent : Barrit's reagent.

Equipment : Bunsen burner, Inoculating loop, glass ware marking pencil

Procedure

Three tubes of given media were taken and two of them were inoculated with E.coli culture.

Tubes were incubated at 37°C for 48 hours.

After the incubation period 15 drops from Barritt’s reagent A and 5 drops of Barritt’s reagent B
was added to each tube.

Results

Test sample Observation- MR Observations - VP

Given bacterial culture Deep red colour Copper colour
Control Yellow colour Copper colour
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Discussion

Negative results have been obtained for the test. This indicates the absence of acetoin producing
bacteria.

5.4. Citrate utilization test (Simmon" Citrate slant)

When fermentable glucose or lactose is absent in the presence of citrate permease, some
microorganisms are capable of using citrate as carbon source for their energy. The citrate
utilization test is used to determine the ability of an organism, using the enzyme citrate permease
to use citrate as its sole carbon source. Citrate agar is a medium containing sodium citrate as the
sole carbon source and the ammonium ion as the sole nitrogen source. An indicator
bromothymol blue is added to the medium, which changes colour based on pH, and will turn
from green at neutral pH (6.9) to blue when pH higher than 7.6 is reached (basic or alkaline).

Materials

Culture : Bacterial cultures.

Media :Simmon’s citrate agar slant (slope).

Indicator : Bromothymol blue

Equipment : Bunsen burner, inoculating loop, glassware marking pencil

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Procedure

Three tubes of given media were taken and two of them inoculated with E.coli culture.

Inoculated media were incubated at 37°C for 48 hours. After 48 hours colour change was
observed.

Results

Test sample Observation

Given bacterial culture Green

Control Green

Conclusion

Test sample Indole Methyl Red VP Citrate Microorganism

present

Given + + - - E.Coli
Bacterial
Culture