The Enterobacteriaceae is a family of non-spore-forming Gram-negative bacilli. They are widely distributed in soil, water and plants. The bacteria belonging to this family are normally present in the gastrointestinal tract of humans and animals and are among the most significant pathogens. The family includes some important genera like Escherichia, Enterobacter, Klebsiella, Salmonella, Shigella, Citrobacter and Yersinia.

The IMViC tests are a group of biochemical tests used to identify the members of the Enterobacteriaceae family. IMViC is an abbreviation for the four tests, namely;

I: Indole test

M: Methyl red test.

V: Voges- Proskauer test

i (added for the ease of pronounciation)

C: Citrate test.

Let’s discuss about each of these test:

• Indole test:

In this test, the pure culture of the test bacteria is incubated for 24 hrs at 37°C into Tryptophan broth medium. This media contains tryptophan as a source of protein. The bacterial cells which are able to produce tryptophanase enzyme, can convert tryptophan to indole and other byproducts.

Fig 1: A represents positive indole test result and B negative result.

Hence if the bacteria can utilize tryptophan, the broth will contain indole. Indole dissolves in xylene, therefore on addition of xylene to the broth indole gets extracted and concentrates in the form of a immiscible ring as a top layer in the test tube (fig 1). Then the broth is treated with Kovac’s reagent or Ehrlich’s reagent, which contains para-dimethylaminobenzaldehyde (p-DMAB), in presence of heat. Indole reacts with these reagents to give a pink or red colouration.

Therefore, if the broth containing the pure culture of certain bacteria turns the red or pink ring, it confirms that the bacteria can utilize tryptophan and the test is said to be positive (fig 1A). No ring or a yellow ring formation means the test is negative and the bacteria is unable to utilize tryptophan (fig 1B).

E. coli is indole positive and Enterobacter aerogenes (also known as Klebsiella aerogenes) is indole negative (fig 5)

• Methyl Red Test:

The micro-organisms from Enterobacteriaceae family carry out fermentation of the sugars in the media, of two different types. One type of fermentation called as ‘mixed acid fermentation‘ produces acidic products and the the other type known as butanediol fermentation produces butanediol and other neutral products.

Fig 2: A. represents methyl red negative result and B. methyl red positive.

The media used in this test is glucose phosphate broth, which contains buffered peptone water. The bacteria carrying out mixed acid fermentation will utilise glucose and convert it into pyruvate, and later to acids like lactate, acetate, succinate, formate, ethanol and the gases H2 and CO2. These acids make the broth acidic. The ratio of each acid may vary among the different bacterial species.

For this test the pure culture of test organism is incubated in the glucose phosphate broth for 24 hours at 37°C. This incubated broth can be divided into two parts and used for both the methyl red test and Voges-Proskauer (V-P) test.

In case of methyl red test, the pH indicator, methyl red is added to the broth, which turns red at acidic conditions (pH 4.4) and yellow at around pH 6.2.

Hence if the test bacteria is capable of carrying out mixed acid fermentation, the broth becomes acidic and turns red, that is the methyl red test is positive (fig 2B). If the test bacteria unable to carry out mixed acid fermentation, the broth will remain yellow, that is negative (fig 2A).

E. coli is methyl red positive, that it carries out mixed acid fermentation, and Enterobacter aerogenes (also known as Klebsiella aerogenes) is methyl red negative (fig 5)

Voges-Proskauer (V-P) Test:

As already mentioned the members of Enterbacteriaceace family can carry out mixed acid fermentation or butanediol fermentation. Methyl red is used to detect the mixed acid fermentation, wherein the broth turns very acidic. Voges-Proskauer test is used to detect the butanediol fermentation, having neutral products. The Voges-Proskauer test is also carried using the glucose phosphate broth. Alpha-naphthol and potassium hydroxide added to the broth incubated with the pure culture of the test organism.

Fig 3: A represents Voges-Proskauer positive and B. Voges-Proskauer negative.

During the butanediol fermentation, one of the intermediate compound formed is acetoin. Acetoin is converted to diacetyl on addition of alpha-naphthol and KOH solution. The diacetyl produced then reacts with the guanidine nucleus of arginine present in the peptone from the broth to give a pink colouration.

Fig 4: The flowchart for VP test.

Thus the formation of pink colour indicates the presence of acetoin and confirms bacterial Butanediol fermentation and indicates VP test as positive (fig 3A). The absence of the pink colouration (fig 3B) signifies that the test bacteria is unable to carry out butanediol fermentation.

E. coli is VP test negative and Enterobacter aerogenes (also known as Klebsiella aerogenes) is VP test positive (fig 5).

Citrate Utilization Test:

Some members of the Enterobacteriaceae family can utilize citrate as source of carbon and thus energy. This characteristic is used to identify these organisms using Simmons’ citrate agar. Simmons’ citrate agar contains citrate as a sole source of carbon. This media also contains, ammonium phosphate as a source of nitrogen and bromothymol blue as a pH indicator which is yellow at acidic pH and turns blue at alkaline pH.

Fig 5: The positive and negative Citrate test results.

When the bacteria utilizes citrate, it liberates CO2, which in turn reacts with water and sodium to form sodium carbonate. Also as mentioned earlier, the media also contains ammonium salt as sole nitrogen source, which is metabolised by the bacteria to release ammonia gas. The production of both sodium carbonate and ammonia causes the pH of the media to increase. As the media turns alkaline, bromothymol blue present in the media turns blue.

Hence if the bacteria turns the Simmons’ citrate agar blue from green, it is able to bring about butanediol fermentation and helps identify the organism.

E. coli is citrate test negative and Enterobacter aerogenes (also known as Klebsiella aerogenes) is citrate test positive (fig 5).

The cumulative results of these four tests along with other tests can help identify bacteria, depending on their ability to utilize tryptophan, citrate and carry out fermentation. The table (adapted) below shows the results of IMViC tests carried out in few bacteria belonging to Enterobacteraceae family. Other tests like Motility Test, Triple Sugar Iron (TSI) Agar Test, Urease Test should also be performed to give more information.

Table 1: List of test results of few members of Enterobacteriaceae family

(Just for info: Have a look at this paper which involves isolation and identification of bacteria form fish sample)

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Krieg & Padgett (2011) 3 – Phenotypic and Physiological Characterization Methods. Methods in Microbiology 38: 15-60.

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Fig 5: Malviya (2012). Ecofriendly treatment of axons dyes: biodecorization using bacterial strains isolated from textile wastewater. 10.13140/RG.2.1.3214.6083.