IMViC Test

Theory:

This is a group of tests used particularly in the identification of the family Enterobacteriaceae. This group consists of Escherichia coli, Salmonella sp, Shigella sp, Enterobacter sp, Proteus sp, Eruinia sp. 

IMViC test contains several tests such as;

• I = Indole test
• M = Methyl red test
• V = Voges Proskauer test
• C = Citrate utilization test

Indole test carried out in sim agar media;

Methyl red test carried out in MR-VP Broth;

Reaction with E.coli;

E.coli can change the pH of the medium into an amount of 4.

Reaction with E.aerogenes;

E.aerogenes can change the pH of the medium into an amount of 6.5.

Voges Proskauer test carried out in MR-VP Broth;

This changes the pH around 6.0.

Citrate test carried out in Simmons citrate medium;

Color change in Indole test is from pale yellow to red and methyl red test pale yellow to red. In Voges Proskauer test is from yellow to red and in citrate test, it is from green to blue.

Procedure:

Indole Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with tryptone broth in separate test tubes. Next, these were incubated at 37⁰C for 2-5 days. Finally, the indole was tested by Kovac’s method.

Kovac’s reagent was added carefully to the test medium dropwise to form a layer at the top of the medium. Finally, the tube was gently agitated with a rotary motion.

Kovac’s reagent: 5g of para dimethylamino benzaldehyde dissolved in a mixture of 75ml of amyl alcohol and 25ml of concentrated HCl.

Methyl red Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with MR-VP (dextrose phosphate medium) in separate test tubes. And this was incubated at 37⁰C for 48 hours. Finally, a few drops of an alcoholic solution of methyl red were added to the tube.

Voges Proskauer Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with MR-VP (dextrose phosphate medium) in separate test tubes. And this was incubated at 37⁰C for 2-5 days. In order to test Acetylmethylcarbinol, 0.5ml of naphthol solution (5% alcoholic) and 0.5ml of 40% KOH containing 0.3% creatine were added to about 3ml of the test medium. Next, the tubes were stoppered and shaken vigorously and were placed in a slanted position for 5-15 minutes. 

Citrate Utilization Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with Koser’s citrate medium. And Bromothymol blue was added as an indicator.

Observations:

	Test	Observation	Conclusion
	Indole Test	A red ring was formed on top of the peptone solution which was incubated with E.coli and the pale yellowish color of the solution remained the same. No such formation of a red ring has occurred in the broth which was incubated with E.aerogenes.	E.coli present.
	Methyl red Test	MR-VP medium with E.coli was formed a red ring on top of the solution and color of the solution remained the same. MR-VP medium with E.aerogenes has not formed a red ring.	E.coli present.
	Voges Proskauer Test	MR-VP medium with E.aerogenes turned the yellow solution into dark red whereas no specific change in MR-VP medium with E.coli.	E.aerogenes present.
Citrate Utilization Test		Koser’s citrate medium with E.aerogenes turned the green solution into blue whereas no change in color in Koser’s citrate medium with E.coli	E.aerogenes present.

Discussion:

In order to provide definite biochemical proof that the isolated organism is indeed E.coli a series of test, usually referred to as the IMViC tests are used. These can be used both to characterize E.coli and to differentiate it from Enterobacter aerogenes. Colonies from the completed test used to inoculate several different media. And this test can be used to identify other species in family Enterobacteriaceae.

A peptone medium rich in the amino acid tryptophan is inoculated and allowed to grow for 24 hours. E.coli makes the enzyme tryptophanase which forms indole, pyruvic acid, and ammonia from tryptophan. Since E.aerogenes cannot catabolize tryptophan, testing for the presence of indole differentiate these two organisms. Formation of red ring indicates the presence of E.coli.

P-dimethyl aminobenz-aldehyde     +    Indole ------> Cherry-red compound

Methyl red is an acid-base indicator that turns red in a slightly acidic medium. Thus if methyl red is added to a culture medium containing glucose in which an organism has grown for 18 to 24 hours, a red color will be observed which would indicate that organic acids had been formed as a result of fermentation of the glucose. E.coli forms large amounts of acids, which make methyl red positive. E.aerogenes on the other hand, carries out a 2,3-butylene glycol type of fermentation and thus produces only small amounts of organic acids which makes methyl red negative.

The Voges Proskauer test detects the presence of acetoin (Acetylmethylcarbinol), the immediate precursor of 2, 3-butylene glycol fermentation which is positive for E.aerogenes and negative for E.coli and the positive result is indicated by a pink ring.

Acetyl methyl carbonyl  +  Napthol ------>  Diacetyl +Guanidine (group of  peptone)

This reaction is carried out in the 40% KOH oxidation. The pink color is obtained both by Diacetyl and guanidine.

Citrate test merely determines whether the organism in question can grow using citrate as its sole source of carbon. Although both E.coli and E.aerogenes possess the necessary enzymes to metabolize citrate, only E.aerogenes can use it as a carbon source. E.coli cannot use citrate because citrate cannot enter the cells of E.coli. This test, therefore, determines the difference in the ability of these two organisms to transport citrate across their cell membranes.

                       Citrase

Citric acid -------> Oxalic acid + acetic acid + pyruvic acid + CO₂ excess

2CO₂ +  2 Na⁺  + 2 H₂O ----> Na₂CO₃ ----> Colour change from green to blue

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