IMViC Test; Indole Production Test

 

IMViC test

The groups of bacteria that can be found in the intestinal tract of humans and lower mammals are classified as members of the family Enterobacteriaceae.  They are short, gram negative, non spore forming bacilli.  Members included in this family are:

1.       Pathogens such as members of the genera Salmonalla and Shigella.

2.       Occasional Pathogens such as members of the genera Proteus and Klebsiella.

3.       Normal intestinal flora such as members of the genera Escherichia and Enterobacter,      which are saprophytic inhabitants of the intestinal tract.

Differentiation of the principal groups of Enterobacteriaceae can be accomplished on the basis of their biochemical properties and enzymatic reactions in the presence of specific substrates.  For this purpose, IMViC series of tests can be used.  The IMViC tests consist of four different tests: (i) Indole production (ii) Methyl-red (iii) Voges-Proskauer and (iv) Citrate utilization test.  The name IMViC stands for the first letter of the name of each test in the series, with the lower case ‘i’ included for ease of pronunciation.

  

Indole production test

Aim

To determine the ability of microorganisms to degrade the aminoacid tryptophan.

Principle

Tryptophan, an essential aminoacid, is oxidized by some bacteria by the enzyme tryptophanase resulting in the formation of indole, pyruvic acid and ammonia.  Hydrolysis of tryptophan with the production of indole serves as a biochemical marker as this characteristic is not shown by all the microorganisms. 

In this experiment, peptone broth, which contains the substrate tryptophan, is used.  The presence of indole is detected by adding Kovac’s reagent, which produces a cherry red reagent layer.  This color is produced by the reagent, which is composed of p-dimethylaminobenzaldehyde, butanol and hydrochloric acid.  Indole is extracted from the medium into the reagent layer by the acidified butyl alcohol component and forms a complex with the of  p-dimethylaminobenzaldehyde, yielding a cherry red color. 

Tryptophan      tryptophanase              Indole +  Pyruvic acid +  Ammonia    

Indole + Kovac’s reagent       (HCl + butanol)           Rosindole + H₂O

                                                                                    (cherry red compound)

Cultures producing a red reagent layer following addition of Kovac’s reagent are Indole positive and its absence indicates negative reaction.

Requirements

24 hr nutrient broth cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Peptone broth (5ml / tube), Kovac’s reagent, Bunsen burner, inoculating loop, glass marking pencil, dropper etc.

 Procedure

1.  Using sterile techniques, inoculate each given organisms into appropriately labeled tubes containing peptone broth.  One uninoculated tube kept as the control.

2.  Incubate all the tubes at 37^oC for 24-48 hrs.

3.  After incubation, 1 ml of Kovac’s reagent is added to all tubes and shake gently. 

4.  Allow the tubes to stand to permit the reagent to come to the top.

Observations

E.coli produced a cherry red reagent layer following addition of Kovac’s reagent. Absence of red coloration in the cultures of species Bacillus, Streptococcus, Staphylococcus and Pseudomonas on addition of  Kovac’s reagent.

Result

E.coli is Indole positive, whereas, Bacillus, Streptococcus, Staphylococcus and Pseudomonas sp. Are Indole negative.

§  Peptone broth

Peptone                                               10.0 g

Sodium chloride                                  5.0 g

Distilled water                                    1 litre

pH                                                       7.2