What is Indole Test?

Indole test is one of the biochemical tests which is conducted on the bacterial species to detect the ability of pathogen to produce indole from tryptophan in the presence of a group of enzymes known as tryptophanase.

Generally, it is a qualitative test, which is performed as part of the IMViC test and is used to differentiate the members of the Enterobacteriaceae family.

It is very important in identifying the different species of bacteria like Escherichia coli, proteus, Morganella, etc.

This plays a central role in characterizing the coliforms and it is denoted by various differences of the indole test and also in combination with other biochemical tests.

It is used as a traditional method to distinguish Indole negative Enterobacter and Indole Positive E. coli and Klebsiella.

Variation of the test is known by Ehrlchs reagent that is used when the test has been performed in non-fermenters and anaerobes.

Indole Test Purpose

Indole test is used to detect the ability of the organism to produce enzyme tryptophanase.

Indole Test Principle

As mentioned before, Indole test is one of the biochemical tests which differentiates the coliform from other members of the Enterobacteriaceae; by detecting the ability of the pathogen to produce enzyme known as tryptophanase.

Tryptophanase hydrolyses the amino acid known as tryptophan into indole, ammonia and pyruvic acid.

It is usually known as intracellular enzyme or endoenzyme. Pyruvic acid thus produced can be used by the organism in the Krebs’s cycle or it enters into the glycolysis pathways and it is used to synthesize other necessary compounds for the cell.

The culture medium used for indole test involves Sulphide, Indole, motility (SIM) or nutrient peptone, either of the given media provides necessary amino acid a tryptophan which acts as a substrate the reaction.

Thus, the organism is able to produce indole as a main product and ammonia as a byproduct.

Kovac’s reagent is used as the reagent for this test which reacts as a side product in catabolizing the tryptophan and this results the indole to form a Rosindole dye which is cherry red in color.

Hence the formation of cherry red color denotes the positive indole test and it is not in the sense that E. coli is positive to indole and Klebsiella is negative to it.

Indole Test Materials

• Filter paper

• Watch glass

• Test tubes

• incubator

• Peptone broth-enriched with amino acids and tryptophan

• Bacterial samples of Escherichia coli and Klebsiella species

• Kovac’s reagent.

Indole Test Procedure

Indole test can be performed in two ways such Rapid Spot test and tube test.

1. Rapid Spot Indole Test

 To perform rapid spot test, a piece of filter paper is taken and is it moistened using the reagent.

 An isolated colony from a cultured medium is taken using an inoculated loop and it is rubbed against the moistened filtered paper.

 Then the filter paper is observed for color change

Rapid Spot Indole Test Interpretation

 If the appearance of blue color is observed in the filter paper within 20 minutes, then it indicates the positive result of indole test.

 If there is absence of any colors in the filter paper then it denotes the negative result for indole test.

2. Tube Indole Test

 Either peptone broth or medium is prepared using Sulphide, Indole and motility in the test tubes and the test tubes are autoclaved at 15lbs/inch pressure for about 15 minutes.

 By using the sterile wire, broth is inoculated with the samples that were provided and the test tubes are labelled with the specific name of the organism.

 Further the test tubes are incubated at 37ºC for 24 to 48 hours.

 After the proper incubation, test tubes are taken from the incubator and are added with four to eight drops of Kovac’s reagent in each of the test tubes, in such a way that reagent touches the walls of the test tubes.

 After adding the reagents, the test tubes are placed in between the palms and they are shaken to mix the reagent such that it mixes well with the culture.

 The test tube is left undisturbed till the solution stands and development of cherry red color is observed at the surface of the media.

Tube Indole Test Interpretation

 Indole test is diagnosed as positive if it appears in the cherry red color; it also indicates the presence of E. coli.

 Indole test shows negative result, if there is no formation of red color, and indicates the presence of Klebsiella.

Uses of Indole Test

• Indole test is generally used to test the ability of an organism to utilize tryptophan and to produce indole.

• This test is used to differentiate the members of the Enterobacteriaceae family as a part of IMViC test.

• This test also helps in differentiating the Proteus mirabilis from other species of Proteus.

• This test also differentiates the indole positive E. coli and the Indole negative Enterobacter and klebsiella.

• Along with this, this test also helps in differentiating K. pneumoniae, which is indole positive from K. oxyotoca, an indole positive and also in differentiating Citrobacter freundii, an indole Negative from the Citrobacter koseri, an indole positive species.