Indole Test

Indole test is a biochemical test that helps in the identification and classification of bacteria based on their tendency to decompose amino acid (Tryptophan) into a compound refers as “Indole”. It is a part of a biochemical method popularly known as IMViC test, where ‘I’ stands for indole test. Indole is one of the end product that forms after the deamination or hydrolysis of tryptophan, caused by an enzyme refers as “Tryptophanase”.

Indole test generally includes two methods, namely spot indole test and conventional indole test. A spot tube test does not require incubation time and give rapid results by the formation of the blue-green zone. A conventional tube test involves incubation of the culture medium, and correct conclusions could be drawn only after 24 hours. Therefore, the conventional tube method is quite slower than the spot method.

Content: Indole Test

  1. Definition
  2. Purpose
  3. Principle
  4. Indole Test Media
  5. Indole Test Reagents
  6. Methods and Procedure
  7. Important Facts

Definition of Indole Test

Indole test can define as a part of a biochemical test or IMViC test procedure that aims to distinguish different members of bacteria (indole positive or indole negative) based on the production of indole by the splitting of tryptophan, present in the medium. Indole positive bacteria having an intracellular enzyme (Tryptophanase) that degrade the tryptophan amino acid into indole, whereas indole negative bacteria lacks such enzyme. This test classifies the different members of the Enterobacteriaceae family by one of the rapid and slow methods.

Spot indole test is a rapid method that identifies the indole producing organisms by taking a few minutes. Conventional indole test is a slow method that requires an incubation period of 24 hours to detect the microorganisms producing indole. To classify the group of Enterobacteria, Kovacs reagent is commonly used, but to distinguish non-fermentative and anaerobic bacteria, Ehrlich’s reagent is used.


A purpose of an indole test accounts the following:

  • To detect the organisms, based on their capacity to degrade tryptophan into a compound known as indole.
  • To classify the different members of bacteria belongs to the Enterobacteriaceae family.
  • It is a qualitative method confirms by the appearance of a coloured complex on adding the particular reagent.

Principle of Indole Test

In the indole test, the growth of bacteria is induced in tryptone rich medium. Some bacteria possess an intracellular enzyme “Tryptophase” that results in deamination of tryptophan into three end products, namely indole, pyruvic acid and ammonium. The hydrolysis of tryptophan results in the removal of amine (NH2) group by the addition of water. Addition of Kovacs and spot reagent confirms the production of indole in a conventional tube and spot indole method, respectively.

Principle of indole test

Spot indole method makes the use of p-Dimethylaminocinnamaldehyde reagent that combines with indole to produce a blue coloured complex. In a conventional method, Kovacs reagent contains 4(p)-dimethylaminobenzaldehyde, which reacts the indole and produces a red coloured compound by forming rosindole.

Indole Test Media

Indole test can be performed by using a single media such as “Tryptophan broth” and a combined test medium such as “SIM media”. By using tryptophan broth, we could only distinguish among the indole positive and indole negative organisms. In contrast to this, SIM (Sulfide Indole Motility) is a combined medium, which can differentiate between the microorganisms based on H2S production, indole production and motility.

The composition of tryptone broth includes:
Tryptone: 10g
Sodium chloride: 5g
Distilled water: 1L
pH: 7.5  0.2 (at 25 Degrees Celsius)

The composition of SIM media includes:
Tryptone: 20g
Peptone: 6.1g
Agar: 3.5g
Fe (NH)2(SO4)2.6H2O: 0.2g
Na2S2O3.5H2O: 0.2g
Distilled water: 1L
pH: 7.5  0.2 (at 25 Degrees Celsius)

For the inoculation of bacterial culture, we can select isolated colonies from any of the non-selective medium added with sufficient amount of tryptophan.

Indole Test Reagent

Indole Kovacs reagent: Components of indole Kovacs reagent to prepare 100ml of a solution includes:

preparation of kovacs reagent4(p)-dimethylaminobenzaldehyde: 5g
Amyl alcohol: 75ml
Concentrated hydrochloric acid: 25ml
Distilled water: 100ml

Indole spot reagent: Components of indole spot reagent to prepare 100ml of a solution includes:

preparation of spot reagent(p)-Dimethylaminocinnamaldehyde: 1.0g
Concentrated hydrochloric acid: 10.0ml
Distilled water: 100ml

Methods and Procedure

There are two common methods that differentiate the organisms based on indole production, namely:

  1. Spot indole method
  2. Conventional tube method

Spot Indole Method

Procedure: It includes the following steps

procedure of spot indole test

  1. Saturate Whatman no.1 filter paper with the spot indole reagent.
  2. Allow drying for a few minutes.
  3. Add little bacterial inoculum via wooden applicator from any non-selective medium rich in tryptophan like Blood agar media.
  4. Rub the inoculum or prepare a thin bacterial smear over the reagent saturated zone.
  5. Observe the results for the appearance of a blue coloured zone after 3-5 minutes.
Result Interpretation

Positive result: Appearance of blue-green colour on the bacterial smear.
Negative result: A filter paper remains colourless.

result interpretation of spot tube test

  • Spot indole test is a less sensitive method.
  • Inoculum should be taken from an isolated colony to prevent the diffusion of indole.
  • A bacterial culture must be incubated aerobically.
  • A culture should be taken from the glucose-free medium, as it rapidly breakdown the indole.
  • An inoculum isolated from selective media like MacConkey’s agar and Eosin methylene blue agar can interfere with the result interpretation.
  • Bacteria isolated from Mueller Hinton agar can degrade tryptophan by causing acid hydrolysis of casein.
  • Weak indole positive bacteria like Cardiobacterium hominis cannot be examined by this method.

Conventional Indole Method

Procedure: It includes the following steps:

procedure of conventional indole test

  1. Prepare tryptone broth by accurately weighing the reagents required.
  2. After adding all the components in a sterilized conical flask, add an appropriate amount of sterile water and autoclave at 121 degrees Celsius on 15psi pressure for 15 minutes.
  3. After autoclaving pour 5ml of tryptone broth into each test tubes.
  4. Then, inoculate bacterial pure culture via the inoculating loop.
  5. Incubate the test tubes for at least 24 hours at a temperature of 35 degrees Celsius.
  6. Pour 0.5 ml of Kovacs reagent for the appearance of a red coloured complex on the top of the alcohol layer.
Result Interpretation

Positive result: Confirms by the appearance of the cherry red colour.
Negative result: A medium form a colourless or yellow compound.
result interpretation of conventional tube test

  • A conventional tube method takes much time (Minimum of 24hours) to observe the results.
  • It cannot differentiate among the non-fermentative and anaerobic bacteria, for which an Ehrlich reagent acts as a replacement of Kovacs reagent.
  • During a reaction, a compound forms named as “Skatole” that can obscure the result interpretation.
  • Clostridium species rapidly breaks the indole and can give false results.

Important Facts

Indole test differentiates organisms by the presence of Tryptophanase enzyme, responsible for the breakdown of tryptophan. Therefore, bacteria those can degrade tryptophan into indole must have an enzyme Tryptophanase and refer as “Indole positive bacteria”. Examples of indole positive bacteria are Aeromonas hydrophila, Bacillus alvei, Escherichia coli, Klebsiella oxytoca etc.

Few bacteria lack Tryptophanase enzyme in their intracellular space and cannot split tryptophan into indole and refer as “Indole negative bacteria”. Examples of indole negative bacteria include Proteus mirabilis, Pasteurella ureae, Bacillus sp, Enterobacter sp etc.

Tryptophanase is an intracellular enzyme present in some bacteria that uses tryptophan and water as “Substrates” to produce “Products” like indole, pyruvic acid and ammonium. It belongs to the C-C lyases family, which facilitates the tryptophan metabolism via pyridoxal phosphate and potassium cofactors.

Indole (primary product) forms as a result of tryptophan hydrolysis, which is an aromatic heterocyclic compound. The production of indole can be visualized by the addition of some coloured solution like Kovacs reagent (for tube method) and Spot reagent (for spot method).

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