Indole Test- Principle, Reagents, Procedure, Result Interpretation and Limitations

By Prof Moses Joloba


The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into indole. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as “tryptophanase.”

Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzyme.

Like many biochemical tests on bacteria, results of an indole test are indicated by a change in color following a reaction with an added reagent.

Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24–48 hours before performing the test. Following incubation, five drops of Kovac’s reagent (isoamyl alcohol, para-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) are added to the culture broth.

A positive result is shown by the presence of a red or red-violet color in the surface alcohol layer of the broth. A negative result appears yellow. A variable result can also occur, showing an orange color as a result. This is due to the presence of skatole, also known as methyl indole or methylated indole, another possible product of tryptophan degradation.

The positive red color forms as a result of a series of reactions. The para-Dimethylaminobenzaldehyde reacts with indole present in the medium to form a red rosindole dye. The isoamyl alcohol forms a complex with rosindole dye, which causes it to precipitate. The remaining alcohol and the precipitate then rise to the surface of the medium.

A variation on this test using Ehrlich’s reagent (using ethyl alcohol in place of isoamyl alcohol, developed by Paul Ehrlich) is used when performing the test on nonfermenters and anaerobes.


Some bacteria have the ability to hydrolyse the amino acid tryptophan to indole, as they can produce the enzyme ‘tryptophanase’.Tryptophan is hydrolyzed with the production of indole, pyruvic acid and ammonia. Indole is detected by the colour reagent, paradimethylaminobenzaldehyde (p-DMAB, i.e. Kovac’s reagent), which produces a pink ring. Indole reacts with p-DMAB to produce the compound ‘quinoidal red violet compound (rosindole dye)’, which is pink in colour. The ring is produced at the surface, as the reagent is prepared in amyl alcohol.

In the indole test, the test bacteria is grown in a broth medium containing tryptophan. If the bacteria has the ability to hydrolyse tryptophan to indole, a pink ring is formed at the surface of the broth culture upon addition of p-DMAB.


Reagents And Materials Required

  • Test tubes
  • Conical flask
  • cotton plugs
  • Inoculating loop
  • Autoclave
  • Bunsen burner
  • Laminar flow chamber
  • Dispose jar
  • Incubator
  • Tryptone broth
  • Kovac’s reagent (para­dimethylaminobenzaldehyde)
  • Isolated colonies or pure cultures of bacteria.


  1. The ingredients of tryptone broth medium or its ready-made powder required for 100 ml of the broth is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling. Tryptone acts as a source of tryptophan. Therefore, addition of tryptophan to the broth is optional.
  2. Its pH is determined using a pH paper or pH meter and adjusted to 7.5 using 0.1N HC1 if it is more or using 0.1N NaOH if it is less. The flask is heated, if required, to dissolve the ingredients completely.
  3.  The broth is distributed into five test tubes (approximately 5 ml each), cotton-plugged, covered with craft paper and tied with thread or rubber band.
  4. The broth tubes are sterilized at 121 °C (15 psi pressure) for 15 minutes in an autoclave.
  5. The broth tubes are allowed to cool to room temperature.
  6. The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, into the broth with the help of an inoculating loop sterilized over bunsen flame. The loop is sterilized after each inoculation.
  7. The inoculated broth tubes are incubated at 37°C for 48 hours in an incubator.
  8. The color reagent, para-dimethylaminobenzaldehyde (p-DMAB), (i.e. Kovac’s reagent) is dropped into the broth tubes (0.5 ml each) and observed after 1 minute.


  • Procedure 7: Paper strips soaked in oxalic acid are put at the mouth of the inoculated broth tubes, so that about half of the paper strips remain inside the tubes without touching the broth and the other half remain to outside. For this, filter paper is cut into strips of approximately 35 mm long and 5 mm wide. The strips are soaked in hot saturated aqueous solution of oxalic acid and dried before use.
  • Procedure 8: The inoculated broth tubes are incubated at 37°C for 48 hours in an incubator.


1. Pink ring produced

Indole positive (i.e. the bacteria can convert tryptophan to indole).

2. Pink ring not produced

Indole negative (i.e. the bacteria cannot convert tryptophan to indole).


1. Pink color produced on paper strip

Indole positive (i.e. the bacteria can convert tryptophan to indole).

Examples:  Aeromonas hydrophilaAeromonas punctataBacillus alvei,Edwardsiella sp., Escherichia coliFlavobacterium sp., Haemophilus influenzaeKlebsiella oxytocaProteus sp. (not P. mirabilis and P. penneri), Plesiomonas shigelloides,Pasteurella multocidaPasteurella pneumotropicaEnterococcus faecalis, and Vibrio sp.

2. Pink color not produced on paper strip

Indole negative (i.e. the bacteria cannot convert tryptophan to indole).

Examples: Actinobacillus spp., Aeromonas salmonicidaAlcaligenes sp., most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most Haemophilus sp., most Klebsiella sp., Neisseria sp., Pasteurella haemolyticaPasteurella ureaeProteus mirabilisP. penneriPseudomonas sp.,Salmonella sp., Serratia sp., Yersinia sp.

Limitations of Indole Test

  • Indole tests may be used as an aid in the identification and differentiation of gram-positive and gram-negative organisms. Additional biochemical testing using pure cultures is recommended for complete identification.
  • The tube test is a more sensitive method of detecting indole than the spot test.
  • When performing a spot test, Kovacs Indole Reagent may be used as a substitute for the spot test reagent. However, Kovacs Indole Reagent, when used as the spot test reagent, is less sensitive in detecting indole than the Indole Spot Reagent (DMACA).
  • Kovacs Indole Reagent is not recommended for use with anaerobic bacteria. The Indole Spot Reagent (DMACA) is suitable for anaerobe use.
  • Since peptones have been shown to vary with regard to their suitability for use with indole testing, media selected for indole determination should be tested with known positive and negative organisms to insure suitability.
  • Media containing glucose should not be used for indole testing due to the formation of acid end products which have been shown to reduce indole production. Mueller Hinton Agar should also not be used for this test because tryptophan is destroyed during acid hydrolysis of casein.
  • Media containing dye, such as MacConkey and EMB, are unsuitable sources of inoculum due to possible carryover of dye and subsequent interference of indole color interpretation.
  • Indole-positive colonies have been reported to cause adjacent indole-negative colonies to appear false-positive due to diffusion of indole into the media. To avoid false-positives, select colonies of different morphologies that are separated by at least 5mm for indole testing.

Uses of Indole Test

  • To differentiate Proteus mirabilis (indole negative) from all other Proteus species (indole positive).
  • To differentiate Klebssiella pneumoniae (indole negative) from Klebsiella oxytoca (indole positive).
  • To differentiate Citrobacter freundii (indole negative) from Citrobacter koseri (indole positive).