Effective Date: 
Thu, 08/01/2013
Fri, 01/05/2018
Fri, 08/09/2013

Purpose and Scope:

The indole test is a qualitative procedure for determining the ability of bacteria to produce indole by deamination of tryptophan.

Using Kovacs method, indole combines, in the presence of a tryptophan rich medium, with p-Dimethylaminobenzaldehyde at an acid pH in alcohol to produce a red-violet compound.

Reagents and Supplies:

  • Indole Kovacs Reagent
  • Filter paper
  • Sterile inoculating loop or stick

Quality Control: 

QC is performed on each day the test is performed. Use procedure with stock culture of E. coli ATCC 25922 (indole positive) and P aeruginosa ATCC 27853 (indole negative).

Record QC results on Indole Test QC Log.


  1. Place a small square of filter paper on a parafilm square of the same size.
  2. Place 2-3 drops of Indole Kovacs Reagent on the filter paper.
  3. With a sterile inoculating loop or wooden applicator stick, pick a portion of an 18-24 hr hour isolated colony from a blood agar plate, and rub it onto the reagent saturated area of the filter paper.
  4. A positive Kovacs test reaction is denoted by the appearance of a pink to red .
  5. Record the result in the microbiology culture work-up log.
  6. Dispose of all waste in biohazard bag. Place wooden applicator sticks in sharps container.


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.





Hardy Diagnostics Indole Test Reagents Package Insert 2012

Bailey and Scott. Diagnostic Microbiology, 9th ed. St. Louis, Mo., Mosby. 1994.

Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I. Washington DC. ASM Press. 1992.