Indole Test in Peptone Water Showing Indole Test Positive : Principle and Procedure

Indole test in peptone water showimg indole test positive

Indole Test in Peptone Water Showing Indole Test Positive

Indole test in peptone water is showing pink color ring that indicates indole test positive as shown above picture.

Indole test

This indole test is useful to identify the bacteria on the basis of indole production. The list of indole positive organisms are-

Indole negative list of organisms are-

  • Most Klebsiella species
  • Proteus mirabilis
  • Proteus  penneri
  • Citrobacter species except Citrobacter koseri
  • Salmonella species
  • Serratia species
  • Enterobacter species
  • Alcaligenes species
  • Aeromonas salmonicida
  • Yersinia species
  • Pseudomonas species
  • Actinobacillus species
  • most Bacillus species
  • Bordetella species
  • most Haemophilus species
  • Neisseria species
  • Rhizobium species and
  • Pasteurella ureae.

Principle of Indole test

The ability of an organism to split Indole from the amino acid tryptophan is due to the presence of tryptophanase. Indole, if present, combines with the aldehyde in the reagent to produce a pink to red-violet quinoidal compound (benzaldehyde reagent). The enzyme tryptophanase catalyzes the deamination reaction attacking the tryptophan molecule in its side chain and leaving the aromatic ring intact in the form of Indole.

Requirements for indole test

  • Test organisms
  • Peptone water
  • Incubator
  • Inoculating wire  and Bunsen burner
  • Kovac’s reagent

Procedure of Indole Test

  1. Stab a smooth bacterial colony on peptone water by a sterile stab wire and the inoculated media incubate at 37ºC for 24 hours.
  2. Add  2-3 drops of Kovac’s reagent (paradimethylaminobenzaldehyde in acid ethanol) after 24 hours incubation.
  3. The appearance of red color on the top of media indicates Indole positive.
  4. Indole if present combines with the aldehyde present in the reagent to give a red color to the alcohol layer.
  5. The color reaction is based on the presence of the pyrrole structure present in Indole.

Observation of indole test

Observe for red-colored rink after addition of Kovac’s reagent

Result Interpretation of indole test

Positive: Pink or red-colored rink after addition of Kovac’s reagent

Negative: No color change even after addition of Kovac’s reagent

Point to remember: The indole test can also aid in species differentiation.


  1. Klebsiella species: Klebsiella oxytoca is indole positive whereas Klebsiella pneumoniae is indole negative.
  2. Proteus species: Proteus vulgaris is indole positive whereas Proteus mirabilis is indole negative.
  3. Citrobacter species: Citrobacter Koseri is indole positive where as Citrobacter freundii is indole negative.
  4. Other test media are MIU or SIM or Motility-indole-ornithine ( MIO).

Further Readings

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
[3347 visitors]


© 2023 | All Rights Reserved