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Indole Test for Bacteria

This  indole test is useful for both aerobic and anaerobic gram negative rods. Indole test positive and negative organisms as shown above picture. The list of indole positive organisms are-

Escherichia coli

Klebsiella oxytoca

Proteus vulgaris

Morgenella morganii

Vibrio cholerae

Providencia species

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 quinodal compound (benzaldehyde reagent). The enzyme tryptophanase catalyses the deamination reaction attacking the tryptophan molecule in its side chain and leaving the aromatic ring intact in the form of Indole.

Procedure of Indole Test

1. Stab a smooth bacterial colony  on SIM (Sulphide Indole Motility) medium or motlity indole urea (MIU) medium or 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. 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 in the alcohol layer.
5. The color reaction is based on the presence of the pyrrole structure present in Indole.

Result Interpretation of Indole Test

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

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

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

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. Clinical Microbiology Procedure Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
4. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
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.
6.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
7. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.