Biochemical tests


Written by MicroDok

Indole test (Tryptophan hydrolysis): Indole test is used to identify Enterobacteria that breakdown tryptophan (an amino acid) to produce indole. Tryptophan hydrolysis is catalyzed by the enzyme tryptophanase to produce indole and pyruvic acid. Indole production is a characteristic feature of Enterobacteriaceae (e.g. Escherichia coli). Proteus vulgaris, Moraxella, and Providencia species are also indole positive. Indole test is performed with a reagent called Kovac’s reagent that contains 4 (p)-dimethylamino benzaldehyde. Indole reacts with 4 (p)-dimethylamino benzaldehyde to produce a characteristic pink colouration which indicates a positive result. In addition, the test culture is first cultured in a medium that contains tryptophan (e.g. peptone water) before performing the test.


  1. Prepare peptone water by adding 10 g of tryptone or peptone to 100 ml of distilled water.
  2. Add 0.5 g of sodium to the above solution.
  3. Adjust the final pH of the solution to 7.2.
  4. Dispense 5 ml portions of the solution into clean test tubes with caps and cover loosely.
  5. Sterilize in the autoclave and cool medium to 30o The tubes can be stored in the refrigerator until use.
  6. Inoculate the tube(s) with the test culture and incubate overnight at 37o
  7. Add 0.5 ml of Kovac’s reagent to each of the tube(s).
  8. Shake tube(s) gently and allow standing.
  9. Examine tube(s) for the presence of pink-red colour in the surface layer of the tube(s) within 5 mins. Negative tubes do not produce this colouration, and may remain colourless, amber, brown or green.

Illustration of Indole test

Tube on left side is uninoculated, tube at the center is negative and tube on the right side is positive as indicated by a red/pink colour on the surface of the medium upon addition of Kovac’s reagent.


Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

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