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Indole test is used to identify Enterobacteria that breakdown tryptophan (an amino acid) to produce indole. The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into the 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 organisms. 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 peptone water 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 30oC. The tubes can be stored in the refrigerator until use.
  6. Inoculate the tube(s) with the test culture and incubate overnight at 37oC.
  7. Add 0.5 ml of Kovac’s reagent to each of the tube(s) after incubation.
  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. Presence of pink-red colour on the surface layer of the tube is a positive test result (Figure 1.). Negative tubes do not produce this colouration, and may remain colourless, amber, brown or green.
Figure 1. Indole test. The tube on the left is negative and tube on the right is positive as indicated by a red colour on the surface of the medium upon the addition of Kovac’s reagent. Photo courtesy:

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, 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.

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