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Indole test for bacteria

This indole test is useful for both aerobic and anaerobic gram-negative rods and really very useful in the routine identification of bacteria in Microbiology.

Principle of Indole test

The ability of bacteria to split indole from the amino acid tryptophan is due to the presence of the enzyme, tryptophanase.  Indole,  if present combines with an aldehyde in the reagent to produce a pink to red-violet quinoidal compound if using benzaldehyde reagent or blue to green when using cinnamaldehyde reagent.

Requirements for Indole test

• Fresh growth of gram-negative rods (test organism)
• Sulphide indole motility (SIM) medium/ Motility indole urea (MIU) medium/ peptone water
• Kovac’s reagent
• Incubator
• Inoculating  wire  and Bunsen burner

Quality control strains

•  Positive control-E. coli (ATCC25922)
• Negative Control-Pseudomonas aeruginosa (ATCC27853)

 Test Procedure of Indole test

1. Inoculate liquid tube medium or stab agar medium with the colony.
2. Incubate for 18 to 24 hours in a BOD incubator.
3. Add 3 drops of Kovac’s reagent down the side of the tube and observe color change at the meniscus.
4. If the test is negative, repeat the test after additional 24 hours of incubation if desired.

Result Interpretation of Indole Test

• Development of a brown-red to purple-red color:  Test  Positive (presence of indole)
• Colorless or slightly yellow: Negative test
• Tests positive (left)
• Test negative (Right) as shown above picture.

Indole test positive organisms are-

• Escherichia coli
• Klebsiella oxytoca
• Proteus vulgaris
• Citrobacter koseri
• Morgenella morganii
• Vibrio cholerae
• Providencia species
• Aeromonas species
• Plesiomonas species
• Pasteurella species
• Cardiobacterium hominis
• Propionibacterium acenes

While indole test negative organisms are–

• Klebsiella pneumoniae
• Enterobacter species
• Proteus mirabilis
• Citrobacter freundii

Limitation of indole test

• Do not use media that contain dyes (e.g. Eosin methylene blue, MacConkey agar).

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. 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.
6. 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.
7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.