Indole test: Introduction, Principle, Test Requirements, Procedure, Result -Interpretation, and Keynotes

Indole Production test

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

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

Requirements for test

• Test organisms
• Control strains
• positive control (PC): E. coli (ATCC 25922)
• Negative control (NC): Klebsiella pneumoniae (ATCC )
• Test medium MIU or SIM or Motility-indole-ornithine (MIO)

Procedure

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

Observation of test

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

Result Interpretation

Positive	red-colored rink after the addition of Kovac’s reagent
Negative	No color change even after the addition of Kovac’s reagent

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

Keynotes

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 whereas Citrobacter freundii is indole negative.

Motility indole urease (MIU) test showing motile organism, indole positive and urease positive

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 Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
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.
6. 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.
7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.