Indole test: Introduction, Principle and procedure and Result Interpretation

Indole test

Indole Production test

This  indole test  is useful to identify the bacteria on the basis of indole production. 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.

Requirements for indole 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 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.

Observation of indole test

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

Result Interpretation

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.

Key notes

  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 where as Citrobacter freundii is indole negative.


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