Indole test: Introduction,Principle, procedure, result and interpretation

Indole test

Indole test for bacteria

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

Principle of Indole test

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

Requirements for Indole test

Quality control strains

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

 Test Procedure

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

Result Interpretation

  • 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).


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