Indole Test: Principle, Procedure and results

Indole test is used to determine the ability of an organism to split amino acid tryptophan to form the compound indole. Tryptophan is hydrolysed by tryptophanase to produce three possible end products – one of which is indole. Indole production is detected by Kovac’s or Ehrlich’s  reagent which contains 4 (p)-dimethylamino benzaldehyde, this reacts with indole to produce a red coloured compound.
Indole Test Reaction
Indole test is a commonly used biochemical test (e.g. in IMVIC test, SIM test etc). Indole test helps to differentiate Enterobacteriaceae and other genera.

Two methods are in use;

  1. a conventional tube method requiring  overnight incubation, which identifies weak indole producing organisms and
  2. a spot indole test, which detects rapid indole producing organisms
Procedure of  Conventional Tube method for Indole Test

a. Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism in tryptophan broth.
b. Incubate at  37°C for 24-28 hours in ambient air.
c. Add 0.5 ml of Kovac’s reagent to the broth culture.

Photo Courtesy: Karen M. Kiser

Expected results:



It is used to determine the presence of the enzyme tryptophanase. Tryptophanase breaks down tryptophan to release indole, which when reacts with cinnamaldehyde produces a blue-green compound.  The absence of enzyme results in no color production (i.e. indole negative).
Procedure of Spot Indole Test

Note: The bacterial inoculum should not be taken from MacConkey Agar because the color of lactose fermenting colonies on this medium can interfere with test interpretation.


  1. Positive: Development of a blue color within 30 seconds. Most indole positive organism turn blue within 30 seconds.
  2. Negative: No color development or slightly pink color.


  1. Spot indole test along with gram stain result and colony characteristics can assist in the rapid identification of isolates. For example
    A flat, dry lactose fermenting (pink) colony on MacConkey agar that is also spot indole positive and oxidase negative can be reported presumptively as E.coli.
  2. Organisms that swarm on 5% sheep blood agar, exhibit a characteristics odour, and are oxidase negative can be presumptively identified as Proteus spp.  With further testing by spot indole, the positive isolates may be presumptively reported as Proteus vulgaris and the negative ones as Proteus mirabilis.

Indole positive organisms: Most strains of E.coli, P. vulgaris, M. morganii and Providenica are indole positive.

Point to remember: Indole test can also aid in species differentiation.

  1. Klebsiella species:  Klebs‪iella oxytoca is indole positive whereas Klebsiella pneumoniae is indole negative.
  2. Citrobacter species: Citrobacter Koseri is indole positive where as Citrobacter freundii is indole negative
  3. Proteus species: Proteus Vulgaris is indole positive whereas Proteus mirabilis is indole negative 

I used to remember above mentioned information with the help of MNEOMONIC,  which can be useful for you.

For this remember the phrase “OK VIP”

Where: O means: Oxytica (Klebsiella oxytoca), K means (koseri-i.e. Citrobacter koseri), means vulgaris (Proteus vulgaris) and IP means Indole Positive.

In Diagnostic laboratories, Indole test can is performed using multitest agar. Three most commonly used agar medium are:

  1. Sulfide-indole-motility (SIM) medium: The SIM medium is a multitest agar used to test for indole production while simultaneously determining motility and  hydrogen sulfide producing abilities of the isolate.
  2. Motility-indole-urease (MIU) medium: MIU medium is used to test for indole and urease producing characteristics of the organism along with test for motility.
  3. Motility-indole-ornithine (MIO) medium: In addition to testing for indole production, MIO agar is used to test for motility and ornithine decarboxylase.