Indole test

Indole test

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 ReactionIndole 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

Conventional Tube Indole

Procedure

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

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Photo Courtesy: Karen M. Kiser

Interpretation

• Positive: Pink colored rink after addition of appropriate reagent
• Negative: No color change even after the addition of appropriate reagent. e.g. Klebsiella pneumoniae

Spot Indole Test

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

• Saturate a piece of filter paper with the 1% paradimethylaminocinnamaldehyde reagent.
• Use a wooden stick or bacteriologic loop to remove a small portion of a bacterial colony from the agar surface and rub the sample on the filter paper.

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.

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.

Utility

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.

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

Mnemonic “OK VIP”

1. O for oxytica (Klebsiella oxytoca)
2. K for koseri (Citrobacter koseri)
3. V for vulgaris (Proteus vulgaris)
4. IP for indole positive.

Multitest Agar

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 ureaseproducing 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 totest for motility and ornithine decarboxylase.