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Indole test (Left: negative; Right: positive) Indole test is used to determine the ability of an organism to split amino acid tryptophan to form the compound indole. It is used as part of the IMViC procedures to differentiate members of the family Enterobacteriaceae. Principle Tryptophan is hydrolyzed by tryptophanase to produce three possible end products; indole, pyruvate, and ammonia. Indole production is detected by Kovac's or Ehrlich's reagent. Indole, if present, combines with the aldehyde in the reagent to produce a pink to red-violet quinoidal compound (if benzaldehyde reagent is used) or a blue to green color (if cinnamaldehyde reagent is used). The absence of enzyme results in no color production (i.e. indole negative). Indole Test Reaction Indole test is a commonly used biochemical test and helps to differentiate Enterobacteriaceae and other genera. Quality Control Do not use benzaldehyde reagents (including Ehrlich’s and Kova´cs’) if color is not pale yellow.Perform QC on each new lot of reagent prior to using them. QC in-house-prepared reagents weekly, as they can deteriorate, especially if not stored at 4°C. Discard if reactions become weak.OrganismsEscherichia coli ATCC 25922—indole positivePseudomonas aeruginosa ATCC 27853—indole negativeFor Ehrlich’s reagent for use with anaerobic microorganismsPorphyromonas asaccharolytica ATCC 25260—indole positiveBacteroides fragilis ATCC 25285—indole negative Procedure Two methods are in use; a rapid or spot indole test in which indole is detected directly from a colony growing on a medium rich in tryptophan.a conventional tube method requiring overnight incubation, which identifies weak indole producing organisms. Spot Indole Test Spot indole test is performed by using one of the three methods mentioned below. Saturate a piece of filter paper with the 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.Sweep the colony onto a swab. Add a drop of indole reagent to the colony swab.Add reagent directly to the colony growing on the agar surface. Note: The bacterial inoculum should not be taken from media that contain dyes such as MacConkey agar and EMB agar because the color of lactose fermenting colonies on this medium can interfere with test interpretation. Conventional Tube method Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism in tryptophan broth.Incubate at 37°C for 24-28 hours in ambient air.For Ehrlich’s methoda. Add 0.5 ml of xylene to a tube and invert to mix. Let settle.b. Add 6 drops of Ehrlich’s indole reagent down the side of the tube and observe color below the xylene layer.For Kovac's method, Add 0.5 ml of Kovac's reagent to the broth culture down the side of the tube and observe color change at meniscus. Result The development of a brown-red to purple-red color (benzaldehyde reagents) or blue color (cinnamaldehyde reagent) within 20 seconds indicates the presence of indole.A negative test is colorless or slightly yellow. Reporting results E. coli is indole positive, as are many other Enterobacteriaceae, Vibrio, Aeromonas, Plesiomonas, and Pasteurella. Most strains of P. vulgaris, M. morganii and Providenica are indole positive.Several fastidious Gram-negative rods are indole positive, such as Cardiobacterium hominis and Pasteurella bettyae.Propionibacterium acnes is indole positive. Uses Spot indole tests along with gram stain results 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.Indole test can also aid in species differentiation.Klebsiella species: Klebs‪iella oxytoca is indole positive whereas Klebsiella pneumoniae is indole negative.Citrobacter species: Citrobacter koseri is indole positive where as Citrobacter freundii is indole negativeProteus species: Proteus vulgaris is indole positive whereas Proteus mirabilis is indole negative You can remember which species are Indole positive by remembering the phrase "OK VIP". Where: O means: Oxytica (Klebsiella oxytoca), K means (koseri-i.e. Citrobacter koseri), V 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 media are: 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.Motility-indole-urease (MIU) medium: MIU medium is used to test for indole and urease-producing characteristics of the organism along with a test for motility.Motility-indole-ornithine (MIO) medium: In addition to testing for indole production, MIO agar is used to test for motility and ornithine decarboxylase. Limitations Growth medium must contain an adequate amount of tryptophan. Do not use colonies from Mueller-Hinton agar for this test, because tryptophan is destroyed during the acid hydrolysis of casein.Do not use a plate with a nitrate disk, as nitrate can interfere with the spot indole test by inducing false-negative results.Only the cinnamaldehyde reagent can be used for spot testing of anaerobic microorganisms. It is the more sensitive reagent, but it is less stable.If the rapid indole test is negative, perform a tube test as the isolate could be positive in the more sensitive tube test.For fastidious Gram-negative rods, such as C. hominis, a heavy inoculum and extraction are necessary. References and further readings MacWilliams, M. P. (2009, December 8). Indole Test Protocol. https://www.asmscience.org/content/education/protocol/protocol.3202Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814