Indole test: Difference between revisions
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*Tests for the presence of tryptophanase, which metabolizes tryptophan to indole |
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Source: [https://microbeonline.com/indole-test-principle-procedure-results/ Microbe Online] |
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*May be done either as a '''conventional tube indole''' (overnight incubation) or as a '''spot indole''' rapid test |
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**The tube test requires overnight incubation at 37°C in ambient air, then add the reagent; it turns pink if positive |
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**The spot test is quick, testing for indole reacting with cinnamaldehyde to give a '''blue-green''' colour within 30 seconds |
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*Helps to differentiate species within the ''Enterobacteriaciae'' |
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**Specifically can help identify species within a genus: |
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**Notable indole-negative species include [[Salmonella]], [[Hafnia]], [[Enterobacter]] |
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**[[Yersinia enterocolitica]] and some [[Shigella]] are indole-variable |
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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'''. |
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[https://i2.wp.com/microbeonline.com/wp-content/uploads/2012/04/Principle-of-Indole-Test.png [[File:https://i0.wp.com/microbeonline.com/wp-content/uploads/2012/04/Principle-of-Indole-Test-300x57.png?resize=400%2C77|Indole Test Reaction]]]Indole Test Reaction |
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Indole test is a commonly used biochemical test (e.g. in [https://microbeonline.com/imvic-tests-principle-procedure-and-results/ IMVIC] test, SIM test etc). Indole test helps to differentiate [https://microbeonline.com/seven-common-characteristics-family-enterobacteriaceae/ ''Enterobacteriaceae''] and other genera. |
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Two methods are in use: |
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# a conventional tube method requiring overnight incubation, which identifies weak indole producing organisms and |
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# a spot indole test, which detects rapid indole producing organisms |
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= Conventional Tube Indole = |
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== Procedure == |
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# Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism in tryptophan broth. |
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# Incubate at 37°C for 24-28 hours in ambient air. |
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# Add 0.5 ml of Kovac’s reagent to the broth culture. |
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[https://i0.wp.com/1.bp.blogspot.com/-uQI1gFizQKs/T3xd3m8IDhI/AAAAAAAAA10/8JMkQiMEbhU/s1600/indole.jpg [[File:https://i2.wp.com/1.bp.blogspot.com/-uQI1gFizQKs/T3xd3m8IDhI/AAAAAAAAA10/8JMkQiMEbhU/s320/indole.jpg?resize=320%2C250|img]]] |
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''Photo Courtesy: Karen M. Kiser'' |
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== Interpretation == |
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* '''Positive:''' Pink colored rink after addition of appropriate reagent |
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* '''Negative:''' No color change even after the addition of appropriate reagent. e.g. ''Klebsiella pneumoniae'' |
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= Spot Indole Test = |
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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). |
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== Procedure == |
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* Saturate a piece of filter paper with the 1% paradimethylaminocinnamaldehyde reagent. |
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* 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. |
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'''Note:''' The bacterial inoculum should not be taken from [https://microbeonline.com/macconkey-agar-mac-composition-preparation-uses-and-colony-characteristics/ MacConkey Agar] because the color of lactose fermenting colonies on this medium can interfere with test interpretation. |
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== Interpretation == |
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# '''Positive:''' Development of a blue color within 30 seconds. Most indole positive organism turn blue within 30 seconds. |
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# '''Negative:''' No color development or slightly pink color. |
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= Utility = |
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# Spot indole test along with [https://microbeonline.com/gram-staining-principle-procedure-results/ gram stain] result and colony characteristics can assist in the rapid identification of isolates. For example: A flat, dry lactose fermenting (pink) colony on [https://microbeonline.com/macconkey-agar-mac-composition-preparation-uses-and-colony-characteristics/ MacConkey agar] that is also spot indole positive and [https://microbeonline.com/oxidase-test-principle-procedure-and-oxidase-positive-organisms/ oxidase] negative can be reported presumptively as ''E.coli.'' |
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# Organisms that swarm on 5% sheep [https://microbeonline.com/blood-agar-composition-preparation-uses-and-types-of-hemolysis/ 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.'' |
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== Indole positive organisms == |
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Indole test can also aid in species differentiation. |
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== Mnemonic “OK VIP” == |
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# '''O''' for oxytica (''Klebsiella oxytoca'') |
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# '''K''' for koseri (''Citrobacter koseri'') |
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# '''V''' for vulgaris (''Proteus vulgaris'') |
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# '''IP''' for indole positive. |
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= Multitest Agar = |
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In Diagnostic laboratories, Indole test can is performed '''using multitest agar'''. Three most commonly used agar medium are: |
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# '''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. |
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# '''Motility-indole-urease (MIU) medium:''' MIU medium is used to test for indole and [https://microbeonline.com/urease-test-principle-procedure-interpretation-and-urease-positive-organsims/ urease]producing characteristics of the organism along with test for motility. |
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# '''Motility-indole-ornithine (MIO) medium:''' In addition to testing for indole production, MIO agar is used to[https://microbeonline.com/tests-bacterial-motility-procedure-results/ '''test for motility'''] and [https://microbeonline.com/decarboxylation-test-types-uses-principles-procedure-results/ '''ornithine decarboxylase.'''] |
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Latest revision as of 02:55, 8 February 2022
- Tests for the presence of tryptophanase, which metabolizes tryptophan to indole
- May be done either as a conventional tube indole (overnight incubation) or as a spot indole rapid test
- The tube test requires overnight incubation at 37°C in ambient air, then add the reagent; it turns pink if positive
- The spot test is quick, testing for indole reacting with cinnamaldehyde to give a blue-green colour within 30 seconds
- Helps to differentiate species within the Enterobacteriaciae
- Most strains of Escherichia coli, Proteus vulgaris, Morganella morganii and Providencia are indole-positive
- Specifically can help identify species within a genus:
- Klebsiella: Klebsiella oxytoca is indole-positive whereas Klebsiella pneumoniae and Klebsiella aerogenes are indole-negative
- Citrobacter: Citrobacter koseri is indole-positive where as Citrobacter freundii is indole-negative
- Proteus: Proteus vulgaris is indole-positive whereas Proteus mirabilis is indole-negative
- Notable indole-negative species include Salmonella, Hafnia, Enterobacter
- Yersinia enterocolitica and some Shigella are indole-variable
