The ability of an organism to split Indole from the amino acid tryptophan is due to the presence of tryptophanase. Indole, if present, combines with the aldehyde in the reagent to produce a pink to red-violet quinodal compound (benzaldehyde reagent). The enzyme tryptophanase catalyses the deamination reaction attacking the tryptophan molecule in its side chain and leaving the aromatic ring intact in the form of Indole.
Requirements for indole test
positive control (PC): E. coli (ATCC 25922)
Negative control (NC): Klebsiella pneumoniae (ATCC )
Test medium MIU or SIM or Motility-indole-ornithine ( MIO)
Procedure of Indole test
Stab a smooth bacterial colony on SIM (Sulphide Indole Motility) medium or motlity indole urea (MIU) medium or peptone water by a sterile stab wire and the inoculated media incubate at 37ºC for 24 hours.
Add 2-3 drops of Kovac’s reagent (paradimethylaminobenzaldehyde in acid ethanol) after 24 hours incubation.
Appearance of red color on the top of media indicates Indole positive.
Indole if present combines with the aldehyde present in the reagent to give a red color in the alcohol layer.
The color reaction is based on the presence of the pyrrole structure present in Indole.
Observation of indole test
Observe for red colored rink after addition of Kovac’s reagent
Positive :red colored rink after addition of Kovac’s reagent
Negative : No color change even after addition of Kovac’s reagent
Point to remember: Indole test can also aid in species differentiation.
Klebsiella species: Klebsiella oxytoca is indole positive whereas Klebsiella pneumoniae is indole negative.
Proteus species: Proteus vulgaris is indole positive whereas Proteus mirabilis is indole negative.
Citrobacter species: Citrobacter Koseri is indole positive where as Citrobacter freundii is indole negative.
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