Indole Production
DESCRIPTION
Indole is an aromatic heterocyclic organic compound that can be produced by some bacteria as a degradation product of the amino
acid tryptophan. Tryptophanase is the enzyme involved tryptophan degradation resulting indole, pyruvate, and ammonia. Indole test is
most used for coliforms identification.
MATERIALS
Simplest medium for indole testing is Buffered peptone water (Proteose peptone 10g,
NaCl 5g, Disodic phosphate 3.5g, Monopotasic phosphate 1.5g, H2O ad 1000 ml; pH
7.2), but most used for Enterobacteriaceae identification is Motility Indole Urea
medium (MIU).

Kovacs reagent composition:
Amyl alcohol (may be replaced by n-butanol or ethanol) .............................. 750 ml,
p-Dimethylaminobenzaldehyde .......................................................................... 50.0 g,
Hydrochloric acid (conc.) ..................................................................................... 250 ml.
REFERENCES
1. Helgomar Raducanescu, Valeria Bica-Popii,1986. Bacteriologie veterinara,
Ed. Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.
3. Standards Unit, Microbiology Services, PHE. UK Standards for Microbiology Investigations. Identification of Clostridium species.
Bacteriology – Identification, ID 8, Issue no: 4, Issue date: 12.01.15.
Indole positive result: red ring on
MIU medium surface (middle tube)
PROCEDURE
Inoculate a Buffered peptone water medium or a MIU tube and incubate at 37°C. After
24 hours of incubation add few drops of Kovacs reagent. This will combine with the
indole forming a pink-red compound. Reaction is positive if a red layer appears on the
surface of the medium. Reaction is negative if red color does not appear.

NOTES
Indole should not be tested after 4-5 days of incubation because bacteria may further
degrade indole and a false negative result may occur.
Do not use peptone media containing glucose. A decrease in pH towards acid
production may produce a false negative result or the reaction may be weak.
Anaerobes, particularly Clostridium species, form indole but can rapidly break it down
as it is produced; therefore, false negative reactions may occur.
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