Indole test

The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to split indole from the amino acid tryptophan. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as "tryptophanase."

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Biochemistry

Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (NH₂) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid, ammonia (NH₃) and energy. Pyridoxal phosphate is required as a coenzyme.

Performing a Test

Like many biochemical tests on bacteria, results of an indole test are indicated by a change in color following a reaction with an added reagent.

Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48 hours before performing the test. Following incubation, add 5 drops of Kovac's reagent (isoamyl alcohol, p-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) to the culture broth.

A variation on this test using Ehrlich's reagent (using ethyl alcohol in place of isoamyl alcohol, developed by Paul Ehrlich) is used when performing the test on nonfermenters and anaerobes.

A positive result is shown by the presence of a red or red-violet color in the surface alcohol layer of the broth. A negative result appears yellow. A variable result can also occur, showing an orange color as a result. This is due to the presence of skatole, also known as methyl indole or methylated indole, another possible product of tryptophan degradation.

Indole-Positive Bacteria

Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas hydrophilia, Aeromonas punctata, Bacillus alvei, most Citrobacter spp., Edwardsiella spp., Escherichia coli, Flavobacterium spp., Haemophilus influenzae, most Proteus spp. (not P. mirabilis), Plesiomonas shigelloides, Pasturella multocida, Pasturella pneumotropica, and Vibrio spp.

Indole-Negative Bacteria

Bacteria which give negative results for the indole test include: Actinobacillus spp., Aeromonas salmonicida, Alcaligenes spp., most Bacillus spp., Bordtella spp., Enterobacter spp., most Haemophilus spp., most Klebsiella spp., Neisseria spp., Pasturella haemolytica, Pasturella ureae, Proteus mirabilis, Pseudomonas spp., Salmonella spp., Serratia spp., Yersinia spp.

References

• MacFaddin, Jean F. "Biochemical Tests for Indentification of Medical Bacteria." Williams & Wilkins, 1980, pp 173 - 183.
• Example of typical indole reactions