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Theindole test is a biochemical test performed onbacterial species to determine the ability of the organism to converttryptophan intoindole. This division is performed by a chain of a number of different intracellularenzymes, a system generally referred to as "tryptophanase."[citation needed]
Indole is generated by reductivedeamination from tryptophan via the intermediate molecule indolepyruvic acid.Tryptophanase catalyzes the deamination reaction, during which theamine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole,pyruvic acid,ammonium (NH4+) and energy.Pyridoxal phosphate is required as acoenzyme.
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, five drops ofKovács reagent (isoamyl alcohol,para-Dimethylaminobenzaldehyde, concentratedhydrochloric acid) are added to the culture broth.
A positive result is shown by the presence of a red or reddish-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 ofskatole, also known as methyl indole or methylated indole, another possible product of tryptophan degradation.
The positive red color forms as a result of a series of reactions. The para-Dimethylaminobenzaldehyde reacts withindole present in the medium to form a red rosindole dye. The isoamyl alcohol forms acomplex with rosindole dye, which causes it toprecipitate. The remaining alcohol and the precipitate then rise to the surface of the medium.
A variation on this test usingEhrlich's reagent (usingethyl alcohol in place of isoamyl alcohol, developed byPaul Ehrlich) is used when performing the test on nonfermenters andanaerobes.
Bacteria that test positive for cleaving indole from tryptophan include:Aeromonas hydrophila,Aeromonas punctata,Bacillus alvei,Edwardsiella sp.,Escherichia coli,Flavobacterium sp.,Haemophilus influenzae,Klebsiella oxytoca,Proteus sp. (notP. mirabilis andP. penneri),Plesiomonas shigelloides,Pasteurella multocida,Pasteurella pneumotropica,Vibrio sp., andLactobacillus reuteri.
Bacteria which give negative results for the indole test include:Actinobacillus spp.,Aeromonas salmonicida,Alcaligenes sp., mostBacillus sp.,Bordetella sp.,Enterobacter sp., mostHaemophilus sp., mostKlebsiella sp.,Neisseria sp.,Mannheimia haemolytica,Pasteurella ureae,Proteus mirabilis,P. penneri,Pseudomonas sp.,Salmonella sp.,Serratia sp.,Yersinia sp., andRhizobium sp.
The Indole test is one of the four tests of theIMViC series, which tests for evidence of an enteric bacterium. The other three tests include: themethyl red test [M], theVoges–Proskauer test [V] and thecitrate test [C].[1]