Utilization of Amino acid as Carbon Source -Indole Test

 

INDOLE TEST

Aim

• To detect the formation of indole from tryptophan by the enzymatic action of tryptophanese.
• To differentiate members of family Enterobacteriaceae, especially E. coli from Enterobacter and Klebsiella.

 Principle

Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that express tryptophanase enzyme.  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, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzyme.

 

When indole is combined with Kovac’s Reagent (which contains hydrochloric acid and p-dimethylaminobenzaldehyde in amyl alcohol) the solution turns from yellow to cherry red. Because amyl alcohol is not water soluble, the red coloration will form in an oily layer at the top of the broth.

Indole production test is important in the identification of Enterobacteria. Most strains of E. coli, P. vulgaris, P. rettgeri, M. morgani and Providencia species break down the amino acid tryptophan with the release of indole. This is performed by a chain of a number of different intracellular enzymes, a system generally referred to as “tryptophanase.” It is used as part of the IMViC procedures,a tests designed to distinguish among members of the family Enterobacteriaceae.

Materials required

• Peptone broth ( a nutrient enriched with Amino acid tryptophan)
• Bacterial sample (Escherichia coli and Klebsiella spp)
• Kovac’s reagent

Procedure

1. Prepare peptone broth in test tubes and the tubes were autoclaved at 15 lbs/inch2 pressure for 15 mins.
2.  using sterile wire, inoculate the broth with the given samples of organism and label the tubes with name of organism
3. Incubate the tubes at 37°C for 24-48 hours.
4. After proper incubation, add 4-8 drops of kovac’s reagent to the tube touching the wall of glass tube
5. Roll each tube between your palms to mix the reagent through the culture.
6. Let stand for a while and observe for the development of cherry red color at the surface of media.

Result

Positive: Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the medium within seconds of adding the reagent.

Negative: No color change even after the addition of appropriate reagent.

Indole test positive organisms

Escherichia coli, Klebsiella oxytoca, Proteus vulgaris, Citrobacter koseri, Morgenella morganii, Vibrio cholera, Providencia species, Aeromonas species, Plesiomonas species, Pasteurella species, Cardiobacterium hominis, Propionibacterium acenes

Indole test negative organisms

Klebsiella pneumoniae, Enterobacter species, Proteus mirabilis, Citrobacter freundii

Uses of Indole Test

• The indole test is used to test an organism’s ability to utilize tryptophan and produce indole.
• The test is used to differentiate members of the Enterobacteriaceae family as a part of the IMViC test.
• The test also differentiates Proteus mirabilis from other Proteus species.
• This test differentiates indole-positive E. coli from indole-negative Enterobacter and Klebsiella.
• The test further differentiates K. pneumoniae(indole negative) from K. oxytoca (indole positive) and Citrobacter freundii (indole negative) from Citrobacter koseri (indole positive).

Write left side

Indole Kovacs Reagent:
p-Dimethylaminobenzaldehyde	50.0 gm
Hydrochloric Acid, 37%	250.0 ml
Amyl Alcohol	750.0 ml

 

Reference

1. Indole Test- Principle, Reagents, Procedure, Result Interpretation and Limitations (microbiologyinfo.com)
2. Indole test: objective, principle, procedure and result - Online Biology Notes
3. Indole test: Introduction,Principle, procedure, result and interpretation (universe84a.com)