SENTHIL PRABHU SIVASAMY: August 2017

Objective:
-To determine the organism is producing indole from amino acid tryptophan
-To realize that amino acid can be used by microorganisms as energy source
-To identify the tryptophanase enzyme producing organism

Principle:

Tryptophan is an amino acid that can undergo deamination and hydrolysis by tryptophanase enzyme which is produced by bacteria. 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(p-DMAB) 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.

Materials required:

Cultures : E.coli, Klebsiella sp.,

Medium : Tryptone/Peptone broth

Indicator : Kovac’s reagent

Procedure :

1 -Inoculate one loopful of test culture into the peptone broth tube.

2 -The tubes are incubating at 37 ͦ C for 24 hrs.

3 -The next day gently add Kovac’s reagent into the culture (ratio 1:10, that means 0.5 ml into 5 ml of cultue)

4 - Mix well and wait for upto 5 minutes to develop the color.

Observation:
- Cherry red color formation on top layer of the E.Coli broth.

- No color change (Yellow color) in Klebsiella sp., broth.

Result :

- E.coli : Indole Positive

- Klebsiella sp., : Indole Negative

Interpretation:

E.coli utilizes tryptophan with the help of tryptophanse enzyme and produces indole positive,it gives cherry red color when react with Kovac’s reagent . The absence of a red color indicates tryptophan was not hydrolyzed by bacteria (Klebsiella sp.,) are indole negative.