Indole Production Test

Objective:

1.     To determine the organism is producing indole from amino acid tryptophan

2.     To realize that amino acids can be used by microorganisms as energy source

3.     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 Regent (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.  Bacteria producing a red layer following addition of Kovacs’ reagent are indole positive; the absence of a red color indicates tryptophan was not hydrolyzed, and the bacteria are indole negative.

  

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 tube with E.Coli

           -  No Cherry red color formation  (Yellow color) in Klebsiella sp., tube.

Result :

-  E.coli  : Indole Positive

 - Klebsiella sp.., Gives Negative Result in Indole Production  

Interpretation:

          E.coli utilizes tryptophan with the help of tryptophanse enzyme and produces indole ,it gives cherry red color when react with Kovac’s reagent. Klebsiella sp., doesn’t produces tryptophanse enzyme so when it react with reagent  its shows only yellow color.