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.
