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
-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.
- 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
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.
Indole Positive Organisms:
E.coli., Proteus vulgaris, Enterococcus faecalis
Peptone Broth Composition:
Peptone
20g
Sodium chloride 5g
Distilled water 1000ml
Kovac’s Reagent:
Amyl alcohol or isoamyl acohol 150 ml
Paradimethyl aminobenzaldehyde 10g
Conc. Hcl
50 ml
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