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.
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
- Prepare peptone broth in test tubes
and the tubes were autoclaved at 15 lbs/inch2 pressure for 15 mins.
- using sterile wire, inoculate
the broth with the given samples of organism and label the tubes with name
of organism
- Incubate the tubes at 37°C for 24-48
hours.
- After proper incubation, add 4-8
drops of kovac’s reagent to the tube touching the wall of glass tube
- Roll each tube between your palms to
mix the reagent through the culture.
- 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
- Indole
Test- Principle, Reagents, Procedure, Result Interpretation and
Limitations (microbiologyinfo.com)
- Indole
test: objective, principle, procedure and result - Online Biology Notes
- Indole test:
Introduction,Principle, procedure, result and interpretation (universe84a.com)
