Indole tes
Indol
test is the biochemical test .This test detection of
indoles such as LSD, see Ehrlich's reagent.
The indole
test is a biochemical test performed on bacterial
species to determine the ability of the organism to
convert tryptophan into indole.
This division is performed by a chain of a number of different
intracellular enzymes,
a system generally referred to as "tryptophanase.
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.
A positive
result is shown by the presence of a red or red-violet color in the surface
alcohol layer of the broth. A negative result appears yellow. A variable result
can also occur, showing an orange color as a result. This is due to the
presence of skatole, also known as methyl indole or methylated
indole, another possible product of tryptophan degradation.
The positive red color forms
as a result of a series of reactions. The para-Dimethylaminobenzaldehyde reacts
with indole present in the medium to form a red
rosindole dye. The isoamyl alcohol forms a complex with
rosindole dye, which causes it to precipitate.
The remaining alcohol and the precipitate then rise to the surface of the
medium.
Indole-Positive Bacteria
Bacteria that test positive
for cleaving indole from tryptophan include: Aeromonas
hydrophila, Aeromonas punctata, Bacillus alvei, Edwardsiella sp., Escherichia coli, Flavobacterium sp., Haemophilus
influenzae, Klebsiella oxytoca, Proteus sp.
(not P. mirabilis and P. penneri), Plesiomonas
shigelloides, Pasteurella
multocida, Pasteurella pneumotropica, Enterococcus
faecalis, Vibrio sp., and Lactobacillus
reuteri.
Indole-Negative Bacteria
Bacteria which give negative
results for the indole test include: Actinobacillus spp., Aeromonas
salmonicida, Alcaligenes sp., most Bacillus sp., Bordetella sp., Enterobacter sp., most Haemophilus sp., most Klebsiella sp., Neisseria sp., Mannheimia haemolytica, Pasteurella ureae, Proteus mirabilis, P. penneri, Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp., and Rhizobium sp.
The Indole test is one of the
four tests of the IMViC series, which
tests for evidence of an enteric bacterium. The other three tests include:
the methyl red test [M],
the Voges–Proskauer
test [V] and the citrate test.
In the spot test, indole combines, in the filter
paper matrix, at an acid pH with p-Dimethylaminocinnamaldehyde (DMACA) to
produce a blue
to blue-green compound. Indole
Spot Reagent has been reported to be useful in detecting indole production by
members of the family Enterobacteriaceae and certain anaerobic species.
Reagents Used in Indole Test
|
Indole Spot Reagent:
|
|
p-Dimethylaminocinnamaldehyde
(DMACA)
|
10.0
gm
|
|
Hydrochloric Acid, 37%
|
100.0
ml
|
|
Deionized Water
|
900.0
ml
|
|
Indole Kovacs Reagent:
|
|
p-Dimethylaminobenzaldehyde
|
50.0
gm
|
|
Hydrochloric Acid, 37%
|
250.0
ml
|
|
Amyl Alcohol
|
750.0
ml
|
Procedure of Indole Test
- Take a
sterilized test tubes containing 4 ml of tryptophan broth.
- Inoculate the
tube aseptically by taking the growth from 18 to 24 hrs culture.
- Incubate the
tube at 37°C for 24-28 hours.
- Add 0.5 ml of
Kovac’s reagent to the broth culture.
- Observe for the
presence or absence of ring.
Indole Spot Reagent (DMACA) Procedure
1.
Place
several drops of Indole Spot Reagent on a piece of filter paper.
2.
With
an inoculating loop or wooden applicator stick, pick a portion of an 18-24 hour
isolated colony from a non-selective media and rub it onto the reagent
saturated area of the filter paper.
3.
Examine
immediately
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.
Examples: Aeromonas hydrophila, Aeromonas
punctata, Bacillus alvei,Edwardsiella sp., Escherichia
coli, Flavobacterium sp., Haemophilus influenzae, Klebsiella
oxytoca, Proteus sp. (not P. mirabilis and P.
penneri), Plesiomonas shigelloides,Pasteurella multocida, Pasteurella
pneumotropica, Enterococcus faecalis, and Vibrio sp.
Negative: No color
change even after the addition of appropriate reagent.
Examples: Actinobacillus spp., Aeromonas
salmonicida, Alcaligenes sp., most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp.,
most Haemophilus sp., most Klebsiella sp., Neisseria sp., Pasteurella
haemolytica, Pasteurella ureae, Proteus mirabilis, P.
penneri, Pseudomonas sp.,Salmonella sp., Serratia sp., Yersinia sp.
Indole Spot Reagent Result
Positive
reaction: The
development of a blue colour within 3 minutes.
Negative
reaction: The
development of a pink colour within 3 minutes.
Method
i.
Place
several drops of 1% p-dimethylaminocinnamaldehyde reagent on a piece of filter
paper until saturation.
ii.
With
an inoculating loop or wooden applicator stick, pick a portion of an 18-24 hour
isolated colony from a non-selective media and rub it onto the reagent
saturated area of the filter paper.
iii.
Observe
for colour development within 1 to 3 minutes.
Expected Results
Ø Positive: A positive
reaction is denoted by the appearance of a blue to blue-green color change on
the bacterial smear within 2-3 minutes.
Ø Negative: Negative
reactions remain colorless or light pink.
Note: Positive reaction is Red-violet in the case of Providencia alcalifaciens.
Note
:-
- Indole tests may be used as an aid in the
identification and differentiation of gram-positive and gram-negative
organisms.
- Additional biochemical testing using pure cultures is
recommended for complete identification.
- The tube test is a more sensitive method of detecting
indole test.
- Kovacs Indole Reagent may be used as a substitute for
the spot test reagent. However, Kovacs Indole Reagent, when used as the
spot test reagent, is less sensitive in detecting indole than the Indole
Spot Reagent (DMACA).
- Kovacs Indole Reagent is not recommended for use with
anaerobic bacteria. The Indole Spot Reagent (DMACA) is suitable for
anaerobe use.
- Since peptones have been shown to vary with regard to
their suitability for use with indole testing, media selected for indole
determination should be tested with known positive and negative organisms
to insure suitability.
- Media containing glucose should not be used for indole
testing due to the formation of acid end products which have been shown to
reduce indole production. Mueller Hinton Agar should also not be used for
this test because tryptophan is destroyed during acid hydrolysis of
casein.
- Media containing dye, such as MacConkey and EMB, are
unsuitable sources of inoculum due to possible carryover of dye and
subsequent interference of indole color interpretation.
- Indole-positive colonies have been reported to cause
adjacent indole-negative colonies to appear false-positive due to
diffusion of indole into the media. To avoid false-positives, select
colonies of different morphologies that are separated by at least 5mm for
indole testing.
