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.