Spot Indole Test Introduction

Many of the biochemical tests are used to perform the characteristics of an organism and its reaction towards enzyme in a medium.

Spot indole test is used to determine the presence of an enzyme tryptophanase.

Tryptophanase breaks down the tryptophan to release the indole. So, this test is commonly known as indole test.

It is one of the biochemical tests, that is performed on the specific species of bacteria that helps in determining the ability of the organism for the conversion of tryptophan into indole.

This division is generally performed by the chain of the different intracellular enzymes. This system is commonly referred as Tryptophanase.

According to biochemical experiments indole is usually generated by the reductive deamination from the tryptophan through the intermediate compound known as Indole pyruvic acid.

Here, the Tryptophan catalyzes the reaction of deamination, when the amide group of the tryptophan molecule is removed. The final products of this process involve indole, pyruvic acid, energy, and ammonium. Here pyridoxal phosphate is required as a coenzyme.

Spot Indole Test

Spot Indole test is used to determine the presence of an enzyme tryptophanase, which helps in breaking the tryptophan to release indole which on reacting with cinnamaldehyde forms a blue-green compound.

However, the absence of an enzyme results in no change in color, which represents the indole negative.

Indole test is one such biochemical tests, which is basically performed on the species of bacteria to determine the ability of the organism in conversion of the tryptophan into indole.

This division is usually performed by a numerous chain of reaction that contains variety of intracellular enzymes, which are generally referred to as tryptophanase.

Indole is generated here by a reducing deamination from tryptophan through the intermediate molecule known as indole pyruvic acid.

Here Tryptophanase catalyzes the deamination reaction during the process of removing an amine group from the tryptophan. Whereas the final products of the reactions are indole, pyruvic acid, ammonium, energy.

Spot Indole Test Objective

The main aim of the test is to determine the ability of the organism to produce the indole by the action of an enzyme tryptophan.

Spot Indole Test Principle

Principle of the spot indole test helps in mediating the enzyme intracellular enzyme produces the indole by hydrolytic activity against the amino acid Tryptophan.

Bacteria produces the enzyme tryphtophase plays a role in degrading this amino acid tryptophan and converts it into pyruvic acid, along with ammonia and indole.

Indole is usually detected by its ability of combine with some aldehydes and its results information of a colored substance.

Whereas in positive indole test, bacteria forms a blue-green compound which is formed by the reaction of the indole with the cinnamaldehyde which is visualized easily.

This reaction is usually happening during the process of condensation, which is formed by the acid, that helps in splitting of the protein.

Whereas the absence of the enzyme results in no change in color and implies the negative reaction of the indole.

Spot Indole Test Procedure

• Initially a piece of filter paper is saturated using 1% of p-dimethylaminocinnamaldehyde reagent.

• With the help of a wooden stick or using a bacteriologic loop a small portion of the bacterial colony is removed from the small portion of the colony of bacteria present in the agar and the sample is rubbed on the surface of the filter paper.

• Then the change in color in the medium is observed within one to three minutes.

Spot Indole Test Results

Positive Spot Indole Test: If the result is positive it results in development of a blue color within 3 minutes.

Negative Spot Indole Test: If the result is negative, there will be no change in color. In some cases, there will be a development of pale pink color.

Spot Indole Test Limitation

 Colonies are only tested if the cultured media doesn’t contain any glucose as glucose inhibits production of indole.

 It should also be noted that the bacterial inoculum should not be selected from the Macconkey agar and the EMB agar, as the color of the lactose fermenting colonies present in the medium has a maximum of chances to interfere with the test interpretation along with the indicators used in the media, which all results in false positive results.

 On the other hand, Some strains like proteus, vulgaris, Providencia and Aeromonas species gives the false negative reaction during spot indole test.

 As the adjacent colonies are likely to take up the indole in the diffused form, positive tests are only considered as a valid one, if only the pure cultures are cultured.

 While undergoing these tests, Kovacs indole reagent is generally used as a substitute for the spot test reagent, which is usually used as a substitute for the reagent in the spot test. Anyhow this reagent that is being used in the spot test is detected as a less sensitive one for detecting the role of indole in an Indole spot reagent.