Indole test - Biochemistry, Procedure, Result, Interpretation, Introduction

Introduction TO Indole test

The indole test is a biochemical test used to determine if the test bacteria are able to convert the α-amino acid tryptophan into indole, an aromatic heterocyclic organic compound. The conversion of tryptophan by the microorganism takes place with the help of a number of intracellular enzymes collectively known as tryptophanase.

This is one of the most important biochemical tests as it differentiates between the genera and sometimes even species of bacteria, especially microorganisms of the Enterobacteriaceae family.

The indole test is one of the important groups of biochemical test series called IMViC- I = indole test, M = Methyl red test, Vi = Voges–Proskauer test, C = citrate test.

Biochemistry of Indole test

From the intermediate molecule indolepyruvic acid, an amine (-NH₂) is removed (deamination) by reduction, and the process is catalyzed by the enzyme tryptophanase in presence of water. For tryptophan to be reduced, a coenzyme- pyridoxal phosphate is essential.

As a final product of the reaction, indole, pyruvic acid, ammonium (NH₄+), and energy are released. The pyruvic acid can be metabolized by undergoing the glycolytic cycle or Kreb's cycle which releases CO₂, H₂O, and energy. By obtaining energy for an anabolic reaction, ammonium can be used to make amino acids.

The indole molecule, which has been split from tryptophan can be detected by the use of Kovac's reagent or Ehrlich's reagent which contains aldehyde to produce a red color. This whole biochemical process is based on the presence of pyrrole structure on indole. The pyrrole's CH₂ group permits condensation with aldehyde, which is present in the reagent giving a red pigmented ring on the top layer as a positive reaction.

Procedure of Indole test

The procedure of Indole test is as follows:

1. Take a sterile test tube and add approximately 4 ml of sterile tryptophan broth.

2. Using aseptic conditions, inoculate the tryptophan broth with the test organism with has been freshly cultured.

3. Incubate the inoculated tube at 35-37°C for 18-24 hours.

4. If Kovac's reagent is used:

   Add 4-5 drops of Kovac's reagent to the incubated test tube containing the test organism.

   Shake the tube gently and observe for the development of red color.

5. If Ehrlich's reagent is used:

   Add 0.1 ml of ether to the incubated test tube containing the test organism

   After ether rises to the surface of the medium, gently add 0.5 ml of Ehrlich's reagent to the tube by tilting the tube

Result, Interpretation of Indole test

The result and interpretation of Indole test is as follows:

Positive

Red color develops on the top layer of broth containing test bacteria.

Negative

No brick red color development on the top layer of the broth containing test bacteria. The broth will retain the yellow color of the reagent (Ehrlich or Kovac) itself.

Intermediate

Orange color is seen on the surface of the broth due to the formation of skatone.

* The methylated compound skatone may be a precursor of indole

Indole-Positive Bacteria

The list of Indole-positive bacteria are:

• Aeromonas hydrophila

• Aeromonas punctate

• Bacillus alvei

• Edwardsiella sp.

• Escherichia coli

• Enterococcus faecalis

• Flavobacterium sp.

• Haemophilus influenzae

• Klebsiella oxytoca

• Lactobacillus reuteri

• Pasteurella multocida

• Pasteurella pneumotropica

• Proteus sp. (Proteus mirabilis, Proteus penneri not included)

• Plesiomonas shigelloides

• Vibrio sp.

Indole-Negative Bacteria

The list of Indole-negative bacteria are:

• Actinobacillus spp.

• Aeromonas salmonicida

• Alcaligenes sp.

• Bacillus sp.

• Bordetella sp.

• Enterobacter sp.

• Haemophilus sp.

• Klebsiella sp.

• Neisseria sp.

• Mannheimia haemolytica

• Pasteurella ureae

• Proteus mirabilis

• Proteus penneri

• Pseudomonas sp.

• Salmonella sp.

• Serratia sp.

• Yersinia sp.

• Rhizobium sp.