Indole Test

IMViC Test Full Form

IMViC Test stands for Indole, Methyl red, Voges-Proskauer, Citrate Test. This as a set of four biochemical tests used to differentiate between Escherichia coli, Klebsiella Enterobacter, and Enterobacteriaceae family organisms.

Indole Test

The indole test is a biochemical test used to determine the ability of bacteria to produce the enzyme tryptophanase, which can break down the amino acid tryptophan into indole, pyruvic acid, and ammonia. The test is commonly used in microbiology to identify and classify bacteria based on their metabolic properties.

Organisms produce indole from the amino acid tryptophan by means of the enzyme tryptophanase. The source of tryptophan in the medium can be peptone or tryptone (which has an especially high content of tryptophan). The reaction of breakdown of tryptophan to indole is as shown in the figure below:

Figure – Enzymatic degradation of tryptophan

Indole reaction with Kovac’s/Ehrlich’s reagent:

The chemical reaction for detection of indole is based on the fact that when a pyrrole (indole = benzo-pyrrole) and a weakly acid alcoholic solution of p-dimethylaminobenzalaldehyde – HCL are mixed (in the presence of heat), a red-violet color develops. The reaction will occur without heat if the reagent is made with conc. HCL.

The indole produced by the organism can be detected with several different reagents, but the basic chemical reaction in the detection is the same.

The Ehrlich-Boehme reagent consists of two parts, p-dimethylaminobenzalaldehyde HCL in alcohol and potassium persulfate; each reagent is added, and a red colour (rose-indole dye) develops if indole is present.

Indole Test Definition

  1. An alternative procedure is to add a solvent, such as xylene, ether or chloroform, to extract and concentrate the indole: to this is then added the p-dimethylaminobenzalaldehyde reagent, and red colour is formed in the solvent layer, if indole is present.
  2. The latter procedure with xylene is the one usually used in the Ehrlich test.
  3. Kovac’s modified the Ehrlich-Boehme reagent by using amyl alcohol in place of ethyl alcohol and found that extraction and concentrate of indole occurred with addition of one reagent. Kovac’s reagent is probably the most widely used today.
  4. A more rapid test used by taking heavy inoculums from growth on a solid medium and inoculating a small amount (0.3-0.5ml) of tryptone broth.
  5. The tryptophanase already present in the organisms will break down the tryptophan in the broth within 4 hours, and indole may be detected in this short amount of time.         
  6. A rapid spot for indole production has also been used.
  7. In this test, a piece of filter paper moistened with p-dimethylaminobenzalaldehyde- reagent is placed in a petri dish.
  8. A loopful of growth from a medium containing tryptophan is placed on the filter paper; development of a red color within a few seconds indicates the presence of indole.

Requirements of Indole Test

Sodium chloride5.0g
Distilled water1000 ml
pH 7.4

Dissolve 10g of p-dimethylaminobenzalaldehyde in 150 ml of amyl, isoamyl or butyl alcohol. Heat it in a 56oC water bath until dissolved. Cool. Slowly add 50 ml of conc. HCL. Store it in a glass-stoppered brown bottle in the refrigerator. This reagent should be light yellow in color.

  • Ehrlich’s reagent

Dissolve 1 g of p-dimethylaminobenzalaldehyde in 95 ml of 95% ethyl alcohol. Slowly add 20 ml of conc. HCL acid. Store in a glass stoppered brown bottle in the refrigerator. This reagent should be light yellow in color.

Procedure of Indole Test

  1. Inoculate the tryptone broth (or 2% peptone broth) with the test culture. 
  2. Incubate at 37oC for 24 hours. 
  3. Add 1ml of xylene in the test tube and shake vigorously. 
  4. Let the tube stand for 1-2 minutes. 
  5. Slowly add about 0.5 ml of Kovac’s or Ehrlich’s reagent down of the side of the tube. Do not shake. 
  6. A reddish pink color imparted to the xylene layer indicates a positive test.

Indole Test Results

Indole Positive BacteriaIndole Negative Bacteria
Escherichia coliKlebsiella pneumoniae
Klebsiella oxytocaProteus mirabilis
V. choleraeSalmonella spp.
Proteus vulgarisShigella spp.
Porphyromonas asaccharolyticaPseudomonas aeruginosa
Vibrio spp.Bacteroides fragilis
Flavobacterium spp.Staphylococcus aureus
Haemophilus influenzaeCitrobacter freundii