Biochemical Test

Indole test – Principle, Procedure, Uses.

THIS BLOG INCLUDES: hide 1 Principle of Indole Test 2 Procedure of Indole Test 3 Uses of Indole Test 4 List of...

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Indole test is a biochemical process,  which is used to identify the indole producing organism from tryptophan. Tryptophan is an important amino acid which is found in most bacterial cell protein.

Bacteria those have tryptophanase enzyme will hydrolyze tryptophan into different metabolic products such as indole, pyruvic acid, and ammonia. Then the bacterial cell will utilize the pyruvic acid and ammonia as a nutrient and indol will started to accumulate in bacterial surrounding medium. The presence ofindol can be detected by using the indol test.

Principle of Indole Test

Tryptophan converted into indol by reductive deamination, via the intermediate molecule indolepyruvic acid. This reaction is catalyzed by the enzyme Tryptophanase, which helps in the removal of  amine (-NH2) group from the tryptophan molecule.

Then, the presence of Indol  can be detected by adding Kovacs’ reagent (4 (p)-dimethylamino benzaldehyde) to the medium containing indol forming microorganism. This Kovacs’ reagent will form a bright red compound on the surface of the medium by reacting with indol. 

Indole test


Figure: Conversation of Tryptophan to Indole With the help of Tryptophanase Enzyme

In the spot Indol test, p-Dimethylaminocinnamaldehyde (DMACA) combined with the indole on filter paper matrix and form a blue to blue-green compound.


Procedure of Indole Test

There Are present two different procedure for indole test;

    1. Conventional Tube method for Indole Test.
    2. Spot indole test

Conventional Tube method for Indole Test


    • Nutrient broth culture of the test organism ( E coli,  enterobacter aerogenes, P. vulgaris )
    • test tubes
    • 1% tryptone broth
    • Kovac’s reagent (4 (p)-dimethylamino benzaldehyde)
    • Inoculating loops

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1% tryptone broth preparation: Dissolve 10 grams of peptone in 1 liter distilled water and then sterilize it using an autoclave.

Indole Kovacs Reagent: Mix 50.0 gm p-Dimethylaminobenzaldehyde with 250.0 ml of 37% Hydrochloric Acid and 750.0 ml of Amyl Alcohol.

Procedure of Conventional Tube method

    1. First of all, take two sterilized test tube,  and then add 5 ml nutrient broth culture to each test tube.
    2. After that added selective organism in one test tube and another one leave blank.
    3. Label the first test tube with the organism name And the second test tube labeled as control.
    4. After that incubate all these test tubes at 35-degree centigrade for  24-28 hours.
    5. After incubation add 5 ml Kovac’s reagent to each test tube including control.
    6. Keep them all in a static condition,  to allow them for reaction.

Result of  Conventional Tube method for Indole Test

  1. Positive Indol test: If within 30 seconds the color changes pink to red (“cherry-red ring”), it indicates a positive result. It means the test organism produces indole in medium.
  2. Negative Indol test: If no color change occurs it indicating a negative result.  The test organism has no ability for indole production.

Spot Indole Test

spot indole test


Figure: Spot Indole Test | Image Source:

This test only perform to detect the presence of tryptophanase enzyme. Tryptophanase enzyme convert the tryptophan into Indol, which then reacts with p-Dimethylaminocinnamaldehyde (DMACA) and form a blue-green compound. 

Requirement for spot indole test:

  • Indole Spot Reagent
  • Filter paper

Composition of Indole Spot Reagent: Mix 10.0 gm p-Dimethylaminocinnamaldehyde (DMACA) with 100.0 ml of 37% Hydrochloric Acid and 900.0 ml of Amyl Alcohol.

Procedure of Spot Indole Test

  1. Take 3-5  drop of Dimethylaminocinnamaldehyde (DMACA)  on a filter paper
  2. Then transfer a portion of an 18-24 hour isolated colony from a non-selective media On this reagent saturated filter paper using an inoculating loop.
  3. Then observe the color change

Result of Spot Indole Test:

    • Positive Indol test: If Within 2 to 3 minutes it develops a Blue color then it indicating a positive result, which means the microorganism has the ability of Indol production. 
    • Negative Indol test: If no color change occurs then it indicating a negative result, which means the test organism has no ability for indole production.

Uses of Indole Test

    1. Used in wastewater treatment for the detection of coliform bacteria.
    2. Used for the differentiation between Klebsiella species, For example,  Klebs‪iella oxytoca shows  indole positive and Klebsiella pneumoniae shows indole negative result in indol test.
    3. Used for the differentiation between Citrobacter species, For example,  Citrobacter Koseri shows indole positive and Citrobacter freundii shows indole negative result in indol test.
    4. Used for the differentiation between Proteus species, For example,  Proteus Vulgaris shows indole positive and Proteus mirabilis shows indole negative result in indol test.

List of Indole positive And Negative Organisms

Microorganism NameIndole PositiveIndole Negative
Actinobacillus spp.NoYes
Aeromonas hydrophilaYesNo
Aeromonas punctataYesNo
Aeromonas salmonicidaNoYes
Alcaligenes sp.NoYes
Bacillus alveiYesNo
Bacillus sp.NoYes
Bordetella sp.NoYes
Edwardsiella spYesNo
Enterobacter sp.NoYes
Enterococcus faecalisYesNo
Escherichia coliYesNo
Flavobacterium sp.YesNo
Haemophilus influenzaeYesNo
Haemophilus sp.NoYes
Klebsiella oxytocaYesNo
Klebsiella sp.NoYes
Lactobacillus reuteriYesNo
Mannheimia haemolyticaNoYes
Neisseria sp.NoYes
P. penneriNoYes
Pasteurella multocidaYesNo
Pasteurella pneumotropicaYesNo
Pasteurella ureaeNoYes
Plesiomonas shigelloidesYesNo
Proteus mirabilisNoYes
Proteus sp. (not P. mirabilis and P. penneri)YesNo
Pseudomonas sp.NoYes
Rhizobium sp.NoYes
Salmonella sp.NoYes
Serratia sp.NoYes
Vibrio spYesNo
Yersinia sp.NoYes
List of Indole positive And Negative Organisms

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