Indole test – Principle, Procedure, Uses.

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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.

Indole Test Definition

  • Indole test can be described as a test that uses biochemistry on bacterial species to test their capacity to create indole by combining tryptophan with in combination with a class of enzymes referred to as “tryptophanase”.
  • It is a test of qualitative nature which tests how tryptophan is converted indole.
  • The test is conducted as part of the IMViC test which can be used to distinguish between individuals belonging to the Enterobacteriaceae family.
  • It’s crucial in the detection of various bacteria such as Escherichia coli Proteus, Morganella, etc.
  • It is a key element in the analysis of coliforms, which is evident by the many variations of the test. It is also used in conjunction of other test biochemicals.
  • It’s still employed as a method of separating indole-positive E. E. coli from indole-negative Enterobacter or Klebsiella.
  • A different version of the test is called Ehrlich’s reagent (using the ethyl alcohol in place of alcohol isoamyl) is utilized in cases where the test must be carried out on non-fermenters or anaerobes.
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Purpose of Indol Test

The Indol Test checks the capacity of an organism to break down the amino acid tryptophan to create indole. It is component of the IMViC tests which is a series of tests that are designed to differentiate from the members of the group Enterobacteriaceae.

Principle of Indole Test

indole test 1

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.

Reagents Used in Indole Test

Reagents Used

  • For the spot test, 5% p-dimethylaminobenzaldehyde or 1% pdimethylaminocinnamaldehyde is prepared in 10% (v/v) concentrated HCl.
  • For the tube method, Kovac’s reagent is used for aerobic organisms, and Ehrlich’s reagent is used for anaerobes and weak indole producers.

Kovac’s reagent

p-dimethylaminobenzaldehydre50 gram
Hydrochloric acid250 ml
Amyl Alcohol750 ml
  • The medium used with this reagent is either broth containing tryptophan, motility- indole-ornithine agar, or sulfide-indole-motility agar (SIM).

Ehrlich’s reagent

p-dimethylaminobenzaldehydre1 gram
Hydrochloric acid20 ml
Ethyl alcohol95 ml
  • The medium is utilized with Ehrlich’s reagent is heart infusion or anaerobic medium with tryptophan.

Other Supplies Erquired

  • Sterile loop, swab, or stick for inoculation
  • Filter paper (optional)

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.

Quality Control for Indole Test

  • Do not use benzaldehyde-based agents (including Kova’cs’ and Ehrlich’s) in the event that the color isn’t light yellow.
  • Perform QC on every new batch of reagent before using them. QC on reagents that are prepared in-house since they may degrade particularly if they are not kept at temperatures of 4 degrees Celsius. Refrain from using if the reaction becomes weak.
  • Organisms
    • Escherichia coli ATCC 25922—indole positive
    • Pseudomonas aeruginosa ATCC 27853—indole negative
    • For Ehrlich’s reagent for use with anaerobic microorganisms
      • Porphyromonas asaccharolytica ATCC 25260—indole positive
      • Bacteroides fragilis ATCC 25285—indole negative

Limitations of Indole Test

  • Indole tests can be utilized as a tool for determination of the difference between gram-positive and Gram-negative organisms. Additional biochemical testing with pure cultures is suggested to confirm the identity of the organism.
  • Tube tests are more accurate in detecting indole than a spot test.
  • If you are performing a spot test Kovacs Indole Reagent could be used in lieu to the spot test test reagent. It is however, Kovacs Indole Reagent, in the case of being used as the spot test reagent is not as sensitive to detect indole than Indole Spot Reagent (DMACA).
  • Kovacs Indole Reagent isn’t advised for use in conjunction for anaerobic bacteria. Its Indole Spot Reagent (DMACA) is appropriate for use with anaerobes.
  • Peptones have been proven to differ in their ability to be used for indole testing, the media used to be used for determination of indole should be tested using recognized positively and negatively disposed organisms in order to confirm their the suitability.
  • It is not recommended that glucose-containing media be used in the testing of indoles due to the production of acid-end products that have been found to reduce the production of indoles. Mueller Hinton Agar should also not be used in this test as tryptophan gets removed during the acid hydrolysis process of casein.
  • Media that contain dye like MacConkey and EMB are not appropriate sources for inoculum due to possible transfer of dye and the subsequent interfering with the indole color processing.
  • Indole-positive colonies are known to cause indole-negative colonies to appear as false positive due to the spread of indole through the media. To prevent false positives, select colonies with different morphologies which have a distance of at least 5mm to allow for indole tests.

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

Indole-Positive Bacteria

Bacteria with a positive test for the ability to cleave indole from tryptophan are: 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 that produce negative results on the indole test are: Actinobacillus spp., Aeromonas salmonicida and Alcaligenes sp. Most Bacillus species., Bordetella sp., Enterobacter sp., the majority of Haemophilus sp. The most Klebsiella isp., Neisseria sp., Mannheimia haemolytica, Pasteurella ureae and Proteus mirabilis. P. penneri Pseudomonas Sp., Salmonella sp., Serratia sp., Yersinia sp., and Rhizobium sp.

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