Spot Indole Test- Objective, Principle, Procedure, Results, Limitations

Objective of Spot Indole Test

To determine the ability of organism for the production of indole by the action of tryptophanase enzyme.

Principle of Spot Indole Test

Intracellular enzyme tryptophanase mediate the production of indole by hydrolytic activity against amino acid Tryptophan. Bacteria that produce the enzyme tryptophanase are able to degrade the amino acid tryptophan into pyruvic acid, ammonia, and indole.  Indole is detected by its ability to combine with certain aldehydes to form a colored compound. For indole-positive bacteria the blue-green compound formed by the reaction of indole with cinnamaldehyde is easily visualized. The reaction occurs by a condensation process formed by an acid splitting of the protein. The absence of enzyme results in no color production implying indole negative reaction.

Procedure of Spot Indole Test

  1. Saturate a piece of filter paper with the 1% p-dimethylaminocinnamaldehyde reagent.
  2. Use a wooden stick or bacteriologic loop to remove a small portion of a bacterial colony from the agar surface and rub the sample on the filter paper.
  3. Observe for colour development within 1 to 3 minutes

Result Interpretation of Spot Indole Test

Result Interpretation of Spot Indole Test

Positive test: Development of a blue color within 1 to 3 minutes

Negative test: No color development or slightly pink color

Limitations of Spot Indole Test

  • Test only colonies cultured on media without glucose, as glucose inhibits indole production.
  • The bacterial inoculum should not be selected from MacConkey agar and EMB agar, because the color of lactose-fermenting colonies on this medium can interfere with test interpretation and also indicators in these media may cause a false positive result.
  • Certain strains of Proteus vulgaris, Providencia and Aeromonas spp. will give false negative reaction with the spot indole test.
  • Because adjacent colonies are likely to take up diffused indole, positive tests are only valid if pure cultures are tested.
  • When performing a spot test, Kovacs Indole Reagent may be used as a substitute for the spot test reagent. However, Kovacs Indole Reagent, when used as the spot test reagent, is less sensitive in detecting indole than the Indole Spot Reagent (DMACA).

Quality Control of Spot Indole Test

Positive: Escherichia coli (ATCC25922)

Negative: Klebsiella pneumoniae (ATCC13883)


  1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
  2. Miller JM, Wright JW. 1982. Spot Indole Test: Evaluation of Four Reagents. Jounal Of Clinical Microbiology. 589-592 Vol. 15, No. 4 0095-1137/82/040589-04$02.00/0.

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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