Indole test : Principle, Procedure, Results and Interpretation

Summary :

◉ Overview

As early as 1889, the indole test was used as a means of distinguishing between Escherichia coli and Enterobacter aerogenes , the test looks for the ability of an organism to degrade tryptophan and produce indole, Indole production is demonstrated by the addition of Kovac's reagent which acts with the indole giving a red color.

◉ Principe of indole test

● The indole test determines the ability of an organism to produce indole from the breakdown of the amino acid tryptophan. Tryptophan is hydrolyzed by tryptophanase to produce three possible products: indole, pyruvate and ammonium ion :


Indole test reaction

Detection of indole is based on the chemical reaction between indole and Kovac's reagent (isoamyl alcohol, para-dimethylaminobenzaldehyde, concentrated hydrochloric acid) under acidic conditions:

  1. Para-Dimethylaminobenzaldehyde reacts with the indole present in the medium to form a rosindole red dye.
  2. Isoamyl alcohol forms a complex with the "rosindole" dye, causing it to precipitate.
  3. The remaining alcohol and the precipitate then rise to the surface of the medium.

◉ Procedure and results

◍ Two methods are described:

1- Conventional tube method

The main requirement for a suitable indole test medium is that it contains a sufficient amount of tryptophan (Tryptone broth, indole-free peptone water medium, Urea-indole medium, Tryptophan peptone broth, Sulfide-indole motility medium ( SIM) ...).

  1. Inoculate the tryptophan (or peptone) broth with the organism to be tested and incubate at 37 ° C for 24 to 48 hours.
  2. Add 0.5 ml (5 drops) of Kovác's reagent and shake gently.
  3. Examine the top layer of liquid after about 1 min.

◉ Results : A positive result is indicated by the presence of a red or red-violet color in the alcohol layer on the surface of the broth. A negative result appears in yellow. A variable result may also occur, displaying an orange color as a result. This is due to the presence of skatole, also known as methyl indole or methylated indole, another possible breakdown product of tryptophan.


Indole positive and negative test

2- Spot test

  1. Place several drops (1 -1.5 ml) of Indole Spot reagent (1% or 5% p-methylaminobenzaldehyde or 1% p-dimethylaminocinnamaledhyde) on a piece of filter paper.
  2. Spread an isolated pure colony (from an 18-24 hour culture) on the saturated surface of the filter paper using a sterile loop.
  3. Examine immediately

Indole Spot test

◉ Result : Depending on the reagent used for the indole spot test, the resulting colors differ. If you are using p-methylaminobenzaledhyde the presence of indole is indicated by a red color and if you are using p-dimethylaminocinnamaledhyde a blue-green color is observed.

◉ Indole test positive and negative

◍ Indole positive and negative bacteria (9, 10, 11)

Bacteria Indole positive Variable indole Indole negative
Escherichia spp. Escherichia coli , Escherichia fergusonii / Escherichia albertii
Shigella spp. / Shigella boydii , Shigella dysenteriae , Shigella flexneri Shigella sonnei
Salmonella spp. / / Salmonella spp.
Klebsiella spp. K. oxytoca / klebsiella pneumoniae
Raoultella spp. R. ornithinolytica R. planticola R. terrigena
Serratia spp. / S. odorifera biogroup 1, S. odorifera biogroup 2 S. entomophila, S. ficaria, S. fonticola, S. liquefaciens group, S. marcescens, S. plymuthica, S. rubidaea
Citrobacter spp. C. amalonaticus, C. farmeri, C. koseri, C. murliniae, C. braakii ,C. freundii , C. sedlakii, C. youngae C. gillenii , C. werkmanii
Edwardsiella tarda Edwardsiella tarda / /
Proteus P. hauseri , P. vulgaris / Proteus mirabilis , P. penneri
Providencia P. stuartii , P. rettgeri , P. rustigianii , P. alcalifaciens / P. heimbachae
Morganella M. morganii / /
Yersinia spp. / Y. enterocolitica Y. pestis , Y. pseudotuberculosis
Bacilles à Gram négatif non fermentaire Elizabethkingia ,Chryseobacterium , Balneatrix, Bergeyella, Empedobacter, Wautersiella, Weeksella / Acinetobacter , Moraxella, Paracoccus, Sphingobacterium, Shewanella, Rhizobium, Ochrobactrum, Psychrobacter, Sphingomonas, Pseudochrobactrum
Actinobacillus spp / / A. lignieresii, A. equuli , A. suis, A. ureae, A. hominis
Actinobacillus spp / / A. lignieresii, A. equuli , A. suis, A. ureae, A. hominis
Aggregatibacter spp / / A. actinomycetemcomitans , A. aphrophilus, A. segnis
Capnocytophaga spp / / C. ochracea , C. sputigena, C. gingivalis, C. granulosa, C. haemolytica, C. canimorsus, C. cynodegmi
Cardiobacterium spp C. hominis (faible) C. valvarum /
Chromobacterium spp / C. violaceum C. haemolyticum
Dysgonomonas spp D. hofstadii , D. mossii D. capnocytophagoides , D. gadei /
Eikenella spp / / E. corrodens
Kingella spp / / K. kingae , K. denitrificans, K. negevensis, K. oralis , K. potus
Pasteurella spp P. multocida , P. canis , P. dagmatis , P. oralis , P. stomatis / /
Streptobacillus spp / / S. moniliformis
Suttonella spp S. indologenes / /
Haemophilus influenzae Biotype I II V VII / Biotype III IV VI VIII
Actinomyces spp. / / Actinomyces spp.
Bacteroides spp. B. ovatus , B. thetaiotaomicron / B. fragilis , B. vulgatus
Fusobacterium spp. F. necrophorum , F. nucleatum F. varium F. mortiferum
Clostridium spp. C. sordellii , C. tetani C. tertium , C. septicum, C. ramosum, C. perfringens, C. innocuum, Clostridium botulinum

◉ Limits and technical advice


  1. The Hardy Diagnostics-USA-2/16 : Indole Test Reagents Kovacs, DMACA, Spot test
  2. MICHAEL MILLER : Spot Indole Test: Evaluation of Four Reagent
  3. Biorad manuel : PEPTONE WATER INDOLE FREE
  4. Biorad manuel : PEPTONE WATER INDOLE FREE
  5. Public health England : Identification of Enterobacteriaceae
  6. Public health England : Indole test
  7. Biorad manuel : UREA INDOLE
  8. American society for microbiology : Indole Test Protocol
  9. ASM MicrobeLibrery
  10. Color Atlas of Medical Bacteriology (ASM Books) 3rd Edition
  11. Jin-hyung Lee, Jintae Lee : Indole as an intercellular signal in microbial communities.
  12. UK Standards for Microbiology Investigations