Indole Test- Principle, Procedure, Results and Interpretation

 

Aim

To determine the ability of an organism to split amino acid tryptophan to Indole, which is important in the identification of Enterobacteriacea such as E.coli, Proteus vulgaris, P.rettgeri, Morganella morganii and Providencia sps., which have the capacity to produce indole.

Principle

Indole, a benzyl pyrrole, is one of the metabolic degradation products of the amino acid tryptophan. Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid, and ammonia. Indole production is an important characteristic in the identification of many species of microorganisms, being particularly useful in separating Escherichia coli (positive) from members of the Klebsiella-Enterobacter-Hafnia-Serratia group (mostly negative). The indole test is based on the formation of a red complex when indole reacts with the aldehyde group of p-dimethylaminobenzaldehyde. This is the active chemical in Kovac’s and Ehrlich’s reagents. A medium rich in tryptophan must be used. In practice, combination media such as sulfide indole motility (SIM), motility indole ornithine (MIO), or indole nitrate are used. Rapid spot tests, using filter paper strips impregnated with pdimethylaminocinnamaldehyde reagent, are useful in screening for bacteria that are prompt indole producers.

Image source: Koneman’s Color Atlas and Text book of Diagnistic Microbiology

Media and Reagents

Tryptophan Broth (1% tryptophan)

          Peptone or pancreatic digest of casein (trypticase)-  2 g

          Sodium chloride-  0.5 g

          Distilled water-  100 mL

Kovac’s Reagent

Pure amyl or isoamyl alcohol      -150 mL

p-Dimethylaminobenzaldehyde  - 10 g

Concentrated HCl                        - 50 mL

Ehrlich’s Reagent

p-Dimethylaminobenzaldehyde -  2 g

Absolute ethyl alcohol                - 190 mL

Concentrated HCl                       - 40 mL

 

Quality Control

Each new batch of medium or reagent should be tested for positive and negative indole reactions. The following organisms serve well as controls:

Positive control: Escherichia coli

Negative control: Klebsiella pneumoniae

Test Procedure

Inoculate tryptophan broth  with the test organism and incubate at 35°C for 18–24 hours. 

After incubation, add 15 drops of reagent down the inner wall of the tube.

If Ehrlich’s reagent is used, this step should be preceded by the addition of 1 mL xylene. This is not necessary with Kovac’s reagent.

Results and Interpretation

The development of a bright fuchsia red color at the interface of the reagent and the broth (or the xylene layer) within seconds after adding the reagent is indicative of the presence of indole and is a positive test.

Positive result: The presence of a cherry red ring formation in the alcohol layer on the surface of the broth.

Negative result: No cherry red ring formation appears.

Indole Test

Reference

1. Blazevic DJ, Ederer GM. Principles of Biochemical Tests in Diagnostic Microbiology. New York, NY: Wiley,
2. 1975:63–67.
3. Isenberg HD, Sundheim LH. Indole reactions in bacteria. J Bacteriol 1958;75:682–690.
4. MacFaddin JF. Biochemical Tests for Identification of Medical Bacteria. 2nd Ed. Baltimore, MD: Williams
5. & Wilkins, 1980:173–183.
6. Miller JM, Wright JW. Spot indole test: evaluation of four reagents. J Clin Microbiol 1982; 15:589–592.
7. Vracko R, Sherris JC. Indole-spot test in bacteriology. Am J Clin Pathol 1963; 39:429–432.
8. Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s Diagnostic Microbiology, 12th Ed., Mosby Elsevier.
9. Winn et al. Koneman’s Color Atlas and Text book of Diagnistic Microbiology. 6th Ed., Lippincott Williams & Wilkins, Wolters Kluwer.
10. Bile solubility Plate test: (A) Colony lysed (Positive). (B) Intact colony (Negative).