Rapid Indole Broth

Cat. no. Z23 Rapid Indole Broth, 15x45mm Tube, 0.5ml 10 tubes/vial


Hardy Diagnostics Rapid Indole Broth, is recommended for use in the detection of indole production by microorganisms, especially Haemophilus spp., Cardiobacterium spp., Suttonella spp. and other fastidious bacteria isolated from clinical specimens.


Detection of indole in a tryptophan enriched medium was first described by Kovac in 1928. (5) Organisms possessing the enzyme tryptophanase degrade tryptophan to produce indole and other metabolic products. Fastidious organisms are normally tested with Ehrlich's Reagent to detect weak indole production, but this requires a 24-48 hour incubation followed by an extraction with xylene.

Indole production can be determined by the production of a pink-red color complex upon application of Kovac's Reagent (Cat. no. Z67). If present, indole reacts with the aldehyde group of p-dimethylaminobenzaldehyde, the active chemical in Kovac's Reagent. Microorganisms that do not possess tryptophanase will not produce a pink-red color change upon application of the reagent. Hardy Diagnostics' Rapid Indole Broth produces a simple color change result in two to four hours and does not require a xylene extraction.


Ingredients per liter of deionized water:*

L-Tryptophan 10.0gm
Sodium Phosphate 0.35gm
Potassium Phosphate 0.35gm

Final pH of 6.6 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.



Specimen Collection: This medium is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Method of use:

1. Prior to inoculation, allow the medium to equilibrate to room temperature.

2. Obtain organisms from an 18-24 hour pure culture from an appropriate plated medium and heavily (equivalent to a 4.0 McFarland turbidity standard or higher) inoculate the broth tube.

3. Incubate the inoculated broth aerobically at 35ºC. for 2-4 hours; incubate with loosened caps.

4. Following incubation, use a sterile pipet to add 0.5ml (approximately 20 drops) Kovac's Reagent (Cat. no. Z67) to the tube, shake gently.

5. Allow the tube to stand for 1-2 minutes.

6. Observe for the production of a bright pink to red color.


A positive indole test reaction is indicated by the appearance of a bright pink to red color.

A negative reaction is indicated by the medium remaining colorless or light yellow due to the color of the reagent.



Standard microbiological supplies and equipment such as loops, slides, staining supplies, Kovacs Reagent (Cat. no. Z67), other culture media, microscopes, incinerators, incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Haemophilus influenzae
ATCC ® 49766**
E 2-4hr 35°C Aerobic Positive; turns pink to red after the addition of 0.5ml Kovac's Reagent and agitation
Aggregatibacter aphrophilus
ATCC ® 7901**
E 2-4hr 35°C Aerobic Negative; no pink color after adding Kovac's Reagent

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control


Rapid Indole Broth should appear clear, and colorless to light yellow in color.

Rapid Indole Broth

Left - Negative Indole Test:
Haemophilus parainfluenzae (ATCC ® 7901) was incubated in Rapid Indole Broth (Cat. no. Z23) under aerobic conditions for 2 hours at 35ºC. 0.5ml of Indole Kovac's Reagent (Cat. no. Z67) was added directly to the broth and the tube was gently shaken.

Uninoculated tube of Rapid Indole Broth (Cat. no. Z23).

Right - Positive Indole Test:
Haemophilus influenzae (ATCC ® 49766) was incubated in Rapid Indole Broth (Cat. no. Z23) under aerobic conditions for 2 hours at 35ºC. 0.5ml of Indole Kovac's Reagent (Cat. no. Z67) was added directly to the broth and the tube was gently shaken.


1. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

2. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

5. Kovac, N. 1928. Zeit. Immunitest, Exper. Therap.; 53:311.

ATCC is a registered trademark of the American Type Culture Collection.