Cat. no. C5870 CRITERION™ Indole Nitrate Medium 50gm
Cat. no. C5871 CRITERION™ Indole Nitrate Medium 500gm
Cat. no. C5872 CRITERION™ Indole Nitrate Medium 2kg
Cat. no. C5873 CRITERION™ Indole Nitrate Medium 10kg
Cat. no. C5874 CRITERION™ Indole Nitrate Medium 50kg


Hardy Diagnostics CRITERION™ Indole Nitrate Medium is recommended for the detection of indole production and nitrate reduction by microorganisms.

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.


CRITERION™ Indole Nitrate Medium is a combined test medium for indole and or nitrate determination for bacteria other than the Enterobacteriaceae. The low agar concentration creates varying degrees of anaerobiosis in the media. As a result, this semisolid media satisfies the oxygen requirements for aerobes, as well as facultative and obligate anaerobes.

The indole test is a qualitative procedure for determining the ability of bacteria to produce indole. Casein peptone provides the source of tryptophan in the media. By producing the enzyme tryptophanase, certain microbes can deaminate tryptophan to indole. Indole is detected when it reacts with p-Dimethylamino-benzaldehyde (Kovacs Reagent) under acidic conditions to produce a red color, indicative of a positive reaction.(4)

The nitrate reduction test is a qualitative procedure for determining the ability of bacteria to reduce nitrate. Organisms which possess the enzyme nitroreductase vary in their ability to reduce nitrate. In the reaction, potassium nitrate is reduced to nitrite, which may then be further reduced to nitrogen gas or ammonia. The end product of nitrate reduction is dependent upon the bacterial species.(5)

The reduction of nitrate to nitrite is determined by the development of a red color complex upon the addition of Reagent A (sulfanilic acid solution, Cat. no. Z71) and Reagent B (N,N-dimethyl-1-naphthylamine, Cat. no. Z72). The sulfanilic acid reacts with nitrite to form a diazonium salt which then couples with N,N-dimethyl-1-naphthylamine to produce a red dye complex. Absence of a red color reaction indicates that the organism has further reduced nitrite to ammonia or nitrogen gas, or that unreduced nitrate is present, thus indicating the organism does not possess the nitroreductase enzyme.

If an organism does not possess the enzyme, nitrate will remain present in the medium. Application of Reagent C (zinc dust, Cat. no. Z73) will convert nitrate to nitrite to form a red-dye complex. This test reaction is considered negative for nitrate reduction. If, however, the organism has reduced nitrate beyond nitrite to nitrogen gas, application of zinc dust will not produce a color change. The test is then considered positive for nitrate reduction.


Gram weight per liter:* 25.0gm/L
Pancreatic Digest of Casein 20.0gm
Disodium Phosphate 2.0gm
Glucose 1.0gm
Potassium Nitrate 1.0gm
Agar 1.0gm

Final pH 7.2 +/- 0.2 at 25ºC.

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


Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original beige.

Store the prepared culture media at 2-8ºC.



1. Suspend 25.0gm of the dehydrated culture media in 1 liter of distilled or deionized water. Stir to mix thoroughly.

2. Heat, to boiling, to dissolve completely.

3. Sterilize in the autoclave at 121ºC. for 15 minutes.

4. Cool to 45-50ºC.


For information on procedures and interpretation of results, consult listed references or refer to the prepared media Instructions for Use (IFU) for Cat. No. K147.


CRITERION™ Indole Nitrate Medium is not recommended for the determination of indole production by coliforms and other enterics as these organisms reduce nitrate to nitrite, hindering the indole reaction.

Test isolates must be from pure culture and 18-24 hours old.

Interpretation of nitrate reduction color reactions should be made immediately, as color reactions with a positive test may fade rapidly. To avoid false-negative nitrite reduction reactions, negative nitrite reactions must be verified by the addition of Reagent C to the medium.


Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Clostridium sordelli
ATCC ® 9714
A 24-48hr 35°C Aerobic Growth; nitrate positive, indole negative
Propionibacterium acnes
ATCC ® 29399
A 24-48hr 35°C Aerobic Growth; nitrate negative, indole positive

User Quality Control


CRITERION™ Indole Nitrate Medium powder should appear homogeneous, free-flowing, and beige in color. The prepared media should appear opalescent, and light amber in color.


1. 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.

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

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

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

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

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