Cat. no. C6450 CRITERION™ Nitrate Broth 18gm
Cat. no. C6451 CRITERION™ Nitrate Broth 500gm
Cat. no. C6452 CRITERION™ Nitrate Broth 2kg
Cat. no. C6453 CRITERION™ Nitrate Broth 10kg
Cat. no. C6454 CRITERION™ Nitrate Broth 50kg


Hardy Diagnostics CRITERION™ Nitrate Broth is recommended for differentiating microorganisms based upon nitrate reduction.



The ability of bacteria to reduce nitrate is an important biochemical characteristic which aids in the identification of many microorganisms, particularly members of the family Enterobacteriaceae, and members of the Haemophilus, Neisseria, and Branhamella genera.(2,3,5,6)

Organisms which possess the enzyme nitroreductase vary in their ability to reduce nitrate. Some microorganisms reduce nitrate to nitrite while others further reduce the nitrate to form other end products such as ammonia, nitrogen gas, hydroxylamine, etc. The end product of nitrate reduction is dependent upon the bacterial species.(7)

The reduction of nitrate to nitrite is determined by the development of a red color complex upon the addition of sulfanilic acid solution (Nitrate Reagent A, Cat. no. Z71) and N, N-dimethyl-1-naphthylamine (Nitrate Reagent B, 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 the nitrites 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 zinc dust (Nitrate Reagent C, 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: 9.0gm/L
Pancreatic Digest of Gelatin 5.0gm
Beef Extract 3.0gm
Potassium Nitrate 1.0gm

Final pH 6.9 +/- 0.2 at 25ºC.




Store the prepared culture media at 2-30ºC.



1. Suspend 9.0gm of the dehydrated culture media in 1 liter of distilled or deionized water until evenly dispersed.

2. Heat as necessary to dissolve completely.

3. Distribute into test tubes (with durham tubes if desired) and sterilize in the autoclave at 121ºC. for 15 minutes.




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

Interpretation of color reactions should be made immediately, as color reactions with a positive test may fade rapidly.

If air bubbles are present in the durham tube prior to inoculation, the tube should be inverted until the air is released from the durham tube. Failure to remove air bubbles prior to inoculation may result in reading the result as a false-positive reaction for gas reduction.

A faint pink color may be produced following addition of the nitrate reagents. This is not a positive result. Development of a deep red color is indicative of a positive reaction.

A negative zinc reduction (no color change) test, in combination with a negative nitrite reaction, is presumptive indication that the nitrate was reduced beyond the nitrite stage. Although a very common end product of nitrite reduction is nitrogen gas, other end products may be formed. Additional testing may be required to determine the final end products of the reaction.

To avoid false-negative nitrite reduction reactions, negative nitrite reactions must be verified by the addition of Nitrate Reagent C (zinc dust) to the medium.

Excess zinc dust has been reported to cause false-positive nitrite reduction reactions due to complete reduction of previously unreduced nitrate to ammonia.


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


Test Organisms Reaction
Escherichia coli
ATCC ® 25922
Positive nitrate reduction;
deep red color seen after the addition of Reagents A and B
Acinetobacter baumannii
ATCC ® 19606
Negative nitrate reduction;
no color change seen after the addition of Reagents A and B and red color forms after addition of Reagent C

User Quality Control


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



2. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier Science Publishing Co., Inc., New York.




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

7. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

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