January 2001

A. Sulfanilic acid reagent

B. N-(l-naphthyl)ethylenediamine reagent

C. α-Naphthol reagent

To prepare 5 N acetic acid, add 28.75 ml glacial acetic acid to 71.25 ml distilled water.

Store reagents in glass-stoppered brown bottles. To perform test, add 0.1-0.5 ml each of reagent A and either reagent B or reagent C (as specified in method) to culture grown in liquid or semisolid medium. Development of red-violet color with reagents A and B or orange color with reagents A and C indicates that nitrate has been reduced to nitrite. Since color produced with reagents A and B may fade or disappear within a few minutes, record reaction as soon as color appears. If no color develops, test for presence of nitrate by adding small amount of zinc dust. If color develops, nitrate has not been reduced.

Nitrate reduction test for enteropathogenic E. coli. To 3 ml of 18-24 h culture in indole-nitrite medium, add 2 drops each of reagents A and B.

Red-violet color indicates that nitrate has been reduced to nitrite. Check negative tests by adding small amount of zinc dust; if red-violet color does not appear, nitrate has been reduced.

D. Alternative test reagents.

5-Amino-2-naphthylene sulfonic acid (Cleve's acid) and N,N-dimethyl- 1-naphthylamine have been recommended as substitutes for preparation of reagent B. Absolute ethanol may be substituted for acetic acid in reagent C. However, comparative evaluations should be conducted before substitution of these alternative reagents.

CAUTION: The α-naphthylamine reagent recommended in previous editions of the Bacteriological Analytical Manual should not be used because of the possible hazard to laboratory personnel. Any supplies of α-naphthylamine on hand should be inventoried and secured by supervisory personnel because of the strict rules governing the use of carcinogenic substances in laboratories of the U.S. Department of Health and Human Services.

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Original Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.