Nitrate reductase is the enzyme utilized by many facultative anaerobe bacteria to substitute NO3 for O2 during their cellular respiration.
Reduction may result in nitrites production or may go further to ammonia or nitriogen gas. Testing nitrates reduction requires growing
bacteria in nutritive medium that contains potassium nitrate (KNO3).
Nitrate broth composition: peptone 10g, KNO3 10g, distilled water ad 1000 ml.
Reactive 1: α-naphtylamine 1g, distilled water 22ml. Heat solution, filter, then add
acetic acid 1ml (diluted 400 ml in 700 ml distilled water).
Reactive 2: sulfanilic acid 0.5g, diluted acetic acid 150 ml.
Harvest a well isolated colony and inoculate a nitrate broth tube. Incubate at 37 ºC,
min. 24 hours. After incubation add 5 drops of reactive 1 and reactive 2.
A red color appears within 5-10 minutes if reaction is positive. Reaction is negative if
no color changes.
If reaction is negative then it is possible that bacteria may have further reduced the
nitrite to ammonia or nitrogen. Add a small quantity of zinc in the tube. Reaction is
positive if medium remains unchanged and negative if turns red.
For determining nitrate reductase activity of mycobacteria, it is preferable to use Ehrlich's aldehyde reagent, a 2% solution of
p-dimethylaminobenzaldehyde in 10% HCl.
1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.
3. Michio Tsukamura. A Review of the Methods of Identification and Differentiation of Mycobacteria. Reviews of Infectious Diseases,
Vol. 3, No. 5, International Conference on Atypical Mycobacteria (Sep. - Oct., 1981), pp. 841-861.
(c) Costin Stoica