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
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 °C, min. 24 hours. After incubation add 5 drops of reactive 1 and
A red color appears within 5-10 minutes if reaction is positive. Reaction is
negative if no color changes.
1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.
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.
(c) Costin Stoica