Nitrates Reduction
DESCRIPTION
Nitrate reductase is the enzyme utilized by many facultative anaerobe bacteria to substitute NO
3 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 (KNO
3).
MEDIUM PREPARATION
Nitrate broth composition: peptone 10g, KNO
3 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.

PROCEDURE
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.

NOTES
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.
REFERENCES:
1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.
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