5 Jan 2012



This test is used to differentiate members of the Enterobacteriaceae that produce the enzyme nitrate reductase, from gram negative bacteria that do not produce the enzyme. The test is also helpful in differentiating Mycobacterium species.
A heavy inoculum of the test organism is incubated in a broth containing nitrate. After 4 hours, the broth is tested for the reduction of nitrate to nitrite by adding sulphanilic acid reagent. If nitrite is present, the acid reagent is diazotized and forms a pink-red compound with alpha-naphthylamine.
When nitrite is not detected it is necessary to test whether the organism has reduced the nitrate beyond nitrite. This is done indirectly by checking whether the broth still contains nitrate. Zinc dust is added which will convert any nitrate to nitrite. If no nitrite is detected when the zinc dust is added, it can be assumed that the entire nitrate has been reduced beyond nitrite to nitrogen gas or ammonia by a nitrate reducing organism.


            Nitrate broth can be prepared by adding 0.1 gram of potassium nitrate to 100 ml of        peptone water.
            The medium is used at a concentration of 0.9 grams in every 100 ml of distilled water.
            Sterilize by autoclaving (with loose caps) at 121oC for 15 minutes. When the medium      has cooled, stopper tightly.
Sulphanilic acid reagent
Alpha- naphthalamine reagent.
Zinc dust.

1.         Inoculate 0.5 ml of sterile nitrate broth with a heavy growth of the test organism.
2.         Incubate at 35-37oC for 4 hours.
3.         Add 1 drop of sulphanilic acid reagent and 1 drop of alpha- naphthylamine reagent.
4.         Shake to mix and look for a red color.


Red color………………………………Positive test(Nitrate reduced)
If no red color is produced, add a very small amount (knife point) of zinc dust powder. Look again for a red color and interpret as follows:
Red color…………………………Negative test(No reduction of nitrate)
No red color…………………………    Positive test  (Nitrate reduced)

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