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 nitrate. If no nitrite is detected when the zinc dust is added, it can be assumed that all the nitrate has been reduced beyond nitrite to nitrogen gas or ammonia by a nitrate reducing organism.
Sulphanilic acid reagent
Inoculate 0.6 ml of sterile nitrate broth with a heavy growth of the test organism. Incubate at 35-37OC for 4 hours. Add 1 drop of sulphanilic acid reagent and alphanaphthylamine reagent. Shake to mix and look for a red colour. drop of 1
Red colour ----------------------------- Positive test
Nitrate reduced If no red colour is produced, add a very small amount (knife point) of zink dust powder. Look again for a red colour and intrpret as follows: