The development of the red colour on addition of Zinc indicates that nitrate was not reduced by the organism which suggests that the test organism is not capable of reducing nitrate. Zinc powder catalyses the reduction of nitrate to nitrite. This can be done indirectly by adding small amount of Zinc powder to the culture. Thus, when nitrite is not detected, it is necessary to test if the organism has reduced nitrate beyond nitrite. The nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite the strain is nitrate-positive.The nitrate may not have been reduced the strain is nitrate-negative.
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There is two explanations for this observation. If there’s no red color in the medium after you’ve added sulfanilic acid and α-naphthylamine means only that nitrite is not present in the medium. However, only when nitrate is present in the medium, red color will be produced. The nitrate reduction test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye. The organisms capable of producing the nitrate reductase enzyme reduce the nitrate, present in the broth, to nitrite which may then be further reduced to nitric oxide, nitrous oxide, or nitrogen.
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