To perform this test, a heavy inoculum of the test organism is collected and it is incubated in the broth containing nitrate.

The organisms which have the capability to produces the enzyme, nitrate reductase reduces the presence of nitrate in the broth, thus it forms its reduced form known as nitrite.

Nitrite further reduces to nitric oxide, nitrous oxide or nitrogen.

This test is purely based on the detection of nitrite and its ability to form a red colored compound on reacting with sulfamic acid and forms a complex known as nitrite sulfamic acid.

Sulfanilic acid further reacts with the alpha-naphthylamine which results in the formation of the red precipitate known as Prontosil, this prontosil is completely soluble in water and it is called as azo dye.

It is also noted that only when nitrate is present in the medium, formation of red color can be observed.

If there is no formation of red color then it can be proceeded by adding sulfanilic acid and Alpha-naphthylamine , when nitrite is absent in the medium.

Usually, this observation is explained in two ways, Nitrate does not have reduces, if the strain is noted as nitrate-negative.

The nitrate reduces into nitrite, which is further reduced into nitrous oxide or nitrogen, which on this reduced form does not reacts with nitrite, then the strain is said to be nitrate-positive.

However, if nitrate is not detected, it is important to test if the organism has the capability to reduce nitrate beyond nitrite.

This is usually done by adding a small amount of zinc powder catalyzes in order to reduce the nitrate into nitrite.

On adding zinc, the formation of red color can be observed, which indicates that nitrate was not reduced to nitrite.

In other case, where is no change in color even addition of zinc powder, then it indicates that the organism is reduced into any of the nitrogen compounds.