Nitrate Agar Slant

Principle And Interpretation

Nitrate Agar is prepared in accordance with the formula published in Pure Culture Study of Bacteria of the Society of American Bacteriologist (1). The ability to reduce nitrate is valuable for differentiating and identifying various types of bacteria especially Enterobacteriaceae family (2). Non-fermenters and other miscellaneous gram-negative bacilli vary in their ability to reduce nitrates. Some members of this group are capable of denitrification which is the reduction of nitrate to nitrogen gas. For the glucose fermenting gram-negative bacilli, the production of nitrogen gas from nitrate is an important differential test (3).

Potassium nitrate in the medium acts as a substrate for determining nitrate reduction by bacteria. Certain bacteria convert nitrate to nitrite, ammonia or nitrogen gas. The presence of nitrites can be detected by the addition of 0.5 ml each of sulphanilic acid (R015) and alpha-naphthylamine solution (R009). The development of red violet colour, due to the formation of a red diazonium dye i.e. p-sulfobenzene-azo-a-naphthylamine, indicates nitrate reduction to nitrite. If no colour develops, it means that either nitrate is not reduced or further reduction to ammonia or nitrogen gas has taken place. This can be verified by adding a pinch of zinc dust to the tube. Zinc reduces nitrate to nitrite resulting in a red colour. The red colour indicates that nitrate is still present and was not reduced previously. An absence of red colour after the addition of zinc dust indicates that no nitrate is present and thus the nitrate was reduced further than nitrite. Therefore the nitrate reduction test is evidenced by either the presence of a catabolic end product or the absence of nitrate in the medium.

Members of Enterobacteriaceae characteristically reduce nitrate to nitrite which reacts with sulfanilic acid and N, N-dimethyl-1-naphthylamine to produce the red colour. This reaction is known as Griess reaction. If an organism grows rapidly and reduces nitrate actively, the test should be performed after an early incubation period since the nitrite may be further reduced to nitrogen.

For the test: Add few drops of each reagent i.e. sulphanillic acid (R015) and a-naphthylamine solution (R009) into the tube containing culture to be tested. A distinct red or pink colour indicates nitrate reduction. A control (un-inoculated) tube should also be tested. If there is no pink colour formation, add a pinch of zinc dust to confirm the absence of nitrate in the medium (3).