Nitrate ReductionTest



Nitrate Broth: some bacteria (e.g., Pseudomonas aeruginosa) have respiratory enzyme systems that can use nitrate as a terminal electron acceptor. The product of the reaction is nitrite. Some of the organisms that reduce nitrate to nitrite will then reduce the nitrite further. In the scheme below, first test for nitrite by a colorimetric test. If this test is negative, it can mean either that nitrite was not reduced, or that it was reduced beyond the nitrite stage. This can be resolved by the addition of zinc dust; if nitrate is still present, the zinc will reduce it chemically to nitrite, which will then by revealed by the colorimetric reaction.

Procedure: To the nitrate broth, after 48 hours of incubation, add 0.2 ml of acid reagent (Solution A), a mixture of acetic acid and sulfanilic acid, and then 0.2 ml of dimethyl-alpha-napthylamine reagent (Solution B). If nitrite is present you will get a red color: this is a positive test for nitrate reduction. If there is no color, pick up some zinc dust on the end of an applicator stick, and add it to the tube. ZINC DUST SUSPENDED IN AIR CAN BE EXPLOSIVE; KEEP AWAY FROM FLAMES! If you get a red color at this stage, what can you conclude? What if no color is obtained?


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