The nitrate reduction test tests for a bacterial strain’s ability to reduce nitrate to nitrite or further compounds. To perform the test, first inoculate a tube of nitrate broth with a pure culture of your test organism and incubate it at 37 degrees Celsius for 5-10 days. After incubation, the tube should be turbid with growth. Before incubation, there is only nitrate in the tube. The first step of nitrate reduction is the reduction of nitrate to nitrite. We can test for the presence of nitrite using two reagents. First, add 5 drops of Reagent A to the tube and then add 5 drops of Reagent B, bending the droppers to break the ampules inside. If nitrite is present, the liquid in the tube will turn red within five minutes of adding both reagents. This means the organism was able to reduce nitrate to nitrite, a positive result for nitrate reduction.
Reagents A and B have already been added to these tubes, but they didn’t turn red, which means nitrite is not present in the broth. This does not necessarily mean that the organism can’t reduce nitrate to nitrite, though. It might also be able to reduce nitrite to other compounds and has simply used up all of the nitrite. To test for this, we can add nitrate reagent C, which is zinc powder. If nitrate is still present in the broth, adding zinc powder will cause the liquid to turn red. This means the nitrate was never reduced in the first place and is still present in the liquid – a negative result for nitrate reduction. However, if the broth does not turn red even after adding zinc powder, the organism must have reduced nitrate to nitrite and then redued nitrite to other nitrogen compounds such as nitric oxide, nitrous oxide, nitrogen gas, or ammonium. This is also a positive result for nitrate reduction. The Durham tube gives a clue as to which compound was formed – it will trap nitrogen gas, forming a bubble.
The nitrate reduction test tests for a bacterial strain’s ability to reduce nitrate to nitrite or further compounds.