Nitrate Reduction Test
Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen.
Nitrate broth contains nutrients and potassium nitrate as a source of nitrate.
After incubating the nitrate broth, add a dropperful of sulfanilic acid and α-naphthylamine. If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid. This reacts with the α-naphthylamine to form a red-colored compound. Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction.
If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable to reduce the nitrate, or it could mean that the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test.
If the medium does not turn red after the addition of the nitrate reagents, add a small amount of powdered zinc. Be careful, as powdered zinc is hazardous! If the tube turns red after the addition of the zinc, it means that unreduced nitrate was present. Therefore, a red color on the second step is a negative result. The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby produced reacted with the α-naphthylamine to create the red complex.
If the medium does not turn red after the addition of the zinc powder, then the result is called a positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the medium, either, that means that denitrification took place and ammonia or molecular nitrogen were formed.
We do not use Durham tubes in our nitrate broth.