In order to determine if a bacteria can reduce nitrate, the test organism is inoculated into nitrate reduction broth, an undefined medium that contains large amounts of nitrate (KNO₃). After incubation, alpha-napthylamine and sulfanilic acid are added . These two compounds react with nitrite and turn red in color, indicating a positive nitrate reduction test.  (Tube 2 in image below.) 

If there is no color change at this step, nitrite is absent. (Tube 1 below.) If the nitrate is unreduced and still in its original form, this would be a negative nitrate reduction result. However, it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas. This would be recorded as a positive nitrate reduction result.  

To distinguish between these two reactions, zinc dust must be added. Zinc reduces nitrate to nitrite. If the test organism did not reduce the nitrate to nitrite, the zinc will change the nitrate to nitrite. The tube will turn red because alpha-napthylamine and sulfanilic acid are already present in the tube. (Tube 4 below.) Thus a red color after the zinc is added indicates the zinc found the nitrate unchanged. The bacteria was unable to reduce nitrate. This is recorded as a negative nitrate reduction test.  

If however, the tube does not change color upon the addition of zinc, then the zinc did not find any nitrate in the tube. (Tube 3 below.) That means the test organism converted the nitrate to nitrite and then converted the nitrite to ammonia and/or nitrogen gas. Thus no color change upon the addition of zinc is recorded as a positive nitrate reduction test.  

 

nitrate reduction test