Most facultatively anaerobic bacteria produce nitrate reductase, an enzyme which adds electrons to or removes oxygen atoms from nitrate. Use of this enzyme allows facultative anaerobes to use nitrate instead of oxygen as the final electron acceptor in cellular respiration. Once an unknown bacterium is isolated and diluted in liquified agar medium, nitrate reduction can be tested. Using aseptic technique, a loop of bacteria is transfered to a tube of nitrate broth. After 48 hours of incubation, 15 drops of sulfanilic acid and 15 drops of dimethyl-alpha-naphthylamine are added to the nitrate broth. The test tube is rolled between the hands to mix the two reagents into the broth. If during a period of 15 minutes the solution becomes red, the culture is positive for nitrate reduction. If however the solution is not red, it can be concluded that there is no nitrite present. This could be due to three reasons. First of all, the bacteria may not reduce nitrate. Secondly, the bacteria may reduce nitrate to nitrite, and in the presence of nitrite reductase reduce nitrite to ammonia. Finally, through denitrification bacteria may reduce nitrate to nitrite to nitrogen gas. To further understand the absence of nitrite, a small portion of zinc powder is added to the broth using a toothpick and mixed into the solution. If during a period of 15 minutes the solution becomes red, the culture is negative for nitrate reduction by bacteria. The zinc is reduced from nitrate to nitrite. If no red color is formed, the culture is positive for nitrate reduction by bacteria. Nitrate no longer remains in the broth.