Nitrate reduction

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Add a small amount of zinc (a toothpick full) to each broth. Zinc catalyzes the reduction of nitrate to nitrite. a. At this point, a color change to RED indicates a ...
Nitrate Reduction Test (WINTER 2014 version) Introduction Some facultative anaerobes are capable of using nitrate as an electron acceptor at the end of the Electron Transport Chain. Enteric bacteria that reduce nitrate (NO3) usually produce nitrite (NO2). Other bacteria reduce nitrate and form products such as ammonia (NH3), nitric oxide (NO), and nitrous oxide (N2O). The Nitrate Reduction Test uses the presence or absence of nitrite (after incubation) as an indicator of nitrate reduction. The results are complicated by the possibility that nitrite might be further reduced to other compounds. Methods 1. Obtain two Nitrate Broths from the back shelf. 2. Inoculate one broth using aseptic technique. Leave the other broth uninoculated (this will be a negative control). 3. Incubate at an appropriate temperature (whatever temperature your organism grows well at). Incubate for 24 to 48 hours (do not exceed 48 hours for this test). 4. Obtain bottles of sulfanilic acid and -naphthylamine from the refrigerator (ask instructor). 5. Add one dropperfull of sulfanilic acid and one dropperfull of -naphthylamine to each broth. a. At this point, a color change to RED indicates a POSITIVE nitrate reduction test. If you get a red color, then you can stop at this point. b. No color change indicates the absence of nitrite. This can happen either because nitrate was not reduced or because nitrate was reduced to nitrite, then nitrite was further reduced to some other molecule. If you DO NOT get a red color, then you must proceed to the next step. 6. Add a small amount of zinc (a toothpick full) to each broth. Zinc catalyzes the reduction of nitrate to nitrite. a. At this point, a color change to RED indicates a NEGATIVE nitrate reduction test because this means that nitrate must have been present and must have been reduced to form nitrite. b. No color change means that no nitrate was present. Thus no color change at this point is a POSITIVE result. Results + = Positive (Red after sulfanilic acid + -naphthylamine; no color after zinc) - = Negative (No color after sulfanilic acid + -naphthylamine followed by Red after zinc) Photographs

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