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26: Nitrate Reduction

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    Learning Objectives
    • Objective 1
    • Objective 2

    Nitrate Test

    This test determines the production of an enzyme called nitrate reductase, resulting in the reduction of nitrate (NO3): the test is also called the nitrate reduction test. With this enzyme, nitrate is reduced to nitrite (NO2). It then forms nitrous acid that reacts with the first reagent sulfanilic acid, and that reacts with the other reagent naphthylamine to form a red color.

    Reduction of nitrate is generally an anaerobic respiration in which an organism derives its oxygen from nitrate.

    Lab Instructions

    1. Inoculate the nitrate broths with your bacterial unknown.
    2. Incubate at the optimal temperature, 30º C or 37º C, for your organisms.
    3. After Incubation: Look for N2 gas first before adding reagents.
      • Add 6-8 drops of nitrite reagent A.
      • Add the same number of drops of nitrite reagent B.
      • You should see a reaction within a minute or less.
      • If you have not seen either nitrite or N2 gas, you need to add a bit of powdered zinc.
      • A bit of zinc is about the amount that sticks to the end of a wood stick.
      • The reduction of unused nitrate by zinc takes a couple of minutes.


    There are various ways that a bacterium can utilize nitrate as the final electron acceptor in anaerobic respiration. The first obvious product of reduction to look for is reduction to N2 gas, called denitrification, within the Durham tube. This is looked for FIRST before any reagents are added.

    nitrate reduction to N2

    Figure 1: Nitrate can be reduced to nitrogen gas (N2). If this occurs, there will be a bubble located inside of the Durham tube.

    If there is no nitrogen gas, there are still a couple of possible interpretations---nitrate reduction to nitrite (NO2), reduction to ammonia, or no reduction of nitrate at all.

    A red color will be produced in the medium only when nitrite is present in the medium.

    nitrate reduction to nitrite

    Figure 2: Red coloration indicates that nitrate was reduced to produce nitrite.

    There may be 2 explanations for the lack of nitrite:

    1. The nitrate may not have been reduced; the bacterium cannot use nitrate (nitrate reduction negative)
    2. The nitrate may have been reduced to nitrite which has then been completely reduced to ammonia.

    To differentiate between the above two possibilities, powdered zinc is added:

    1. If the bacterium has not used the NO3, it will still be present in the tube. The Zn will reduce the nitrate, forming nitrite, which then reacts with the 2 reagents already added to the tube. A pink-red color will form as confirmation of a negative nitrate reduction.
    2. The last possibility, production of ammonia, is indicated when no pink forms.

    nitrate reduction negative

    Figure 3: If no bubble is present in the Durham tube and the medium did not turn red, it is still possible that nitrate was reduced to ammonia. Zinc is added to determine whether the nitrate was reduced or not. If the nitrate was not reduced by the microbes in the tube, the zinc will cause the medium to turn pink and indicate that the bacteria are nitrate reduction negative. If the medium does not turn pink when zinc is added, this indicates that nitrate was reduced to ammonia by the microbe and is therefore nitrate reduction positive.

    REACTION N2 gas Color after adding reagents Color after adding zinc
    NO3 to NO2 none red (Zn not added)
    NO3 to N2 yes no color (Zn not added)
    NO3 to ammonia none no color no color
    NO3 - no reaction none no color pink-red


    1. Name the 2 major end products of nitrate reduction.
    2. Why add zinc powder?
    3. When do you add zinc powder?
    4. How do the definitions of nitrate reduction and denitrification differ?
    5. Is nitrate reduction an aerobic pathway or an anaerobic pathway?


    Microbiology Labs I by Delmar Larsen and Jackie Reynolds has an undeclared license.

    This page titled 26: Nitrate Reduction is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Rosanna Hartline.

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