3.5.5: Nitrate Reduction Test

OER - BIOL 2420 Laboratory Manual · LibreTexts

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    From LibreTexts, under CC BY 4.0, as the source states it; its equations are written in TeX, which the PDF shows typeset. This page at LibreTexts.

    The nitrate reduction test is performed to determine if a bacterium can reduce nitrate, using the enzyme nitrate reductase. Nitrate reductase is an enzyme located within the electron transport chain of some bacteria that perform anaerobic cellular respiration. Bacteria containing nitrate reductase can utilize nitrate as a final electron acceptor in their electron transport chains. Nitrate can be reduced to produce nitrite ions, nitrous oxide, or nitrogen gas.

    This test utilizes a nitrate broth, which is composed of beef extract, peptones, and potassium nitrate. The tube will have a Durham tube present to trap any gas produced during nitrate reduction. The broth is inoculated, incubated, and then observed for changes.

    After 48 hours the tube is observed for gas production in the Durham tube. Reagents (sulfanilic acid and alpha-naphthylamine) are then added to the broth to detect the presence of nitrite. After 2 minutes if a red color appears the sample contains nitrite, indicating that nitrate has been reduced. If the tube is clear after two minutes, zinc dust is added. If nitrate is still present in the medium zinc will reduce it to nitrite causing the medium to change red/pink. This is a negative result for nitrate reduction. If there is no color change after the addition of zinc, then the nitrate has been reduced to some form other than nitrite.

    Nitrate Reduction Test Results
    Positive for Nitrate Reduction Negative for Nitrate Reduction
    Examples:

    Enterobacter aerogenes

    Escherichia coli

    Staphylococcus aureus    		 Examples:

    Staphylococcus epidermidis

    Enterococcus faecalis

    Alcaligenes faecalis
    Positive nitrate reduction test indicating nitrate reductase activity.
    “A positive nitrate reduction test suggests that the bacterium possesses the enzyme nitrate reductase, which is responsible for the reduction of nitrate to nitrite. It’s important to note that some bacteria, including Mycobacterium tuberculosis, are slow nitrate reducers, and the test may require extended incubation periods up to several weeks) to yield a positive result.” Source: Ajay Kumar Chaurasiya, Wikimedia Commons, CC BY 4.0
    Nitrate reductase test procedure.
    Nitrate reductase test procedure. Credit: Sherry Smith

    Materials Needed:

    • Bunsen burner
    • Inoculating loop
    • Bacterial cultures (3 different species - instructor will provide names of species to be tested)
    • Nitrate broth tubes (3 tubes)
    • Nitrate reagent A
    • Nitrate reagent B
    • Zinc powder
    • Test tube rack

    This page titled 3.5.5: Nitrate Reduction Test was last modified on Sat, 25 Jul 2026 03:43:03 GMT and is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by Sherry Smith.