Nitrate reductase test
The nitrate reductase test is a test to differentiate between bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...

 based on their ability or inability to reduce nitrate
The nitrate ion is a polyatomic ion with the molecular formula NO and a molecular mass of 62.0049 g/mol. It is the conjugate base of nitric acid, consisting of one central nitrogen atom surrounded by three identically-bonded oxygen atoms in a trigonal planar arrangement. The nitrate ion carries a...

 (NO3) to nitrite
The nitrite ion has the chemical formula NO2−. The anion is symmetric with equal N-O bond lengths and a O-N-O bond angle of ca. 120°. On protonation the unstable weak acid nitrous acid is produced. Nitrite can be oxidised or reduced, with product somewhat dependent on the oxidizing/reducing agent...

 (NO2) using anaerobic respiration
Anaerobic respiration
Anaerobic respiration is a form of respiration using electron acceptors other than oxygen. Although oxygen is not used as the final electron acceptor, the process still uses a respiratory electron transport chain; it is respiration without oxygen...



Various assays for detecting nitrate reduction have been described. One method is performed as follows:

  1. Inoculate
    Inoculation is the placement of something that will grow or reproduce, and is most commonly used in respect of the introduction of a serum, vaccine, or antigenic substance into the body of a human or animal, especially to produce or boost immunity to a specific disease...

     nitrate broth with an isolate and incubate for 48 hours.
  2. Add 10-15 drops each of sulfanilic acid
    Sulfanilic acid
    Sulfanilic acid is a grey crystalline solid produced from sulfonation of aniline.It readily forms diazo compounds and is used to make dyes and sulpha drugs....

     and N,N-dimethyl-1-naphthylamine
    N,N-Dimethyl-1-naphthylamine is an aromatic amine. It is formally derived from 1-naphthylamine by replacing the hydrogen atoms on the amino group with methyl groups. N,N-Dimethyl-1-naphthylamine is used in the nitrate reductase test to form a red precipitate of Prontosil by reacting with a...

    . If the bacterium produces nitrate reductase
    Nitrate reductase
    Nitrate reductases are molybdoenzymes that reduce nitrate to nitrite .* Eukaryotic nitrate reductases are part of the sulfite oxidase family of molybdoenzymes....

    , the broth will turn a deep red within 5 minutes at this step.
  3. If no color change is observed, then the result is inconclusive. Add a small amount of zinc
    Zinc , or spelter , is a metallic chemical element; it has the symbol Zn and atomic number 30. It is the first element in group 12 of the periodic table. Zinc is, in some respects, chemically similar to magnesium, because its ion is of similar size and its only common oxidation state is +2...

     to the broth. If the solution remains colorless, then both nitrate reductase and nitrite reductase
    Nitrite reductase
    Nitrite reductase refers to any of several classes of enzymes that catalyze the reduction of nitrite. There are two classes of NIR's. A multi haem enzyme reduces NO2 to a variety of products. Copper containing enzymes carry out a single electron transfer to produce nitric oxide.- Iron based...

    are present. If the solution turns red, nitrate reductase is not present.
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