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The nitrate or nitrite reduction test uses to determine the ability of an organism to reduce nitrate to nitrite. Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen. Many gram-negative bacteria use nitrate as the final electron acceptor. The nitrate reduction test is a test that determines the production of an enzyme called nitrate reductase, which results in the reduction of nitrate. This test is useful to differentiate organisms on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases.
Incubate nitrate broth with a heavy inoculum of test bacteria. If the bacteria are capable of producing the nitrate reductase enzyme, it reduces the nitrate, present in the broth, to nitrite. It may be further reduced to nitric oxide (NO), nitrous oxide (N2O), or nitrogen (N2). This test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex i.e. nitrite-sulfanilic acid which then reacts with an α-naphthylamine to give a red precipitate i.e. prontosil, that is a water-soluble azo dye.
The red color will see only when nitrate is present in the medium. If there’s no red color in the medium after the addition of sulfanilic acid and α-naphthylamine, nitrite is not present in the medium. When the nitrate may not have been reduced, the test organism is nitrate-negative. If the nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite, the test bacterium is nitrate-positive.
It is helpful when nitrite is not detectable and therefore, it is necessary to test if the organism has reduced nitrate beyond nitrite. Add a pinch of Zinc powder to the culture. It catalyzes the reduction of nitrate to nitrite. The development of the red color with the addition of Zinc powder indicates that nitrate was not reduced by the test organism which suggests that the test organism is not capable of reducing nitrate. If there is no color change after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and hence is a nitrate reducer.
Durham tube is placed in the nitrate broth in order to detect gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways.
Ingredients | Gram weight |
Peptone | 2.0 |
Potassium nitrate | 0.2 |
Distilled water | 100 ml |
Final pH | 7.0 ± 0.2 at 25°C |
Note: Heart infusion broth uses in the case of fastidious organisms in place of peptone( 25 grams per liter)
1. Reagent A ( 0.8% Sulfanilic acid )
Sulfanilic acid: 0.8 g
Distilled water: 70 ml
Glacial acetic acid: 30 ml
2. Reagent B (0.5% N, N-Dimethyl-alpha-naphthylamine)
Glacial acetic acid :30 ml
Distilled water:70 ml
N,N-dimethyl-α-naphthylamine :0.5 g
3. Zinc metal dust
Nitrate reduction to nitrite test is sometimes completed in two steps method.
First method
The reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B.
Second method
The reduction of nitrate beyond nitrite determination with the help of zinc powder
Nitrate/Nitrite reduction test: Positive
Development of a cherry red color after the addition of reagents A and B
Lacking red color development on adding Zinc powder
Nitrate/Nitrite reduction test: Negative
Development of red color with the addition of Zinc powder
Control strains
Escherichia coli ATCC 25922—nitrate positive, gas negative
Pseudomonas aeruginosa ATCC 27853—nitrate positive, gas positive
Acinetobacter baumannii ATCC 19606—nitrate negative
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