Nitrate Reduction Test – Principle, Procedure, Uses and Interpretation

Nitrate Reduction Test Results

Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen (O₂). Many gram-negative bacteria use nitrate as the final electron acceptor.

Nitrate reduction test is a test that determines the production of an enzyme called nitrate reductase, which results in the reduction of nitrate (NO3).

Bacterial species may be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases.

Objectives

To determine the ability of an organism to reduce nitrate to nitrite.
Identify the different ways that nitrate can be reduced by bacteria

Principle

Principle of Nitrate Reduction Test

A heavy inoculum of test organism is incubated in a broth containing nitrate. The organisms capable of producing the nitrate reductase enzyme reduce the nitrate, present in the broth, to nitrite which may then be further reduced to nitric oxide, nitrous oxide, or nitrogen.

The nitrate reduction 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 (nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye.

However, only when nitrate is present in the medium, red color will be produced. If there’s no red color in the medium after you’ve added sulfanilic acid and α-naphthylamine means only that nitrite is not present in the medium.

There is two explanations for this observation.

The nitrate may not have been reduced; the strain is nitrate-negative.
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 strain is nitrate-positive.

Thus, when nitrite is not detected, it is necessary to test if the organism has reduced nitrate beyond nitrite. This can be done indirectly by adding small amount of Zinc powder to the culture. Zinc powder catalyses the reduction of nitrate to nitrite. The development of the red color on addition of Zinc indicates that nitrate was not reduced by the organism which suggests that the test organism is not capable of reducing nitrate. If no color change occurs after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and thus is a nitrate reducer.

Note: A Durham tube is placed in the nitrogen broth in order to detect deterioration of the broth before inoculation, as evidenced by gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways.

Media:

Nitrate Broth

Peptone 5 g/l, Meat extract 3 g/l, Potassium nitrate 1 g/l.

Final pH 7.0 ± 0.2 at 25°C

Method

Determination of nitrate reduction to nitrite is a two step process. First, the reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B, then if necessary, the reduction of nitrate beyond nitrite is determined by the addition of Nitrate Reagent C (zinc dust).

Inoculate the nitrate broths with bacterial suspension.
Incubate the tubes at the optimal temperature 30°C or 37°C for 24 hours.
After incubation look for N2 gas first before adding reagents.
Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B.
Observe for the reaction (color development) within a minute or less.
If no color develops add zinc powder.
Observe for at least 3 minutes for a red color to develop after addition of zinc.

Expected Results

Positive Test:
– Development of a cherry red coloration on addition of reagent A and B
– Absence of a red color development on adding Zn powder
Negative Test:
– A development of red color on addition of Zn powder