Nitrate Reduction Test: Result, Principle, Procedure, and Reagents

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Nitrate Reduction Test Introduction

Biochemical tests are generally used to determine the ability of the microorganism to be stable in the enzymatic field. One such test is Nitrate reduction test, which helps us to differentiate between the species of the bacteria based on the ability or their inability to reduce nitrate into nitrite using an anaerobic respiration.

This anaerobic metabolism usually required an electron acceptor other than the atmospheric oxygen.

Many of the gram-negative bacteria use of the nitrate as the final electron acceptor.

Nitrate reduction test is one of vital test which helps us to determine the production of an enzyme known nitrate reductase, which helps us to reduce the nitrate.

Bacterial species is generally used to differentiate bacteria on the basis of their ability to reduce nitrate into nitrite or nitrogenous gases.

Nitrate Reduction Test Objective

• The main aim of the Nitrate Reduction Test is to determine the ability of an organism to reduce nitrate to nitrite.

• Nitrate Reduction Test also helps us to identify the different ways in which the nitrate is reduced in the bacteria.

Nitrate Reduction Test Principle

A heavy inoculum of the test organism is incubated into a broth, that contains nitrate. The organism which are all capable of producing a nitrate reductase enzyme has the capability to reduce nitrate to nitrite that is present in the broth which is further reduced in nitric oxide or nitrogen or nitrous oxide.

The nitrate reduction test is completely based on how the nitrate is reduced by the organism and its ability to form a red compound on it reacts with a sulfonic acid to form a complex known as nitrite-sulfanilic acid which then reacts with the alpha-napthylamine and produces a red precipitate known as Prontonisil which has an ability to soluble in water.

However, when the nitrate is alone present in the medium, there will be production of red color. If there is no formation of red color in the media in which the sulfamic acid and the alpha-naphthylamine is added and nitrite will not be present.

There are generally two explanations for the observation;

1. The nitrate should not be reduced, the strain is considered as nitrate-negative.

2. The nitrative can be reduced into nitrite that have been completely reduced into the nitric oxide or nitrous oxide or in the form of nitrogen which on reacting with nitrogen produces a nitrate-positive strain.

In case, when nitrate is not detected it is significant for the test that the organism is reduced into nitrate beyond the nitrite. This can be done my indirect means by adding a small amount of zinc powder into the culture.

Zinc powder helps in catalyzing the reduction of nitrate into nitrite. The development of red color can be seen on addition of the zinc which indicated that the nitrate has not been reduced by the particular organism as it does not have its capability to reduce nitrate.

In case if there is no change in color after adding zinc powder, it shows that the organisms reduced the nitrate present in the medium into nitrite or other compounds of nitrogen, and shows that it is a nitrate reducer.

Nitrate Reduction Test Reagents


IngredientsGram per liter
Meat extract3.0
Potassium nitrate1.0

Nitrate Reduction Test Procedure

• Here the determination of nitrate reduction involves two steps of processes as follows

• Initially, the reduction of nitrate into nitrite is usually determined by addition of reagents of the nitrate A and B, if it is necessary, the reduction of nitrate beyond the nitrite is usually determined by adding the nitrogen reagent as zinc powder.

• First, the nitrate broths are inoculated in a bacterial suspension.

• The tubes are usually inoculated at a temperature of 30 to 37ºC for about 24 hours.

• After completing the process of incubation, the release of nitrogen gas is noted before addition of the reagents.

• Then about 6 to 8 drops of nitrogen reagents A and B are adding into the medium.

• After addition of the ingredients a medium is observed for any color change.

• In case, if there is no color change is noted, then zinc powder is added into the medium.

• Then the medium is being observed for at least 3 minutes for a change or formation of red color.

Nitrate Reduction Test Results

Positive Nitrate Reduction Test:

• In case of positive results, there will be a formation of a cherry red color while adding the reagents A and B.

• There will be no change in color or production of red color after adding the zinc powder.

Negative Nitrate Reduction Test:

• In case of negative result, there will a formation of red color only after adding the zinc powder into the medium containing the reagents

Nitrate Reduction Test Citations


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