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

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Nitrate Reduction Test Introduction

Many of the biochemical test is usually used to detect the ability and to identify the species of bacteria b differentiating them based on the characteristics and their biochemical activities.

There are many factors such as protein, carbohydrate metabolism and the enzyme production.

These biochemical tests used in many of the clinical test to detect the pathogen and its nature of the disease-causing techniques.

Nitrite is one of the ions consisting molecule that is made up of nitrogen atom which is formed along with the oxygen atoms with the help of nitrogen bonds.

Nitrite is usually known as anion. Nitrite has the capability to inhibit the growth of bacteria hence it is used n many of the biochemical tests and also in clinical test and in food processing units.

In nitrate reduction test is based on the detection of the nitrite that is present in the medium after incubating it along with the organism.

Presence of nitrite in the medium reacts with the sultanic acid and results in the formation of the colourless complex.

This complex helps us to yield a red precipitate when the nitrate reagent is added to the medium.

What is Nitrate Reduction Test?

Anaerobic metabolism usually requires an electron acceptor other than the atmospheric oxygen.

Many of the species of the Gram-negative bacteria uses the nitrate as the final acceptor of electron.

Nitrate reduction test is a test which determines the production of the enzyme known as nitrate reductase, that results in the reduction of nitrate.

Bacterial species are differentiated according to their ability to reduce nitrite or nitrogenous gases.

Nitrate Reduction Test Objective

The main aim of the test is to determine the ability of an organism to reduce the nitrate or nitrite.

To identify the different ways of reduction of bacteria by the nitrate.

Nitrate Reduction Test Principle

To perform this test, a heavy inoculum of the test organism is collected and it is incubated in the broth containing nitrate.

The organisms which have the capability to produces the enzyme, nitrate reductase reduces the presence of nitrate in the broth, thus it forms its reduced form known as nitrite.

Nitrite further reduces to nitric oxide, nitrous oxide or nitrogen.

This test is purely based on the detection of nitrite and its ability to form a red colored compound on reacting with sulfamic acid and forms a complex known as nitrite sulfamic acid.

Sulfanilic acid further reacts with the alpha-naphthylamine which results in the formation of the red precipitate known as Prontosil, this prontosil is completely soluble in water and it is called as azo dye.

It is also noted that only when nitrate is present in the medium, formation of red color can be observed.

If there is no formation of red color then it can be proceeded by adding sulfanilic acid and Alpha-naphthylamine , when nitrite is absent in the medium.

Usually, this observation is explained in two ways, Nitrate does not have reduces, if the strain is noted as nitrate-negative.

The nitrate reduces into nitrite, which is further reduced into nitrous oxide or nitrogen, which on this reduced form does not reacts with nitrite, then the strain is said to be nitrate-positive.

However, if nitrate is not detected, it is important to test if the organism has the capability to reduce nitrate beyond nitrite.

This is usually done by adding a small amount of zinc powder catalyzes in order to reduce the nitrate into nitrite.

On adding zinc, the formation of red color can be observed, which indicates that nitrate was not reduced to nitrite.

In other case, where is no change in color even addition of zinc powder, then it indicates that the organism is reduced into any of the nitrogen compounds.

Nitrate Reduction Test Reagents

Nitrate Broth:

 Peptone – 5 grams per litre

 Meat extract – 3 grams per litre

 Potassium nitrate – 1 gram per litre

Nitrate Reduction Test Procedure

Nitrate reduction is usually determined in two step process. Initially the reduction of nitrate to nitrite is determined by adding the nitrogen reagents into the medium.

If necessary, the reduction of nitrate beyond nitrite beyond is determined by adding nitrate reagent C, which is in the form the zinc dust.

Initially the nitrate broths are inoculated into the nitrate broth into the bacterial suspension.

Incubate the tubes at the optimum temperature of about 30 to 37ºC for 24 hours.

After incubating the formation of nitrogen gas is noted before adding the reagents.

To this medium, about 6 to 8 drops of the nitrate reagent A and the same amount the nitrate reagent B are added, once it is added, color changes can be observed in a minute or some times even less than that.

On the other hand, if there is no color change, then zinc powder is added and formation of any changes can be observed within 3 minutes of adding.

Nitrate Reduction Test Result

Positive Test:

 In positive tests, the development of red color can be detected after addition of the reagents A and B.

 And in Zinc powder addition, the medium should not develop the red color and it results in positive results.

Negative Test:

 Development of a red color when zinc powder is added.

Nitrate Reduction Test Citations


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