Nitrate Reduction Test - Principle, Biochemistry, Reagent, Procedure, Result, Interpretation

Introduction to Nitrate Reduction Test

The nitrate reduction test is a biochemical test with the application to determine the ability of microorganisms to reduce nitrate to nitrite or free nitrogen gas. In other words, this test is used to test the presence of the enzyme nitrate reductase.

Principle, Biochemistry of Nitrate Reduction Test

In anaerobic respiration, oxidation of inorganic substances such as nitrate, sulfate, and sometimes carbonate takes place and oxygen is appointed as an electron acceptor to provide energy. When nitrogen reduction takes place, electrons are transported to specific acceptor molecules by bacterial cytochromes.

An organism has the capacity to derive its oxygen from nitrate by reducing nitrate (NO₃^-) to nitrite (NO₂^-) and to nitrogen gas (N₂) usually in anaerobic conditions. In such cases, the oxygen accepts hydrogen i.e. it is the final electron and proton acceptor. In cases of aerobic and facultatively aerobic bacteria, the nitrate is reduced only in the absence of oxygen.

Biochemistry:

NO₃^- + 2e^- + 2H⁺ → (nitraratase) → NO₂^- + H₂O

Depending upon the bacterial species, the end products of the nitrogen reduction process may vary and includes nitrite (NO₂), ammonia (NH₃), nitric oxide (NO), Nitrous oxide (N₂O), molecular nitrogen (N₂ ; most common), or hydroxylamine (R-NH-OH).

The reduction of nitrate (NO₃^-) to nitrite (NO₂^-) is indicated by the reagents- sulfanilic acid and α-naphthyl amine. These reagents combine with nitrite to form a diazonium compound P-aulfobenzene-azo-α-nepthylamine which is colored.

In cases of absence of nitrate, the organism should be tested for reduction of nitrate beyond nitrite. It is done by checking if nitrate is still present in the broth by adding zinc dust which will convert nitrate to nitrite. Zinc dust, which is a reducing agent reduces diazonium salt in presence of acetic acid to give arhydrazine, which is also a colored compound. If no nitrite is detected after the addition of zinc, it can be assumed that the nitrogen-reducing organism has reduced nitrate to nitrite to nitrogen gas or ammonia.

Reagent of Nitrate Reduction Test

The reagents of the Nitrate Reduction Test are as follows:

• Solution A:

  Dissolve 6 grams of dimethyl α-nepthylamine or 5 grams of α-nepthylamine in 1 liter of acetic acid having a concentration of 5 mol/liter.

• Solution B:

  Dissolve 8 grams of sulphanilic acid in 1 liter of acetic acid having a concentration of 5 mol/liter.

*mix equal volumes of solution A and B immediately before use as a test reagent.

Procedure of Nitrate Reduction Test

The procedures of the Nitrate Reduction Test are as follows:

1. Take a test filled with 1ml of nitrate broth. (tube with slant nutrient agar (NA) can also be used)

2. Heavily inoculate a fresh, pure culture of test bacteria onto the broth. If NA has been used, stab the butt of the media and streak it as a fishtail slant.

3. Incubate at 35-37°C for 18-24 hours. (sometimes incubation of 4 days is required)

4. Phase 1: Directly add 0.1 m of reagent to the nitrate culture. If red color is seen within 30 seconds, the test is positive. Proceed to phase 2 if phase 1 is negative.

   Phase 2 (zinc reduction method): Add 20 grams of zinc dust to the test tube. Observe for color development within 30 seconds.

Result, Interpretation of Nitrate Reduction Test

The rest of the Nitrate Reduction Test is interpreted as follows:

Positive

Phase 1

pink to the deep-red color seen within 30 seconds of adding the reagent indicating nitrate is reduced to nitrite.

Phase 2

No color development after the addition of zinc dust indicates the absence of nitrite. Test microorganism has reduced nitrate to nitrite and further reduced nitrite.

Negative

Phase 1

No color development so proceed to phase 2.

Phase 2

pink to red color is observed indicating the presence of nitrate, which has not been reduced by the test organism.

Nitrate reducing bacteria

Some nitrate-reducing bacteria include:

• E. coli

Nitrate non-reducing bacteria

Some nitrate non-reducing bacteria include:

• Acinetobacter calcoaceticus