Nitrate reduction test:

Objective

•  to detect the ability of the organism to produce Nitrate reductase enzyme.
• to differentiate members of Enterobacteriaceae that produce enzyme nitrate reductase from Gram negative bacteria that do not produce the enzyme  nitrate reductase

Principle of Nitrate reduction test:

Many microorganism can reduce the nitrate salts such as KNO3 to a nitrite salt, KNO2 under anaerobic condition. This is an example of anaerobic respiration in which nitrate molecule act as an electron acceptor in place of O2. This nitrate reacts with the reagent A and B to give red color due to the formation of Azo dye. But some of the organism do have ability to further reduce the nitrites to free nitrogen and ammonia. This clear the two fates of the nitrate positive organism.

A heavy inoculum of test organism is incubated in a broth containing nitrate. After 4 hours, the broth is tested for  reduction of nitrate to nitrite by adding sulphanilic acid reagent. If nitrite is present , the acid reagent is diazotized and forms a cherry-red compound with α – napthylamine.

The culture may not produce red color due to

1. Nitrates not reduced by microorganism OR
2. The organism posses potent nitrate reductase which reduce the nitrate beyond nitrite to ammonia or even molecular N2. Hence, overall fate of the reaction become

When nitrate is not detected, it is necessary to test whether the organism has reduced nitrate beyond nitrite. This is done indirectly by adding small amount of Zinc powder to the culture.  Zinc reduces nitrates to nitrites. The development of red colour on addition of Zinc indicates that nitrate was not reduced by the organism which suggest that organism are capable of reducing nitrate to nitrite while if there is no color change after the addition of Zinc to the culture suggest that nitrate is further beyond nitrite to ammonia or even N2.

Requirements

• Given samples of organism ( E.coli and Pseudomonas aeroginosa)
• Nitrate broth
• Inoculating loop, incubator
• Sulfanilic acid (Reagent A) and α- napthylamine (Reagent B)

Procedure of Nitrate reduction test

1. Prepare nitrate broth
2. Inoculate heavy inoculum of the given samples of organism individually in the medium aseptically
3.  Incubate the test tubes  at 37°C for 4 hours.
4. After 4 hours incubation, add few drops of the reagent A and B in the culture tubes
5. observe the color change due to reduction of nitrate to nitrite
6. To those culture tubes which do not produce red color, add a pinch of Zn powder and look for the development of red color to confirm the fate of organism.

Result interpretation:

Nitrate reduction test positive:

• If cherry red observe after adding reagent A and B
• no red color observe after adding Zn powder

Nitrate reduction test Negative:

• red color observe after adding Zn powder

Nitrate reduction test: Objective, Principle, Procedure and Result