Nitrate Reduction Test: Principle, Protocol, And Reliable Application

Nitrate Reduction Test is used to assess whether an organism is capable of using the enzyme nitratase, also known as nitrate reductase, to convert nitrate (NO3) to nitrite (NO2) or other nitrogenous chemicals. The identification of both Gram-positive and Gram-negative species depends on this test. Microorganisms can be differentiated by different test and it is one of them. Some of the tests, we have already studied in our previous article like amylase test, starch hydrolysis test, indole test, oxidase test etc. Now move into our next test that is Nitrate reduction test.

protocol of nitrate reduction test

What is Nitrate Reduction Test?

The nitrate/nitrite reduction test is used to assess an organism’s capacity to convert nitrate to nitrite and to identify the various ways in which it can do so. Other than ambient oxygen, anaerobic metabolism requires an electron acceptor. Nitrate is frequently used by gram-negative bacteria as the final electron acceptor.

Principle of Nitrate Reduction Test

Nitrate reduction test depends upon the conversion of nitrates to nitrogenous form. This nitrate forms an azo dye when it interacts with reagents A and B, which results in a red color. The nitrites can, however, be further reduced by some organisms to free nitrogen and ammonia. This clarifies the nitrate-positive organism’s two possible outcomes. Ability to reduce nitrate to nitrite.

Reagents Required

The reagents required for this test are:

  1. Sulfanilic acid solution
  2. Alpha-naphthylamine solution

Protocol of Nitrate Reduction Test

  1. First of all we have to prepare nitrate broth.
  2. For this, 9 g of nitrate both powder is dissolved in 1 litre of water.
  3. The ingredients is weighed precisely and is stirred in a gentle heat for any broth substrate. Inverted Durham tubes is added after dispensing into test tubes, 15 minutes at 121 °C and 15 pressure in an autoclave. The Durham tube will be filled with broth thanks to the autoclave’s pressure. Before using, cool.
  4. 4° to 10°C refrigerator is used for storing. The shelf life is roughly six months.
  5. Reagent A and Reagent B are prepared.
  6. The medium is inoculated with a substantial inoculum from well-isolated colonies of the test organism for either substrate, for NO3 or NO2. They are incubated for 12 to 24 hours at 35 °C. Rarely, up to 5 days of incubation may be necessary. The broth is checked for substrate reduction after there is sufficient growth visible in the tube.
  7. Gas production is observed.

Positive Nitrate test

Within two minutes, a red hue that denotes the presence of NO2 in the tube will typically develop if the test organism has converted the NO3 to NO2.

Negative Nitrate test

If no color change is visible in the first two minutes, there are a number of potential causes. It was either (i) completely incapable of lowering NO3, (ii) capable of reducing NO2, or (iii) directly converted NO3 to molecular nitrogen.

Nitrate Reduction Test

Application of Nitrate reduction test

One of the main biochemical assays used in clinical microbiology labs to distinguish M. TB from other members of the M. tuberculosis complex and nontuberculous Mycobacteria is nitrate reductase in conjunction with niacin buildup.



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