Nitrate Reduction Test – Principle, Procedure, Uses and Interpretation

Nitrate Reduction Test Overview

The nitrate reductase enzyme is responsible for reducing nitrate, and the nitrate reduction test measures how much of it is produced (NO3).

Other than ambient oxygen, anaerobic metabolism needs an electron acceptor (O2). Nitrate is frequently used by gram-negative bacteria as the ultimate electron acceptor.

The capacity of different bacterial species to convert nitrate to nitrite or nitrogenous gases can be used to distinguish between them.


to ascertain a living thing’s capacity to convert nitrate to nitrite.

Describe the several ways that bacteria might decrease nitrate.


Principle of Nitrate Reduction Test

The test organism is cultured with a large inoculum in a nitrate-containing broth. The nitrate in the broth is converted to nitrite by organisms that can produce the nitrate reductase enzyme. It can be transformed further into nitric oxide, nitrous oxide, or nitrogen.

The nitrate reduction test is dependent on the identification of nitrite and its capacity to make a water-soluble red precipitate (prontosil) when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid).

Nevertheless, the red colour can only be produced in the presence of nitrate. After adding sulfanilic acid and naphthylamine, the lack of a red tint in the medium solely indicates that nitrite is not present.

For this finding, there are two possible interpretations.

The strain is nitrate-negative; consequently, the nitrate might not have been decreased.

The strain is nitrate-positive because the nitrate might be completely converted to nitric oxide, nitrous oxide, or nitrogen, none of which react with compounds that react with nitrite.

When nitrite cannot be detected, it is essential to ascertain if the organism has converted nitrate to nitrite. This may be accomplished indirectly by adding a little quantity of zinc powder to the culture. Nitrate is reduced to nitrite by the action of zinc powder. The appearance of the red colour with the addition of zinc shows that the organism did not reduce nitrate, suggesting that the creature under study is incapable of doing so. If there is no change in colour after adding zinc, the organism is a nitrate reducer and has transformed nitrate into one of the other nitrogen molecules.

A Durham tube is inserted into the nitrogen broth in order to identify denitrification by organisms that generate gas through different pathways and to detect broth degradation prior to inoculation.


Nitrate Broth

Peptone 5 g/l, Meat extract 3 g/l, Potassium nitrate 1 g/l.

Final pH 7.0 ± 0.2 at 25 °C


There are two steps involved in determining nitrate reduction to nitrite. If necessary, the inclusion of Nitrate Reagent C will determine the reduction of nitrate beyond nitrite after the addition of Nitrate Reagents A and B to determine the reduction of nitrate to nitrite (zinc dust).

  1. Add bacterial suspension to the nitrate broths to inoculate them.
  2. The tubes should be incubated for 24 hours at the ideal temperature of 30 °C or 37 °C.
  3. Check for N2 gas after incubation before introducing any chemicals.
  4. Both the 6-8 drops of nitrite reagent A and nitrite reagent B should be added.
  5. Within a minute or fewer, watch for the reaction (colour development).
  6. Add zinc powder if the colour doesn’t appear.
  7. After adding zinc, wait at least 3 minutes to see whether a red hue appears.

Expected Outcomes

Positive Test:

-The combination of reagents A and B results in the development of a cherry red coloration.

-No red coloration after adding zinc powder.

Negative Test

-A crimson hue develops after adding zinc powder.


  • While every member of the Enterobacteriaceae family reduces nitrate, only a certain number of them can also break down nitrite into other substances. Thus, it is used to differentiate between Gram-negative bacteria that lack the nitrate reductase enzyme and Enterobacteraceae family members that possess this enzyme.
  • In some species, nitrate reduction and anaerobic respiration may go hand in hand.
  • When distinguishing Mycobacterium, it is employed.
  • identifying many kinds of Neisseria and separating them from Moraxella and Kingella When K. denitrificans isolates seem to be gram-negative diplococci on stained smears, the nitrate reduction test is essential for separating N. gonorrhoeae from K. denitrificans.
  • Facilitating species identification of Corynebacterium


  • Bacterial identification may be aided by the nitrate reduction test. For full identification, further biochemical testing using pure cultures is advised.
  • Due to the probable presence of nitrite in the culture media, a low-nitrite medium, like Nitrate Agar or Nitrate Broth, should be employed for the nitrate reduction test.
  • Negative zinc reduction (no colour change) and a negative nitrite reaction are indicators that the nitrate has been reduced beyond the nitrite stage. Nitrogen gas is a common by-product of nitrite reduction, although other by-products are also possible.
  • Finding the reaction’s ultimate byproducts may require additional testing.
  • In order to avoid false-negative nitrite reduction reactions, negative nitrite reduction reactions should be validated by applying zinc dust to the medium.
  • Due to the complete reduction of unreduced nitrate to ammonia, abundant zinc dust has been observed to induce false-positive nitrite reduction reactions.


  • Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
  • D. Skerman, A guide to the identification of the genera of bacteria, The Williams & Wilkins Co., Baltimore, MD, p.218 – 220 (1967)
  • https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/1/73426dat.pdf
  • biologypractical.com/nitrate-reduction-test-objective-principle-procedure-and-result/
  • dcccd.edu/jreynolds/microbiology/2420/files/nitrate.pdf
  • https://microbenotes.com/nitrate-reduction-test-objectives-principle-procedure-and-results/
  • austincc.edu/microbugz/nitrate_reduction.php
  • asmscience.org/content/education/protocol/protocol.3660
  • https://catalog.hardydiagnostics.com/cp_prod/content/hugo/nitratereagent.htm

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