Nitrate is a poly-atomic inorganic molecule and its molecular formula is NO3–  

Nitrate is reduced by micro-organism in two ways.

1. Assimilatory Nitrate reduction.

Assimilatory nitrate reduction mainly occurs during aerobic condition in absence of nitrogen.In this reduction process nitrate and ammonia are used for synthesis of organic nitro-compounds and amino-acids.In assimilatory nitrate reduction the nitrate do not play any role in energy production.

2. Dissimilatory nitrate reduction.

Dissimilatory nitrate reduction is carried out under anaerobic condition where bacteria use nitrate as a electron acceptor.

These assimilatory and dissimilatory nitrate reduction process is carried out nitrate reductase enzyme.This nitrate reductase enzyme converts nitrate to nitrite .The reaction is as follows

NO3 + NADPH + H+       Nitrate reductase           NO2–  + NADP+ + H2O           

Further this nitrite is converted to ammonia by nitrite reductase and the ammonia formed is assimilated.The nitrate reductase test is performed by using two indicators and the reduction of nitrate to nitrite is detected by using two indicators and that are sulphanilic acid and α- napthylamine.


Aim of the experiment is to carry out nitrate reduction test .


  1. Peptone nitrate broth
  2. Test culture.
  3. Reagent A-Sulphanilic acid.
  4. Reagent B- α- napthylamine.
  5. Zinc dust.

Composition of Peptone nitrate broth

  • Peptone            -0.5 gm
  • KNO3                 -0.2 gm
  • Distill water    -100 ml
  • pH                       -7


In nature many micro-organism posses nitrate reductase enzyme  when this micro-organisms are exposed to nitrate as a sole source of energy have a ability to reduce nitrate to nitrite.Here in nitrate reductase test we expose these micro-organism to a media containing nitrate as a sole source of nitrogen and energy and further this nitrite is detected by using two reagents Sulphanilic acid and α- napthylamine.


  1.  Take sterile peptone nitrate broth and  innoculated it with loopful of  test culture  and this innoculated broth is incubated for about 24 hours at about 37 ° C .
  2. After incubation 0.5 ml of reagent A that is  Sulphanilic acid is added and then reagent B α- napthylamine is added observe the test tube for development of red colour.
  3. Further if no red colour develops a pinch of zinc dust is added and mix properly and test tube is observed for development of red colour.

Flow chart of Nitrate reduction test procedure


Reagent A is Sulphanilic acid and Reagent B alpha napthylamine

Reagent A is Sulphanilic acid and Reagent B alpha napthylamine



  • If development of red colour is observed after addition of reagents then it indicates positive nitrate reduction test.
  • If after addition of both reagents there is no red colour formation then it may indicate negative nitrate reduction test or false result  because there is a possibility that the microflora present in the tube may reduce the nitrate to other byproducts like ammonia,molecular nitrogen,nitrous oxide,nitric oxide and as the test reagents detects only nitrates so it can give a false negative result so to clear this doubt a pinch of zinc dust is added and tube is observed for red colour development .Here zinc ions reduce nitrates to nitrites and we can observe development of red colour as zinc ions confirms presence of residual nitrates and this development of red colour indicates negative nitrate reduction test.

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