Nitrate Reduction Test: Principle, Procedure and Results 4.9/5 (10)

Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on the basis of their ability to produce nitrate reductase enzyme that hydrolyze nitrate (NO3) to nitrite (NO2) which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide and ammonia (NH3) depending on the enzyme system of the organism and the atmosphere in which it is growing.

Fig: Nitrate Reduction Pathway
Fig: Nitrate Reduction Pathway

Other uses of Nitrate Reduction Tests are: 

  1. Differentiating Mycobacterium species.
  2. Identifying species of Neisseria and separating them from Moraxella and Kingella species. The nitrate reduction test is a critical test for differentiating between N. gonorrhoeae and K. denitrificans, particularly when strains of K. denitrificans appear to be gram-negative diplococci in stained smears.
  3. Facilitating species identification of Corynebacterium

Principle:Heavy inoculum of test organism is incubated in nitrate broth. After 4 hrs incubation, the broth is tested for reduction of nitrate (NO3) to nitrite (NO2)  by adding sulfanilic acid reagent and α- naphthylamine.

  1. If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid.  When sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid.  This reacts with the α-naphthylamine to form a red-colored compound.  Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction.nitrate reducation test result
  2. If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable to reduce the nitrate, or the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test. Add a small amount of powdered zinc. If the tube turns red after the addition of the zinc, it means that unreduced nitrate was present*.Therefore, a red color on the second step is a negative result.

*Note:  The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby produced reacted with the α-naphthylamine to create the red complex.

Nitrate reducation test experiments

If the medium does not turn red after the addition of the zinc powder, then the result is called a positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the medium, either, that means that denitrification took place and ammonia or molecular nitrogen were formed.

Requirements:

  1. Media: Nitrate Broth with inverted Durhams tube
  2. Reagents: Sulphalinic acid reagent, Alpha napthylamine reagent, zinc dust
  3. Others: Inoculating loop, burner, dropper

Procedure:

  1. Inoculate nitrate broth with a heavy growth of test organism using aseptic technique.
  2. Incubate at an appropriate temperature for 24 to 48 hours
  3. Add one dropperfull of sulfanilic acid and one dropperfull of a α-naphthylamine to each broth.
    1. At this point, a color change to RED indicates a POSITIVE nitrate reduction test. If you get a red color, then you can stop at this point.
    2. No color change indicates the absence of nitrite. This can happen either because nitrate was not reduced or because nitrate was reduced to nitrite, then nitrite was further reduced to some other molecule. If you DO NOT get a red color, then you must proceed to the next step.
  4. Add a small amount of zinc (a toothpick full) to each broth. Zinc catalyzes the reduction of nitrate to nitrite.
    1. At this point, a color change to RED indicates a NEGATIVE nitrate reduction test because this means that nitrate must have been present and must have been reduced to form nitrite.
    2. No color change means that no nitrate was present. Thus no color change at this point is a POSITIVE result.

      Fig. Diagrammatic representation of the detection of nitrite in medium
      Fig. Diagrammatic representation of the detection of nitrite in medium

Result and Interpretation

  1. Nitrate Reduction Positive:  (Red after sulfanilic acid + alpha-naphthylamine; no color after zinc)
  2. Nitrate Reduction Negative:  (No color after sulfanilic acid + alpha-naphthylamine followed by Red after zinc)

Quality Control:

  1. Pseudomonas aeruginosa reduces NO3 (Nitrate) to N2 (Nitrogen).
  2. Escherichia coli reduces NO3 (Nitrate) to NO2 (Nitrite).

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16 thoughts on “Nitrate Reduction Test: Principle, Procedure and Results

    1. Tankeshwar Acharya

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      Thank you for your query. Staphylococcus aurues is nitrate test positive but we do not perform nitrate reduction test for S. aureus in routine laboratory works.

  1. Feri Eko Hermanto

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    Very good. But may you explain how Zn interact with unreduced Nitrate so it’s become red. Thank You.
    I have done some biochemical test for identifying bacteria for my practice laboratory (I’m a undergraduate student) and I don’t know to much how to use it for determining genus of my isolate. May be you have a biochemical identification guide.
    Thanks a lot sir, I hope I can get more knowledge with you.

    1. Tankeshwar Acharya

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      Dear Feri Eko Hermanto
      Thank you for your question. “The addition of the zinc reduce the nitrate to nitrite, and the nitrite in the medium formed nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby produced reacted with the α-naphthylamine to create the red complex.” Hopefully this answers your query. Regarding your second query, there are lots of related information already posted in the site. You can read them. Let us know.

  2. Feri Eko Hermanto

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    And one again, sir. I have do oxydase, urease, and motility test, and each test I have 2 repetition. But, every repetition give different result, one give me positive result and one again give me negative result. whereas, experiments were conducted for the same isolate. According to you, how can it be like that?
    I’m sorry because I have to much question, but it’s because I can understand every explanation you give, so I want to know more.
    Thank you very much, sir, I’m sorry if my English not good. I hope you can understand what I mean.

    1. Tankeshwar Acharya

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      Dear Feri Eko Hermanto
      I am happy to guide you, whatever i can. Firstly, you can get information about these tests in microbeonline too. Read and follow the standard procedure. The result for a particular isolate must be reproducible (whenever/wherever the test performed), but you said that in your laboratory you could not reproduce the results. There are few possibilities;
      1. Isolates might be different, this happens when you take the isolate from confluent growth. Please carry the procedure from well isolated colony.
      2. There might be procedure error that might have affected the result,e.g., if oxidase disc is exposed in environment for longer period of time, it may give false positive result. Use of loop instead of wire while stabbing the motility test medium, may give erroneous result.
      Finally, please try again, let me know. I can understand your English, no problem on this. Keep in touch.

  3. Feri Eko Hermanto

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    Is that possible the problem come from inoculation process? So there is no bacteria put into media?
    And if I conclude from your first possibility, there are many species on my microbe culture. In other words, my isolate is not pure culture, is that right, sir?

  4. The dipstick test for nitrite is little confusing. Can you please tell us about the microbes which will be positive and negative by this test? I ma interested to know if N. gonorrhoea, Chlymadia and Trichomonas will be positive or negative for nitrite test.

Do you have any queries? Please leave me in the comments section below. I will be happy to read your comments and reply.