NITRATE REDUCTION TEST

Nitrate reduction test is used to identify pathogenic bacteria that have the ability to convert nitrate (NO3) to nitrite (NO2) completely.  Certain Gram negative and Gram positive bacteria produce the enzyme, nitrate reductase (nitratase) which reduces nitrate to other nitrogenous compounds including NO2, and this serves as basis for their identification and differentiation from other bacteria that cannot reduce nitrate in the laboratory. Since the test is performed with Durham tube(s), nitrate reduction test can also be used to detect the production of gases (e.g. nitrogen, N2) by non-fermenters which convert nitrate to N2 gas. To actually determine whether or not nitrate has been reduced, nitrate reduction test is performed with two important reagents viz: Reagent A (sulphanilic acid or nitrate I) Reagent B (dimethyl-alpha-naphthylamine or nitrate II) which makes this test unique.

Procedure

  1. Prepare nitrate peptone water by constituting peptone water with the addition of 0.2 % potassium nitrate.
  2. Dispense the prepared medium in test tubes of 10 ml each.
  3. Invert Durham tube(s) into each of the test tubes and sterilize in the autoclave.
  4. After cooling to room temperature, inoculate tube(s) with the test organisms while leaving one tube uninoculated. The uninoculated tube serves as the negative control tube.
  5. Incubate tubes at 37oC overnight or at 18-24 hrs.
  6. Add 1 ml each of the two different reagents (nitrate A and B) to all the test tubes.
  7. First observe the tubes for the presence of gas in the Durham tube.
  8. The tubes turn red if nitrite is present in the medium (i.e. the test bacterium has converted nitrate to nitrite via the production of nitrate reductase). Nitrite in the medium reacts with nitrate A and B to form the red colour and this is considered a positive test result.
  9. Absence of a red colour in the test tubes shows a negative test result. This occurs if NO3 has not been reduced to NO2, or that NO3 was further reduced to another undetectable compound (e.g. N2 or NH3) after been converted to NO2 (which can be a positive result).
  10. To actually ascertain the reliability of the test result, add zinc compound (Zn) to the test tubes. Presence of red colour after addition of Zn indicates a negative result because the Zn compound will convert any remaining NO3 to NO2, and this will allow nitrate A and B to react with NO2 to form the red colour. If tube turns red, it indicates a negative test result. Absence of a red colour after adding Zn compound confirms a positive test result; and the reason is because the test bacterium may have actually reduced nitrate to nitrite and further converted NO2 unnoticeable forms of nitrogen including ammonia and nitrogen. P. aeruginosa is nitrate reduction positive. Streptococcus and Staphylococcus species are nitrate reduction negative. They produces red colour upon the addition of reagent / nitrate A and B as shown in the figures below.

Illustration of Nitrate reduction test, with and without the addition of zinc. From left to right: First tube is uninoculated tube, second tube is inoculated tube (without the addition of zinc), third tube is inoculated tube (with the addition of zinc), and fourth tube is inoculated tube, but showing a negative test result. The tube that remains clear after the addition of nitrate A and B is the positive test result tube. And in this case, the third tube shows a positive test result, because nitrate was reduced completely to nitrite.

Illustration of Nitrate reduction test, with the addition of zinc. Tube on the right hand side is negative while tube on the left hand side is positive. Positive tubes shows complete reduction of nitrate to nitrite, which is the main objective of this test. Nitrate reduction test is used to test an organism’s ability to reduce nitrate completely, thereby converting the former (i.e., nitrate) to nitrite. 

REFERENCES

Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

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