Nitrate Reduction Test: Objective, Principle, Procedure And Result Interpretation

The nitrate reduction test is a qualitative procedure for determining the ability of bacteria to reduce nitrate. In the reaction, nitrate is reduced to nitrite, which may then be further reduced to nitrogen gas or ammonia.Other uses of Nitrate Reduction Tests are:

• Differentiating Mycobacterium species.
• 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.
• Facilitating species identification of Corynebacterium.

Objectives

• To determine the ability organism to produce an enzyme called nitrate reductase, resulting in the reduction of nitrate (NO3).
• It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen.

Principle

Some bacteria have the ability to reduce nitrates, as they can produce the enzyme ‘nitrate reductase’.This enzyme, in presence of suitable electron donor, reduces nitrates (N0₃ – ) to nitrites (N0₂ – ). The presence of N0₂ – is indicated by sulphanilic acid and a-naphthylamine, which change the colour to red.

In the nitrate reduction test, the test bacteria is grown in a broth medium containing nitrate (N0₃ – ). If the bacteria has the ability to reduce nitrates, the broth acquires a red colour upon addition of sulphanilic acid and a-naphthylamine.

If red colour is not produced, it indicates that either N0₃ – is not reduced by the bacteria at all or the bacteria produces highly potent nitrate reductase enzyme, which rapidly reduces N0₃ – beyond N0₂ – to NH₃ and N₂. To confirm this, a small amount of zinc dust is added, which reduces NO₃ – , if available in the medium, to N0₂ – , thereby producing red colour.

Thus, if red colour is produced, it indicates that N0₃ – is present in the medium unchanged and the bacteria does not have the ability to reduce nitrates. If red colour is not produced, it indicates that, the N0₃ – has been reduced beyond N0₂ – to NH₃ and N₂ and the bacteria has the ability to reduce nitrates.

Experiment

Reagents And Materials Required

• Test tubes
• Conical flask
• Cotton plugs
• Inoculating loop
• Autoclave
• Bunsen burner
• Laminar flow chamber
• Lispose jar
• Incubator
• Nitrate broth
• Zinc dust
• Nitrate reagent Solution A
• nitrate reagent Solution B
• Isolated colonies or pure cultures of bacteria.

Procedure

1. The ingredients of nitrate broth medium (containing nitrate as the main component) or its ready-made powder required for 100 ml of the broth is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
2. Its pH is determined using a pH paper or pH meter and adjusted to 7.2 using 0.1N HCI if it is more or using 0.1N NaOH if it is less. The flask is heated, if required, to dissolve the ingredients completely.
3. The broth is distributed into five test tubes (approximately 10 ml each), cotton-plugged, covered with craft paper and tied with thread or rubber band.
4. The broth tubes are sterilised at 121 °C (15 psi pressure) for 15 minutes in an autoclave.
5. The broth tubes are allowed to cool to room temperature.
6. The test bacteria are inoculated aseptically, preferably in a laminar flow chamber, into the broth with the help of an inoculating loop sterilised over bunsen flame. The loop is sterilised after each inoculation.
7. The inoculated broth tubes are incubated at 37°C for 48 hours in an incubator.
8. 0.5 ml each of the nitrate reagent Solution A (containing sulphanilic acid) and nitrate reagent Solution B (containing a-naphthylamine) are added into each test tube, shaken well and colour change is observed after 15 minutes.

Result Interpretation

A positive nitrite reduction is denoted by the appearance of a deep red color change after the addition of Nitrate Reagents A and B. Lack of color development denotes a presumptive negative nitrite reduction test. Development of a red color following the addition of Nitrate Reagent C (zinc dust), confirms the negative nitrite reduction test obtained in the first phase of the test. Lack of color development after the addition of zinc dust indicates that the nitrate was reduced beyond the nitrite reaction to nitrogen gas and constitutes a positive nitrate reduction reaction.

Uses Of Nitrate Reduction Test

• All members of the Enterobacteriaceae family reduce nitrate, but some members further metabolize nitrite to other compounds. It is thus used to differentiate members of Enterobacteriaceae that produce enzyme nitrate reductase from Gram negative bacteria that do not produce the enzyme nitrate reductase.
• The reduction of nitrate may be coupled to anaerobic respiration in some species.
• It is used in differentiating Mycobacterium
• Identifying species of Neisseriaand separating them from Moraxella and Kingella 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.
• Facilitating species identification of Corynebacterium

Limitations

• The nitrate reduction test may be used as an aid in the identification of bacteria. Additional biochemical testing using pure culture is recommended for complete identification.
• Due to the possible presence of nitrite in the culture media, a low nitrite media such as Nitrate Agar or Nitrate Broth should be used for the nitrate reduction test.
• A negative zinc reduction (no color change) test, in combination with a negative nitrite reaction, is presumptive indication that the nitrate was reduced beyond the nitrite stage. Although a very common end product of nitrite reduction is nitrogen gas, other end products may be formed. Additional testing may be required to determine the final end products of the reaction.
• To avoid false-negative nitrite reduction reactions, negative nitrite reactions must be verified by the addition of zinc dust to the medium.
• Excess zinc dust has been reported to cause false-positive nitrite reduction reactions due to complete reduction of previously unreduced nitrate to ammonia.

Further References

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
2. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.
3. Tille, P., et al. Bailey and Scott’s Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.
4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.
6. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

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