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Nitrate Reduction Test Principle and Procedure

In this nitrate reduction test principle and procedure post we have briefly explained nitrate reduction test principle, objectives, requirements, procedure, uses and limitations.

Nitrate Reduction Test

Nitrate Reduction Test in Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. 

Nitrate reduction test also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. Nitrate broth contains nutrients and potassium nitrate as a source of nitrate.

Principle

After incubating the nitrate broth, add a dropper-full of sulfanilic acid and α-naphthylamine. 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.

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 it could mean that the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test.

If the medium does not turn red after the addition of the nitrate reagents, add a small amount of powdered zinc.

Requirements

Chemicals

1. Optimum specimen

2. Sulfanilic acid solution

3. Alpha-Naphthylamine solution

4. Zinc powder

Nitrate broth

Heart infusion broth, 25.0 g, Potassium nitrate, 2.0 g, Distilled water, 1000.0 ml and Dissolve the ingredients in distilled water; adjust solution to pH 7.0.

Dispense 5 ml aliquots of the broth into 16 mm x 100-mm tubes with gas inserts. Autoclave for 15 min at 121⁰C. Store medium at 4C to 10⁰C (refrigerated) until used. Pre-warm the medium to room temperature before inoculation.

Procedure

1. Incubate inoculated media at 35⁰C to 36.5⁰C in a carbon dioxide-enriched atmosphere for 48 h.

2. After 48 h. incubation add, with Pasteur pipettes, 5 drops of Sulfanilic acid solution, followed by 5 drops of Alpha-Naphthylamine solution to each tube. Shake the tube well to mix reagents with medium.

3. Examine the suspension for a pink-red color which should develop within a few minutes if the medium is still warm.  The reaction may take a little longer if the medium is cold when the reagents are added.

4. If no color develops add zinc powder. Observe for at least 3 minutes for a red color to develop after addition of zinc.

Test Result

Nitrate Reduction Test

Nitrate Reduction Test, Image Source: everythingmicro.blogspot.com

A red color will be produced in the medium only when nitrite is present in the medium. Lack of a red color in the medium after the addition of sulfanilic acid and α-naphthylamine means only that nitrite is not present in the medium.

Applications

1. Although all members of the Enterobacteriaceae family decrease nitrate, some of them also metabolise nitrite to produce other chemicals.

2. Nitrate reduction test is thus utilised to distinguish Enterobacteriaceae bacteria that produce the nitrate reductase enzyme from Gram negative bacteria that do not generate the enzyme.

3. Nitrate reduction test some species, nitrate reduction may be linked to anaerobic respiration. It’s used to tell Mycobacterium apart.

4. Identifying Neisseria species and distinguishing them from Moraxella and Kingella When isolates of K. denitrificans appear to be gram-negative diplococci in stained smears, the nitrate reduction test is crucial for distinguishing between N. gonorrhoeae and K. denitrificans.

Limitations

1. The nitrate reduction test can be used to aid in bacterial identification. For a full identification, further biochemical tests using pure culture are recommended.

2. Because nitrite may be present in the culture media, the nitrate reduction test should be performed with a low nitrite media such as Nitrate Agar or Nitrate Broth.

3. When a negative zinc reduction test is combined with a negative nitrite reaction, it is assumed that the nitrate has been reduced beyond the nitrite stage. Although nitrogen gas is a frequent end product of nitrite reduction, it is not the only one. Additional testing may be required to determine the reaction’s final end products.

4. Negative nitrite reduction reactions must be validated by adding zinc dust to the medium to avoid false-negative nitrite reduction reactions.

5. Due to complete reduction of previously unreduced nitrate to ammonia, excessive zinc dust has been reported to generate false-positive nitrite reduction responses.

Further Readings

Reference