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Nitrate Reduction Test

Nitrate positive bacteria are-

• E. coli
• Pseudomonas aeruginosa
• Campylobacter jejuni

whereas nitrate-negative bacterium is Acinetobacter baumannii.

Nitrate reduction test of bacteria as shown above image.

Principle of nitrate/nitrite reduction test

Incubate nitrate broth with a heavy inoculum of test bacteria. If the bacteria capable of producing the nitrate reductase enzyme reduce the nitrate, present in the broth, to nitrite; that may be further reduced to nitric oxide (NO), nitrous oxide(N2O), or nitrogen(N2). This test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex i.e. nitrite-sulfanilic acid which then reacts with an α-naphthylamine to give a red precipitate i.e. prontosil, that is a water-soluble azo dye. The red color will see only when nitrate is present in the medium. If there’s no red color in the medium after the addition of sulfanilic acid and α-naphthylamine, nitrite is not present in the medium. When the nitrate may not have been reduced, the test organism is nitrate-negative. If the nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite, the test bacterium is nitrate-positive.

Use of Zinc Powder

It is helpful when nitrite is not detected and therefore, it is necessary to test if the organism has reduced nitrate beyond nitrite. Add a pinch of Zinc powder to the culture. It catalyzes the reduction of nitrate to nitrite. The development of the red color on the addition of Zinc powder indicates that nitrate was not reduced by the test organism which suggests that the test organism is not capable of reducing nitrate. If there is no color change after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and hence is a nitrate reducer.

Use of  A Durham Tube

It is placed in the nitrate broth in order to detect gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways.

Composition of Nitrate Broth (Medium)

peptone:20 g

potassium nitrate:2 g

distilled water :1,000 ml

Note: Heart infusion broth  uses in the case for fastidious organisms in place of peptone( 25 gram per liter)

Final pH 7.0 ± 0.2 at 25°C

• Dispense 4-ml aliquots in 16- by 125-mm screw-cap tubes with Durham tube.
• Autoclave at 121°C for 15 min.
• Store at 2 to 8°C.

Preparation of Reagents

1. Reagent A ( 0.8% Sulfanilic acid )

Sulfanilic acid: 0.8 g

Distilled water: 70 ml

Glacial acetic acid: 30 ml

• Mix sulfanilic acid with water; heat to dissolve.
• Cool, and then add acetic acid.
• Store at 2 to 8°C.
• Shelf life is 3 months.

2. Reagent B (0.5% N,N-Dimethyl-alpha-naphthylamine)

Glacial acetic acid :30 ml

Distilled water:70 ml

N,N-dimethyl-α-naphthylamine :0.5 g

• Combine acetic acid and water.
• Add α-naphthylamine.
• Store at 2 to 8°C.
• Shelf life is 3 months.

3. Zinc Metal Dust

Requirements for Nitrate/Nitrite Reduction Test

1. Nitrate broth
2. Durham tube
3. Reagent A
4. Reagent B
5. Zinc dust (Powder)
6. Test organisms
7. Bunsen burner
8. Inoculating wire
9. Incubator
10. Control strains

• Escherichia coli ATCC 25922
•  Pseudomonas aeruginosa ATCC 27853
• Acinetobacter baumannii ATCC 19606

Test Procedure of Nitrate Reduction Test

Nitrate reduction to nitrite test is sometimes complete in two steps method.

First Method

The reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B.

Second Method

The reduction of nitrate beyond nitrite  determination with the help of zinc powder

• Inoculate test organism in nitrate broth and incubate the tubes at the optimal temperature of 30°C or 37°C for 24 hours.
• After completion of incubation look for nitrogen gas formation first before adding reagents.
• Add reagent A (6-8 drops).
• Similarly, add reagent B( 6-8 drops).
• Observe for color development within a minute or less.
• If no color develops add zinc powder.
• Observe for at least 3 minutes for a red color to develop after the addition of zinc.

Result Interpretation of Nitrate/Nitrite Reduction Test

Nitrate/Nitrite reduction test: Positive
Development of a cherry red color after the addition of reagent A and B
Lacking  red color development on adding Zinc powder

Nitrate/Nitrite reduction test: Negative
Development of red color on the addition of Zinc powder

Control strains

Escherichia coli ATCC 25922—nitrate positive, gas negative

 Pseudomonas aeruginosa ATCC 27853—nitrate positive, gas positive

Acinetobacter baumannii ATCC 19606—nitrate negative

Uses of Nitrate Reduction Test

1. The nitrate reduction test is a useful to test to differentiate Enterobacteriaceae from other gram-negative bacteria since all the members of the family Enterobacteriaceae reduce nitrate.
2. It is helpful for differentiating Mycobacterium.
3. The Nitrate reduction may be coupled to anaerobic respiration in some species.
4. Identification of Neisseria from Moraxella and Kingella. This test is a critical test for differentiating between N. gonorrhoeae and K. denitrificans, particularly when strains of Kingella denitrificans appear to be the same i.e. gram-negative diplococci in gram-stained smears.
5. It also facilitates species identification of Corynebacterium.

Limitations of Nitrate/Nitrite Reduction Test

1. Failure to recognize that the organism did not grow in the medium will result in possible false-negative test(s).
2. Interpretation of color reactions should be made immediately, as color reactions with a positive test may fade rapidly.
3.  A faint pink color may be produced following the addition of the nitrate reagents. This is not a positive result.
4. 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.
5. A negative zinc reduction (no color change) test, in combination with a negative nitrite reaction, is a 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.
6. To avoid false-negative nitrite reduction reactions, negative nitrite reactions must be verified by the addition of zinc dust to the medium.
7. Excess zinc dust has been reported to cause false-positive nitrite reduction reactions due to the complete reduction of previously unreduced nitrate to ammonia.

Bibliography

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.