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Objectives of Nitrate Reduction Test
- 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 of Nitrate Reduction Test
All members of the Enterobacteriaceae family reduce nitrate to nitrite, but some members further metabolize nitrite to other compounds. Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen (O2). Many gram-negative bacteria use nitrate as the final electron acceptor. The organisms capable of producing the nitrate reductase enzyme then converts the nitrate (NO3) to nitrite (NO2). The reduction of nitrate to nitrite is determined by adding sulfanilic acid and alpha-naphthylamine. Nitrites react with an acid solution of sulfanilic acid and alpha-naphthylamine to form a red azo dye. The sulfanilic acid and nitrite react to form a diazonium salt. The diazonium salt then couples with the alphanaphthylamine to produce a red, water-soluble azo dye. In each of the test reactions the appearance of the red dye indicates the presence of NO 2 – in the test tube, whether as an unreduced primary substrate, a product of the reduction of NO 3 – by the test organism, or a product of the forced reduction of NO3 – with a reducing agent (zinc) for control purposes indicating a negative test result for nitrate reduction by the organism. 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. If no color change occurs after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds. A Durham tube is placed in the nitrogen broth in order to detect deterioration of the broth before inoculation, as evidenced by gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways.
Reactions of Nitrate Reduction Test
- Nitrate reduced to nitrite, red color formation: positive test
2e – + 2H + + NO 3 – → NO 2 –+ H 2O
- Nitrate is unchanged, negative reaction.
NO 3 –
- Nitrate reduced to nitrite to nitric oxide or further to nitrous oxide or further to nitrogen gas
NO 3 – → NO 2 – → NO → N 2O → N 2
- Nitrate reduced directly to molecular nitrogen.
2 NO 3 – + 10e – + 12H + → N 2 + 6H 2O
Media Used in Nitrate Reduction Test
Pancreatic digest of gelatin 20 gm/l, KNO3 2 gm/l
Procedure of Nitrate Reduction Test
- Inoculate the nitrate broths with bacterial suspension.
- Incubate the tubes at the optimal temperature 30°C or 37°C for 24 hours.
- After incubation look for N2 gas first before adding reagents.
- Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B.
- Observe for the reaction (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 addition of zinc.
Result Interpretation of Nitrate Reduction Test
|Reaction||Gas||Color after addition of solution A and B||Color after addition of zinc||Interpretation|
|NO3 → NO2||None||Red||–||NO3+, no gas|
|NO3 → NO2, gas
|None||Red||–||NO3+, no gas|
|NO3 → NO2,
|NO3 → gaseous
|NO3 → no reaction||None||None||Red||Negative|
Limitations of Nitrate Reduction Test
- Nitrate reduction is a supportive test for identification of Enterobacteriaceae to the genus level; however, additional follow-up, confirmatory testing is required for final identification.
Quality control of Nitrate Reduction Test
Positive: NO3+, no gas: Escherichia coli (ATCC25922)
Positive: NO3+, gas: Pseudomonas aeruginosa (ATCC17588)
Negative: Acinetobacter baumannii (ATCC19606)
- Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
- Buxton R. 2011. Nitrate and nitrite reduction test protocols. http://www.asmscience.org/content/education/protocol/protocol.3660
- Becton, Dickinson and Company. BBL nitrate broth with Durham tube package insert. Becton, Dickinson and Company, Sparks, MD. http://bd.com/ds/technicalCenter/inserts/L007480%2807%29%280506%29.pdf
- Reynolds J. 2011. Richland College. BIOL 2421. http://delrio.dcccd.edu/jreynolds/microbiology/2420/files/nitrate.pdf