Nitrate Reduction Test: Principle, Procedure, Results

Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae based on their ability to produce nitrate reductase enzyme that hydrolyzes nitrate (NO3-) to nitrite (NO2-), which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide and ammonia (NH₃₎ depending on the enzyme system of the organism and the atmosphere in which it is growing.

Uses of Nitrate Reduction Tests

1. Differentiating Mycobacterium species.
2. 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.
3. Facilitating species identification of Corynebacterium.

Principle

Heavy inoculum of test organism is incubated in nitrate broth. After 4 hrs incubation, the broth is tested for reduction of nitrate (NO₃^–) to nitrite (NO₂^–)  by adding sulfanilic acid reagent and α- naphthylamine.

1. 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.
2. 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 the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test. Add a small amount of powdered zinc. If the tube turns red after the addition of the zinc, it means that unreduced nitrate was present*. Therefore, the red color on the second step is a negative result.

*Note:  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 the medium does not turn red after the addition of the zinc powder, then the result is called a positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the medium, either, that means that denitrification took place and ammonia or molecular nitrogen were formed.

Requirements:

1. Media: Nitrate broth with inverted Durhams tube
2. Reagents: Sulphalinic acid reagent, alpha napthylamine reagent, zinc dust
3. Others: Inoculating loop, Bunsen burner, dropper

Procedure

1. Inoculate nitrate broth with a heavy growth of test organism using an aseptic technique.
2. Incubate at an appropriate temperature for 24 to 48 hours
3. Add one dropper full of sulfanilic acid and one dropper full of an α-naphthylamine to each broth.
   1. At this point, a color change to RED indicates a POSITIVE nitrate reduction test. If you get a red color, then you can stop at this point.
   2. No color change indicates the absence of nitrite. This can happen either because nitrate was not reduced or because nitrate was reduced to nitrite, then nitrite was further reduced to some other molecule. If you DO NOT get a red color, then you must proceed to the next step.
4. Add a small amount of zinc (a toothpick full) to each broth. Zinc catalyzes the reduction of nitrate to nitrite.
   1. At this point, a color change to RED indicates a NEGATIVE nitrate reduction test because this means that nitrate must have been present and must have been reduced to form nitrite.
   2. No color change means that no nitrate was present. Thus no color change at this point is a POSITIVE result.

Result and Interpretation

1. Nitrate Reduction Positive:  (Red after sulfanilic acid + alpha-naphthylamine; no color after zinc)
2. Nitrate Reduction Negative:  (No color after sulfanilic acid + alpha-naphthylamine followed by Red after zinc)

Quality Control

1. Pseudomonas aeruginosa reduces NO_{3 (Nitrate)} to N_{2 (Nitrogen)}.
2. Escherichia coli reduces NO_{3 (Nitrate)} to NO_{2 (Nitrite)}.