Objective: To determine the ability of an organism to reduce nitrate to nitrite which is then reduced to free nitrogen gas. The nitrogen in nitrate serves as an electron acceptor. The result of the denitrification process is the production of nitrite:
In this case, all the NO3- will be converted to N2 gas which escapes to the atmosphere. We can test for this step by looking for the absence of NO3- through the addition of Zn powder as described below.
1. Inoculate a nitrate agar slant with your pure culture using a sterile loop to transfer a rather heavy inoculum.
2. Incubate at 37°C for at least 48 hours.
3. Add 2-3 drops of Reagent A and 2-3 drops of Reagent B to your tube. Reagent A is 0.8% sulfanilic acid in 30% acetic acid and Reagent B is 0.6% N,N-dimethyl-α-naphthylamine in 30% acetic acid (CAUTION: Reagent B is a potential carcinogen, so work in the hood and avoid inhaling it or allowing for contact with skin; wash hands thoroughly after work).
Reduction of nitrate to nitrite is indicated if a red color develops quickly (within 1-2 minutes). If no color develops, add a very small amount of zinc powder (~20 mg) to the tube containing the reagents. If a pink to dark red color develops after adding the zinc powder within 5 min., the test is negative (nitrate is present and is not reduced by the organism but zinc has reduced it to nitrite). If no color develops, the test is positive (the organism was able to reduce all the nitrate to nitrite and further to N2 which escaped from the tube).
-- If tubes are stored in the refrigerator, they should first be brought back up to the optimum temperature of the growth condition of the organism.
-- When performing the nitrate reduction test using α-naphthylamine, the color produced in a positive reaction may fade quickly. Interpret results immediately, particularly when performing a number of tests.
-- A strong nitrate-reducing organism may exhibit a brown precipitate immediately after the addition of the reagents. This is due to the effect of excess nitrite upon the p-amino group of the azo-dye and may be reduced by using dimethyl-α-naphthylamine.
-- Some organisms are capable of reducing nitrate to nitrite, yet they destroy the nitrite as fast as it is formed, yielding a false negative result. This nitrite destruction is evident in quite a few bacteria, particularly some Salmonella and Pseudomonas spp. and in Brucella suis.
-- Do not use an excess of zinc; if too much Zn is added, the large amount of hydrogen gas produced may reduce the nitrite (formed from unreduced nitrate) to ammonia (NH3) that could result in a false negative reaction or just a fleeting color reaction.