Dissolve 21 grams in 1000 ml distilled water, boil to dissolve the medium completely
and distribute into test tube. Sterilize by autoclaving at 15 lbs. pressure (121 °C) for 15 min, cool
it to 42-45°C and place the tube for preparation of slant. Inoculate test sample aseptically.
After incubation of 24 hours add few drops of Nitrate reagent A and B. Observe for development of a
distinct red or pink color within 1-2 minutes, indicating reduction of nitrate to nitrite.
Preparation of nitrate reagent A and B
Nitrate reagent A: 0.5 g α-naphthylamine (a carcinogen) in 100 ml 5 N acetic acid by gently
heating. Nitrate reagent B: 0.8 g sulfanilic acid in 100 ml 5 N acetic acid by gently heating.
