R37. Methylene Blue Stain (Loeffler's) Solution A Methylene blue (90% dye content) 0.3 g Ethanol (95%) 30 ml
Solution B Diluted potassium hydroxide (0.01%) 100 ml
Mix solutions A and B.
R38. Nitrite Detection Reagents
A. Sulfanilic acid reagent Sulfanilic acid 1 g 5 N acetic acid 125 ml B. N-(l-naphthyl)ethylenediamine reagent N-(l-naphthyl)ethylenediamine dihydrochloride 0.25 g 5 N acetic acid 200 ml C. alpha-Naphthol reagent alpha-Naphthol 1 g 5 N acetic acid 200 ml
To prepare 5 N acetic acid, add 28.75 ml glacial acetic acid to 71.25 ml distilled water. Store reagents in glass-stoppered brown bottles. To perform test, add 0.1-0.5 ml each of reagent A and either reagent B or reagent C (as specified in method) to culture grown in liquid or semisolid medium. Development of red-violet color with reagents A and B or orange color with reagents A and C indicates that nitrate has been reduced to nitrite. Since color produced with reagents A