60 LABORATORY MANUAL OF MICROBIOLOGY 



then slowly to lemon or yellow, and finally becomes a greenish- 

 yellow. 



This test can be used for quantitative determination of 

 nitrates. The test is based upon the final sulphur yellow color 

 rather than upon the initial red coloration. If the solution con- 

 tains much organic matter or ferrous iron, these should be 

 oxidized by a permanganate solution. The nitrous acid is 

 thereby also oxidized to nitric acid and if nitrous acid is deter- 

 mined separately, the results should be subtracted from those 

 of the nitric acid. 



The determinations are made by adding 1 cubic centimeter of 

 the concentrated solution of brucine and 30 cubic centimeters of 

 concentrated sulphuric acid to 20 cubic centimeters of the solu- 

 tion of the unknown. In adding the sulphuric acid, care should 

 be taken to prevent boiling. 



The nitrate is determined in a colorimeter, using for comparison 

 a standard solution containing 0.1872 gram KNO3 in 1 liter of 

 water. This standard gives 0.0001 gram N2O5 or 0.00002594 

 gram nitrate nitrogen per 1 cubic centimeter of solution. A 

 duplicate blank should always be made. 



Phenoldisulphonic Acid^ 



Dissolve 25 grams of pure white phenol crystals in 150 cubic 

 centimeters of pure concentrated sulphuric acid, add 75 cubic 

 centimeters of fuming sulphuric acid (13 per cent SO3), stir well, 

 and heat for 2 hours at about 100°C. The reagent prepared in 

 this way should not contain any mono-acids or any tri-acids. 

 Two cubic centimeters of this reagent give reliable results with 

 not more than 5 milligrams of nitrate nitrogen. 



Since in strongly acid solutions the following reaction takes 

 place, especially by heating, 



3HNO2 = HNO3 + 2N0 + H2O, 



a positive test for nitrate will be obtained, by the phenoldi- 

 sulfonic acid and brucine methods, in solutions containing only 

 nitrite. 



' Chamot, Pratt, and Redfield, /. Am. Chem. Soc, 33, 381-384, 1911. 



