64 LABORATORY MANUAL OF MICROBIOLOGY 



This solution should be alkaline. In the case of soil extracts 

 which had been treated with CaO in filtration no further treat- 

 ment is necessary. In other cases, the solution should be treated 

 with NaOH or limewater to render slightly alkaline before 

 evaporation. 



When evaporated, add 2 cubic centimeters of phenoldisulphonic 

 acid and stir with the rounded end of a glass rod for about 10 

 minutes so as to loosen the residue. 



Note. — Equations for the action of phenoldisulphonic acid on a 

 nitrate : 



H2SO4 + 2KNO3 = 2HNO3 + K,S04. 



C6H3(OH)(S03H)2 + HNO3 = C6H2(OH)(S03H)2(N02) + H2O. 

 C6H2(OH)(S03H)2(N02) + 3NH4OH = 



C6H2(ONH4)(S020NH4)2N02 + 3H2O. 



Dilute with water and add ammonia solution (strong ammo- 

 nium hydroxide diluted with an equal volume of water) until 

 alkaline; a yellow color is formed. This is then diluted to a 

 known volume and compared with the standard. 



Example. — Take 500 cubic centimeters of water to 100 grams of soil, and 

 in order to clarify add about 2 grams of calcium oxide. To secure a fair 

 sample, mix by rubbing in a mortar or by shaking in a wide-mouthed bottle. 

 Filter through folded filter paper until clear. Take a convenient volume, 

 for example, 25 cubic centimeters, and determine the nitrate present. 

 This is equal to 5 grams of soil. Use the colorimeter to compare the 

 standard solution with the unknown. 

 Formula for calculating results: 

 ^ 100 *S , K ,, 

 ^=W-A^'U-^ 

 Where X = Number of milligrams of N as NO3 per 100 grams dry soil. 

 W = Weight of dry soil. 



S = Cubic centimeters of water added to W. 

 A = Aliquot taken for evaporation. 



d = Number of cubic centimeters to which A was diluted. 

 K = Reading on scale of standard solution. 

 U = Reading on scale of unknown solution. 



M = Milligrams of N as NO3 in 1 cubic centimeter of standard 

 solution as diluted for reading. 



Standard Nitrate Solution. — Dissolve 0.722 gram of pure dry 

 potassium nitrate in 1,000 cubic . centimeters of water. Of this 

 strong solution dilute 10 to 100 cubic centimeters, and from this 



