NITRATES AND NITRITES 167 



should be poured back again on the soil so as to keep the latter 

 saturated the whole time. The nitrates are ultimately destroyed 

 by denitrification, when the water coming through shows no 

 reaction with diphenylamine. 



Ex. 85. Inoculate tubes of nitrate bouillon (Ex. 81) with 

 a gram of soil or fresh horse or cow dung, and 



(1) Incubate at room temperature as an aerobe. 



( 2 ) it 35 C. 



(3) 35 C. anaerobe in yeast flask. 



Note (a) time taken to develop froth ; (b) pre- r 

 sence or absence of turbidity and sediment ; (c) j 

 disappearance of nitrate (diphenylamine test). 



Ex. 86. Melt some tubes of nitrate gelatine 

 (Ex. 81) in warm water, and introduce into each 

 a loopful of nitrate bouillon containing any deni- 

 trifying species isolated in Ex. 83. Gently shake 

 the tubes to distribute the bacteria in the medium : 

 allow the latter to solidify and incubate at 18 

 to 20 C. Note the development of gas bubbles 

 in the gelatine (Fig. 31). 



Ex. 87. Prepare the following solution : 

 Calcium tartrate . . 10 gr. 

 Potassium nitrate . . 10 gr. 

 Di-potassium phosphate . .25 gr. 

 Tap water . . . 500 c.c. 

 Sterilize. 



Fill several 50 c.c. sterilized flasks to within an 

 inch of the top with the solution, and introduce F IG . 3I ._Gas bub- 

 into each i to 2 grams of soil, dung, or other S^^Sine 

 material. Then completely fill to the brim and b ? denitrifying 



J organism. 



close with a cork, through which is passed a small 

 piece of glass tube ; some of the solution will run out of the tube, 

 but the flask will be full : inoculate at 28 C. Denitrification soon 

 begins, and the gases (CO 2 and N) evolved drive out some of 



