166 DENITRIFICATION 



(3) Boil the solution after adding the dung ; incubate and test 

 for nitrate as before. Does boiling check denitrification ? 



Ex. 83. Isolate denitrifying organisms from dung, soil, 

 ditch water, and straw. 



(a) Inoculate tubes containing 10 to 15 c.c. of neutral nitrate 

 bouillon (Ex. 81) with small pieces of fresh dung, soil, or 

 other material to be examined, or with watery extracts of them. 

 Incubate at 30 to 35 C. and when frothing is well developed 

 inoculate ordinary agar and gelatine plates from the fermenting 

 medium. Transfer small portions of the colonies which appear 

 on the plates into tubes of nitrate boullion ; observe frothing and 

 test for disappearance of nitrate with diphenylamine (Ex. 74) 

 during a fortnight, or longer if necessary. 



It is generally better to incubate the medium inoculated 

 directly with the dung or its extract anaerobically in a yeast 

 flask at 35 C. (Ex. 140), as this checks the development of 

 aerobic, liquefying, and non-denitrifying species and makes the 

 subsequent isolation of denitrifying species less laborious. 



(b) Similar results are obtained thus : Shake up small pieces 

 of dung, soil, etc., in sterilized water and inoculate agar and 

 gelatine plates with a drop of this extract in the usual way. 

 Test the denitrifying power of the several colonies obtained by 

 transference to nitrate bouillon as before. 



Ex. 84. Take a glass cylinder 2 feet long, 2 or 3 inches 

 in diameter, and close one end with a cork through which a 

 small piece of glass tube is passed. Place in an upright position 

 and half fill the cylinder with garden soil closely packed, first 

 covering the cork on the inside with coarse calico so that the 

 soil does not pass through the small glass tube : close the lower 

 end of the latter with a cork. Now pour water into the wide 

 open end of the cylinder and wait until the soil is saturated; 

 then add enough to fill the empty space above the surface of the 

 soil. Remove the cork from the small glass tube and allow the 

 water to percolate through : test for nitrates with diphenylamine 

 (Ex. 74) every second day. The water which drains through 



