168 DENITRIFICATION 



the solution, but the space in the flask is filled with inert gases 

 and the culture grows under practically anaerobic conditions. 

 From this flask introduce some of the fermenting liquid into 

 another filled completely in the same manner with the culture 

 solution. When frothing has gone on some time in the second 

 flask inoculate a third from it. In this way a culture can be 

 obtained containing denitrifying species almost entirely, the 

 aerobic non-denitrifying species present in the soil or dung being 

 suppressed. Inoculate agar and gelatine plates from the last 

 flask, and isolate and examine the organisms which develop. 



Ex. 88. A good medium in which to grow denitrifying species 

 is : 



Giltay's solution. 



Sodium nitrate . . . . . 2 gr. 



Citric acid . . . . . 5 gr. 



Magnesium sulphate . . . . 2 gr. 



Di-potassium phosphate . . . 2 gr. 



Calcium chloride " . . . . 2 gr. 



Water . . . i litre. 



Neutralize with a solution of sodium carbonate, and add a 

 drop of ferric chloride. 



Cultivate denitrifiers in flasks or tubes of this solution, and 

 note increasing alkalinity as growth proceeds for three or four 

 weeks. Oxygen removed from the sodium nitrate gives rise to 

 caustic soda (NaOH), some of which unites with the CO 2 pro- 

 duced from the oxidation of the citric acid, and forms sodium 

 carbonate (Na 2 CO 3 ). Both the NaOH and Na 2 CO 3 increase 

 the alkalinity of the solution, which may be quantitively estimated 

 with decinormal acid. 



