SPECIES OF AZOTOBACTER 181 



the consistency of thick cream (about eight volumes of gypsum 

 powder to three of water are needed) ; pour it on a plate of 

 glass so as to be about a quarter of an inch thick, and 

 before it has set cut it into narrow strips 3 inches long by 

 J in. to } in. broad, or into circular pieces which will fit easily 

 inside an ordinary Petri dish. 



Prepare the following solution : 



Di-potassium phosphate . . .. . .05 gr. 



Sodium chloride . . . . .05 gr. 



Mannite . . . . . . 2 gr. 



Water . * . . . . . 100 cc. 



Add about 5 c.c. to some test-tubes, and place in each of 

 these a strip of gypsum, so that the lower end dips into the 

 solution : or pour a few c.c. into a Petri dish, and put in a 

 circular plate of the plaster : do not wet the upper surface of the 

 latter. Then place a drop or two of a culture of Azotobacter 

 on surface of the gypsum plate, and incubate at about 28 to 

 30 C. Observe the greyish-white growth, which becomes dark 

 brown generally in three or four days. 



Ex. 93. Isolate Azotobacter from soil by the following 

 method (Gerlach and Vogel) : 



Take 20 grams of fresh garden soil and place in a large 

 Petri dish : pour on it 100 c.c. of the following solution : 



Grape sugar. . , . . . . i gr. 



Di-potassium phosphate . ; . , .25 gr. 



Sodium chloride . . . . . , i .25 gr. 



Calcium carbonate . . . . .25 gr. 



Ferrous sulphate . . . . a trace 



Water . . . . . . 500 c.c. 



Keep it in the dark at 28 C. for two or three days. Examine 

 the scum which arises for cells of Azotobacter : these are usually 

 abundant and crowded into a slimy zooglcea. 



(i) With a platinum needle transfer a small portion of the 

 scum to a drop of water on a glass slide, cover with a slip, and 

 press the latter so as to break up the zoogloea. 



