i8o FIXATION OR ASSIMILATION OF NITROGEN 



Add 100 c.c. of the solution to a conical wide-bottomed flask, 

 and inoculate with .1 to .2 gr. of garden soil. Incubate at 

 28 to 30 C. in the dark. In two or three days a scum or 

 pellicle forms which consists of a mixture or zooglcea of small 

 bacteria and Azotobacter with amoebae and infusoria. Examine 

 a small portion of the scum for stumpy or oval rods of 

 Azotobacter. Transfer a drop or a small portion of the scum 

 from this flask to another flask of the sterilized nutrient solution 

 and incubate : later transfer a drop from the second to the third 

 flask. In this way a tolerably pure culture of Azotobacter may 

 be obtained in three or four generations. From it quite pure 

 cultures may b.e prepared by isolation on mannite agar as 

 indicated in the next Exercise. 



Ex. 91. Prepare the following mannite agar medium : 

 Di-potassium phosphate rh ' . . .02 gr. 



Mannite '",' 2 gr. 

 Agar . ... . . . . 2 gr. 



Distilled water . . . . . 100 c.c. 



Sterilize. Add 10 to 20 c.c. to a Petri dish and slowly cool, 

 turning the dish upside down, so as to allow the water of 

 condensation to collect on the lid and not on the surface of the 

 medium. Add a small portion of the membrane or a drop of 

 the solution from the flasks in the previous Exercise to a small 

 quantity of sterile distilled water, and pour some of it over the 

 surface of the plate. Incubate at 28 C., and note the colonies 

 which grow : examine the size and shape of the organisms from 

 each kind of colony. 



Test the action of the organisms from the different colonies in 

 " bouillon " at 20 C. This medium remains sterile or clear 

 when inoculated with pure Azotobacter^ but generally becomes 

 turbid when other organisms are added. 



Ex. 92. Azotobacter colonies grow well on gypsum plates 

 which are permeated by a mannite phosphate solution. 



Take some plaster of Paris (gypsum) and mix it with water to 



