THE STUDY OF MICROORGANISMS IN THE SOIL 111 



flasks large pans or moist chambers may be used. The object 

 is to use a vessel that will give a large surface exposure. 



After sterilization, inoculate the agar films with a pure culture 

 of Azotobacter. This may be accomplished by using 1-cubic 

 centimeter transfers from a suspension in sterile water. 



Immediately after inoculation remove half of the cultures for 

 control analysis. These may be treated 

 with sulphuric acid or sterilized. 



A few days after inoculation, add 5 cubic 

 centimeters of sterile water to each culture. 



Incubate the cultures in such a position 

 that only a portion of the surface will be 

 covered with water, and from day to day 

 rotate. In this way it is possible to get an 



f,, .1 ,. J. Fig. 11. — Azotobacter 



even film over the entire surface. chroococcum, young cui- 



About 28°C. is a favorable temperature tures. {Krzemieniewski.) 



for growth. 



After 21 days, analyze all of the cultures for total nitrogen. 



Exercise 28 



Effect of Variation in Carbohydrate on the Growth of Azotobacter 



Prepare six tubes of culture Medium 77, without mannitol. 



(a) 1 and 2, control (without mannitol) . 

 (6) 3 and 4, add 1 per cent of mannitol. 

 (c) 5 and 6, add 1 per cent of lactose. 

 Inoculate all cultures from a water 

 suspension of a young culture of Azoto- 

 bacter. Incubate tubes 1, 3, and 5 in 

 the open and tubes 2, 4, and 6 in the 

 anaerobic jar. Examine every 2 or 3 

 days for a period of 14 days. 



Record the growth and pigment for- 

 mation of Azotobacter on the various 

 culture media. If the tubes kept under 

 anaerobic conditions fail to show a brown to black pigment 

 after 2 weeks, open the jar, incubate again and note change 

 in the color. 



Fig. 12. — Azotobacter agile 

 showing flagella, X 660. 

 {Beijerinck.) 



