6 4 



SOIL BACTERIOLOGY 



Exercise 29 

 Nitrogen Fixation by Pure Cultures of Azotobacter 



1. Prepare six i-liter Erlenmeyer flasks with 100 c.c. 

 each of mannit agar. In place of the flasks large pans or 

 moist chambers may be used. The object is to use a vessel 

 that will give a large surface exposure. 



2. After sterilization, inoculate the agar films with a 

 pure culture of Azotobacter. This may be accomplished 

 by using i-c.c. transfers from a suspension in sterile water. 



3. Immediately after inoculation remove half of the 

 cultures for analysis. These may be treated with sulphuric 

 acid or sterilized. 



4. A few days after inoculation add 10 c.c. of sterile 

 water to each culture. 



5. 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 even film over the entire surface. 



6. About 28 C. is a favorable temperature for growth. 



7. After twenty-one days analyze all of the cultures for 

 total nitrogen. 



TABLE 27. Nitrogen Fixation by Pure Cultures of Azotobacter 



