66 SOIL BACTERIOLOGY 



Exercise 32 

 Anaerobic Nitrogen Fixation (Clostridia^) 



1. Prepare four small flasks of Winogradsky's solution 

 (m. 41). Arrange to have the liquid high in the necks of 

 the flasks. 



2. After sterilization inoculate the liquid with a pasteur- 

 ized soil extract. 



Note. — ^Heat 50 grams of soil with 200 ex. of water for fifteen minutes 

 at 80** C. 



3. Allow the coarse particles to settle and pipet 5-c.c, 

 portions into the flasks. 



4. Arrange as follows: 



{a) 1 and 2, sterilize immediately, or add 5 c c. of sulphuric add. 

 Q)) 3 and 4, incubate at 28*^ C. 



5. After twenty-one days analyze for total nitrogen. 

 Transfer the entire contents to a Kjeldahl flask. In order 

 to avoid too rapid evolution of carbon dioxid, the sulphuric 

 acid should be added slowly. 



Exercise 33 

 Isolation of Anaerobic Nitrogen-fixing Organisms 



1. From the cultures obtained in the previous exercise 

 make transfers into tubes of sterile Winogradsky's solution. 



2. Incubate under anaerobic conditions for ten to four- 

 teen days at 28^ C. 



3. Inoculate from these cultures into tubes of sterile 

 water, and from these dilutibns into two tubes (in series) of 

 Winogradsky's agar liquefied and cooled to 40*" C. 



4. Pour plates in the usual way. 



