66 SOIL BACTERIOLOGY 



Exercise 32 

 Anaerobic Nitrogen Fixation (Clostridiae) 



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 c.c. 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) i and 2, sterilize immediately, or add 5 c.c. of sulphuric acid. 

 (6) 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 dilutions into two tubes (in series) of 

 Winogradsky's agar liquefied and cooled to 40 C. 



4. Pour plates in the usual way. 



