ISOLATION OF AZOTOBACTER 6 1 



Exercise 26 

 Nitrogen Fixation in Solution 



1. Measure into four 750-0.0. Erlenmeyer flasks loo-c.c. 

 portions of mannit solution (m. 39). 



2. Weigh accurately just 10 grams of soil into each 

 flask. 



3. To two of the flasks add concentrated sulphuric 

 acid at once, or sterilize. 



4. Incubate the cultures for three weeks at 28 C. 



5. At the end of this time analyze for total nitrogen 

 according to the Kjeldahl method (see page 145). 



6. Subtract the nitrogen in the soil and culture at the 

 beginning, from that in the culture after three weeks' 

 growth. The difference represents the amount of nitrogen 

 fixed by micro-organisms. 



Exercise $27\ 

 Isolation of Azotobacter 



1. Prepare four flasks (loo-c.c. Erlenmeyer) of mannit 

 liquid medium, 20 c.c. in each. 



2. Inoculate with i or 2 grams of soil. 



3. Incubate at 28 C. and note changes occurring in 

 cultures. 



4. Every two days examine the films in hanging-drop 

 preparations and note the predominating type of organism. 

 Also examine some of the surface film in a drop of water 

 mixed with a drop of Meissner's or Gram's iodin solution 

 (see pages 127, 128). 



5. Dilute two loops of surface film in a loo-c.c. sterile 

 water blank containing 50 grams of clean sand. 



Note. The presence of sand in the water blank aids in breaking up the 

 gelatinous clumps of the Azotobacter. 



