116 LABORATORY MANUAL OF MICROBIOLOGY 



dilutions on the surface of the agar offer an easy and rapid method 

 of isolation. Sometimes it is difficult to separate Azotobacter 

 from a small organism known as Bacterium radiobacter. 



After 4 to 6 days examine plates. The Azotobacter colonies 

 are raised, convex, smooth, white, semi-opaque, moist, viscid, 

 often 4 to 8 millimeters in diameter. Make transfers to mannitol 

 agar slants. Stain with carbolated rose bengal or erythrosin. 



Note. — Instead of the enrichment liquid culture described above, 

 isolations may be made directly from the colonies on soil. Exercise 24. 



Exercise 26 



Use of Silica Gel Plate for Isolation of Specific Bacteria 



Silica-gel plates are prepared as described under culture media 

 and dialyzed free from chlorides. A sterile solution containing 

 the necessary mineral salts and the specific substrate is placed 

 upon the surface of the gel. The uncovered plates are then 

 placed in a warm place (at 60°C.) to allow the evaporation of 

 excess liquid, care being taken that medium does not become too 

 dry. 



The silica gel is then inoculated with small particles of soil. 



After a few days incubation, growth will take place around each 

 particle of soil. The nature of the organism developing will 

 depend upon the nature of the medium added to the gel. 



A mannitol medium free from combined nitrogen will allow the 

 development of Azotobacter under aerobic and of CI. pasteur- 

 ianum under anaerobic conditions. 



A medium containing cellulose and an inorganic source of 

 nitrogen will allow the development of Spirochseta and other 

 cellulose decomposing bacteria. 



When the colonies have developed sufficiently, they can be 

 transferred to specific nutrient media. 



Exercise 27 



Nitrogen Fixation by Pure Cultures of Azotobacter 



Prepare four 1-liter Erlenmeyer flasks with 100 cubic centi- 

 meters each of mannitol agar (Medium 77). In place of the 



