NON-SYMBIOTIC FIXATION OF NITROGEN 253 



action of denitrifying organisms in soil and in solution? 

 Why? 



How do denitrification and nitrate reduction differ? 



How may colonies of nitrate-reducing bacteria be 

 detected? 



14. Give all results in full and draw conclusions. Sug- 

 gest any possible practical applications of the above. 



REFERENCES 



LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp. 



35-40. 



MARSHALL: Microbiology, pp. 263-267. 

 LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 98-99. 



EXERCISE 8. TO ILLUSTRATE THE NON-SYMBIOTIC 

 FIXATION OF NITROGEN BY SOIL ORGANISMS, 

 AND ISOLATION OF AZOTOBACTER THROUGH 

 ITS MINERAL FOOD REQUIREMENTS 



Apparatus: Mannit solution for nitrogen-fixing organ- 

 isms; mannit agar; eight sterile 100 c.c. Erlenmeyer 

 flasks; concave slides; cover-glasses; concentrated H2S04; 

 filter paper. 



Culture. From clay loam, sandy loam, manure. 



Method. 1. Place 50 c.c. of the mannit solution in 

 each flask and sterilize (in autoclav). 



2. Inoculate in series as follows: 

 Flasks 1 and 2 



Flasks 3 and 4 0.1 gm. clay loam. 

 Flasks 5 and 6 0.1 gm. sandy loam. 

 Flasks 7 and 8 0.1 gm. manure. 



3. Incubate at room temperature and note the changes 

 taking place. A wrkikled skin, wnite at first, brownish 

 later, is gradually formed. This is composed of the aerobic 

 Azotobacter. 



4. From time to time examine the cultures in the hang- 

 ing drop and note the type of organisms predominating. 

 Single, small, thin bacilli are visible between the large 



