[jONEs] STUDY OF SOME AZOTOBACTER 45 



Examination of slide preparations showed these to be azotobacter, 

 staining golden yellow with iodine solution and being very granular, 

 granules varying in size and mostly spherical and well defined. Cap- 

 sules were not much in evidence. A few contaminating rods as in the 

 surface loam cultures were present. 



Some of the azotobacter colonies were a bright yellow. It was 

 later found that this coloration was due to a yellow pigment-producing 

 rod that was difficult to eradicate from cultures. 



Some colonies of streptothrix and others of the higher bacteria 

 were present, also a few moulds. 



3. Subsoil 30 inches. No azotobacter colonies developed. 



A few very small, very moist, colorless, bacterial colonies, also 

 very small, red, pink and yellow colonies, also some colonies of higher 

 bacteria were present. 



4. Old compost. One azotobacter colony found, giving a count 

 of 100 per gram of soil. This was similar in appearance to those described 

 in surface loam. There were also present a few colorless, viscid colonies 

 of rod bacteria which dissolved calcium carbonate, a few colonies of 

 higher bacteria, and some fungi. 



5. A^ew Compost. Nothing but a few colonies of the streptothrix 

 type and some moulds. 



6. Road Sand. Azotobacter, two colonies, giving a count of 

 200 per gram sand. These were 8 m.m. diameter, raised, glistening, 

 wrinkled, pasty-viscid, colorless, semi-transparent, and similar in 

 every respect to those described from surface loam. 



When three weeks old, the azotobacter colonies from all plates 

 had become brown to a varying degree, the larger, moister colonies 

 being less intensely colored than the smaller, pasty, drier colonies, 

 the latter in some cases being almost black. 



Isolation of Azotobacter. 



For further study, sixteen azotobacter colonies varying more or 

 less in appearance were selected from these cultures, and inoculated into 

 flasks of Ashby's solution and repeatedly replated in Ashby-agar until 

 eventually pure cultures of azotobacter were obtained. A comparison 

 of these pure cultures showed four types, varieties or species of azoto- 

 bacter to be present, and subsequent observation of a large number of 

 reinoculations covering a period of more than two years shows the 

 differential characters of these four types to be practically constant. 

 For convenience, they have been tentatively named Aj, A2, A3, A^. 



