BACTERIA FIXING ATMOSPHERIC NITROGEN 113 



The substances are dissolved, and enough 10 per cent NaOH is added to make 

 faintly pink to phenolphthalein. 



3. Ashby's solution: 42 



Distilled water 1000 cc. NaCl 0.2 gram 



Mannite 15 grams CaS0 4 -2H 2 0.1 gram 



K2HPO4 0.2gram CaC0 3 5.0 grams 



MgS0 4 -7H 2 0.2 gram 



Make neutral to phenolphthalein with NaOH solution. 



4. Omeliansky's solution: 43 



Distilled water 1000 cc. MgS0 4 -7H 2 0.5 gram 



Dextrin 20 grams CaC0 3 10.0 grams 



K2HPO4 1.0 gram 



Solid media are prepared by adding 15 or 20 gm. of agar to the above solutions. 



For the study of pigment formation the last medium is very appropriate. Potato 



and potato agar, milk, mannite-nitrate media are also employed for the study 



of the morphology of the organism. 



A small quantity of fertile garden soil, or well limed and manured field 

 soil is used for the inoculation of the sterile liquid medium. After a few 

 day's incubation at 25° to 30°C, a pellicle is formed on the surface of the 

 liquid, at first gray in color, later becoming brownish. A microscopic 

 examination of the pellicle shows the presence of the typical Azotobacter 

 cells with large slimy capsules. A part of the pellicle is then transferred 

 into a fresh flask with sterile medium. After several transfers, the culture 

 is sufficiently enriched in Azotobacter so that one can proceed to isolate 

 it on agar media. A loopful of material from a young culture, in 

 which no heavy pellicle has as yet been formed, is diluted in a few cubic 

 centimeters of sterile water to separate the Azotobacter cells. A second 

 and third dilution is made and the surfaces of a series of Petri dishes, in 

 which liquefied mannite agar has been placed and allowed to cool, are 

 then streaked out with the suspensions of the various dilutions. At 

 25°C, pale, rounded, raised colonies are formed in a few days. In addi- 

 tion to these, transparent raised colonies of Radiobacter are also found. 

 The Azotobacter colonies are carefully selected by microscopic examina- 

 tion and are transferred into sterile liquid media. These precautions 

 are very important so as not to have the culture contaminated. 



The silica gel plate, to which minerals and mannite have been added, 

 inoculated with small particles of soil will give directly a nearly pure 

 culture of Azotobacter, as shown by Winogradsky. 44 



42 Ashby, S. F. Some observations on the assimilation of atmospheric nitrogen 

 by a free living soil organism, Azotobacter chroococcum of Beijerinck. Jour. Agr. 

 Sci. 2: 35-51. 1907. 



43 Omeliansky, W. L., and Sseverowa, O. P. Die Pigmentbildung in Kulturen 

 des Azotobacter chroococcum. Centrbl. Bakt. II, 29: 643-650. 1911. 



"Winogradsky, 1925 (p. 11). 



