228 Journal of Agricultural Research voi. iv, N0.3 



Since the most important constituents of the cell seem to be the gran- 

 ules, a study of their constitution will be first taken up. So many differ- 

 ent hypotheses have been presented with regard to their constitution 

 that the following studies were undertaken in order to determine it. 



EXPERIMENTS WITH THE ORGANISM 



CULTURE USED 



After having observed the presence of granules in the cells of some 

 cultures of A. chroococcum, microchemical studies were undertaken to de- 

 termine their nature and to ascertain whether they could be stained in 

 such a way as to be easily individualized in future studies. Some micro- 

 scopical preparations had already shown that the Guignard (6, p. 19) 

 method was the one to be depended upon, but to study this point better, 

 a series of slides were prepared and stained from an old stock culture 

 that had been cultivated on ordinary mannit agar for two years without 

 having at any time been rejuvenated in soil. The culture had apparently 

 lost the power to produce pigment, as it had not produced any for the 

 last five or six transfers on solid medium. This culture was used in all 

 the experiments described in this paper. 



The growth on mannit agar was white, transparent, strongly raised 

 at the beginning, flattening with age, smooth, soft, and invading the 

 slant at the bottom. 



The culture had been forwarded to the Ohio Station by the Office of 

 Soil Bacteriology and Plant Nutrition, United States Department of 

 Agriculture, and had been obtained by that institution from the American 

 Museum of Natural History. At the time it was received in this labora- 

 tory it bore the label " Azotobacter chroococcum Beij." and has been kept 

 ever since in pure culture. 



When cultivated on mannit agar, to which had been added i gm. of 

 potassium nitrate per liter, the culture regained its pigment-producing 

 power, assuming a waxy and glossy appearance. 



Since the organisms vary from cocci forms to bacillary ones, no single 

 measure can be given to the species, but a distinction must be drawTi 

 between these two forms. The cocci forms measured i to 2/i in diame- 

 ter and the bacillary forms 3 to 4// by 1.5 to 2//. The nitrogen-fixing 

 power of the organism was very slight, since it fixed only 1.26 mg. of 

 nitrogen in 25 c. c. of solution in 37 days in pure culture. 



STAINING THE ORGANISM 



Many methods of staining the organism were tried in the hope of find- 

 ing one that would be adapted to follow up the growth and role of the 

 granules in the metabolism of the cell. All the methods used gave satis- 

 factory results. The solutions were tested several times on blood as a 



