2 ANNALS OF THE MISSOURI BOTANICAL GARDEN 



[Vol. 6 



tigations of the mechanism of their activities have not been 

 particularly inviting problems to most workers. This con- 

 dition or set of conditions has resulted in the production of 

 a large amount of "soil" and crude culture work and a com- 

 paratively small amount of true physiological work. Many 

 students of these problems have contended that this is as it 

 should be; that pure cultures in completely synthetic media 

 are so unnatural that results can have but little practical 

 bearing. It seems to us that this type of reasoning is unsound 

 and that it can never be productive of thoroughly reliable 

 either practical or purely scientific work. This point has been 

 discussed at length by Allen and Bonazzi (15) with especial 

 reference to the study of nitrification. Existing methods of 

 work were criticized, and the difficulties to be encountered in 

 the improvement of methods discussed. Since then, in line 

 with this method of attack, has appeared the work of Allen 

 (15) and of Davisson and his co-workers (16, 18, 19) on 

 improvement of methods of nitrogen determinations, and of 

 Bonazzi ( 19, 19*) on the nitrifying bacteria. The work re- 

 ported in this paper deals with experiments on Azotobacter 

 chroococcum, and they have proved to be as crude and erratic 

 as were those reported earlier on nitrification, yet just as illus- 

 trative of the difficulty of the problem and just as suggestive, 

 we hope, of possible methods of attack. 



Historical 



To review in detail all the difficulties that have been re- 

 ported in studies of Azotobacter since the organism was dis- 

 covered by Beijerinck in 1901 is wholly unnecessary at this 

 time. From numerous and diverse sources it is evident that 

 ordinary synthetic culture media are lacking in something 

 for pure culture work, and that aqueous soil extract or even 

 tap water is superior to distilled water, but that the addition 

 of a small amount of soil to the culture medium is far better. 



A step forward was made by Krzemieniewski ('08), who 

 found that humus was the important constituent of the soil 

 for Azotobacter, and that the activating substance in the soil 



