1919] 



ALLEN — AZOTOBACTER CHROOCOCCUM 21 



use of crude and cumbersome methods that have not especially 

 invited further work in this direction. 



In the experiments reported above 10-cc. and 100-cc. por- 

 tions of culture media were used. The former did not ren- 

 der the Kjeldahl digestion sufficiently easy, whereas the others 

 were so large that with the containers available it was almost 

 impossible to keep the culture solution shallow enough to 

 permit proper growth. The proper line of improvement 

 seemed to be, therefore, to improve conditions as to methods 

 of digestion for cultures intermediate in size between the 

 above extremes, i. e., having a volume of 25-50 cc. The meth- 

 ods of distilling and recovering the ammonia were not wholly 

 satisfactory, and some studies of refinement of distillation 



methods were made. 



Another source of error or annoyance in the above experi- 

 ments was the matter of a uniform method of inoculating a 

 series of flask cultures. Of course, inoculating directly from 

 an agar slant with a platinum loop is open to considerable ob- 

 jection on the ground of lack of uniformity. Inoculations 

 should preferably be as small as possible where quantitative 

 chemical determinations are to be made on the culture, and 

 to this end attempts were made to inoculate the culture flasks 

 with either one cc. or a spiral of a suspension of 1 spiral of 

 agar slant growth in 50 cc. of sterile water. Results were 

 uncertain, in fact almost wholly negative. The point seemed 

 therefore worthy of further study. 



The work on improvements in methods (1) of sugar deter- 

 mination, (2) of nitrogen methods, and (3) of inoculation, 

 will now be considered seriatim. 



CULTURES 



For the determination of dextrose in cultures of Azotobacter 

 it seemed to us to be worth while to attempt to adopt some of 

 the more modern methods to the problem in hand. The 

 method of Shaffer ( '14) appeared most promising from the 

 standpoint of ease of manipulation and accuracy of results, 

 and, with only very minor modifications, it proved to be ap- 

 plicable to cultures of Azotobacter. The principle of the 











