186 CHARLES OSCAR CHAMBERS 



media slightly and the temperature considerably, some being 

 grown at room temperature, about 20°C. but mostly in the incu- 

 bator at about 29°C. as this had been shown by WilV^ Nitinsky^^ 

 and Czapek^^ to be the optimum temperature for this organism. 

 The criticism might be made that the method failed to get 

 all the nitrogen out of the cultures and that as a result no gain 

 was shown; and this point calls for serious consideration. It is 

 possible that the losses in D, E, G, and H are due to this cause; 

 but Experiments A, B and C return exactly the amount of 

 nitrogen that was supplied, and Experiment I, with Azotohacter, 

 shows a liberal gain or fixation. The large loss shown in Experi- 

 ment F, especially, was doubtless largely due to a change in the 

 method of digestion, by adding a drop or two or H2SO4 and then 

 evaporating until SO2 fumes appeared. There is this feature 

 in favor of the blowing-over method instead of distillation, and 

 of the colorimeter method instead of titration, viz., there is 

 less danger of driving over any of the alkali, which may occur in 

 distillation. Even if this should occur it would have no evil 

 effect, since the Nessler's solution to be added is itself strongly 

 alkaline. For this reason there can be no error on the side of 

 gain from this source, which by the distillation and titration 

 method is possible. A sHght increase in alkahnity from this 

 source would be sufficient to cause the gain shown in some 

 investigations reported. One such instance is cited by Pen- 

 nington. ^^ As to the process of digestion, it is the same in both 

 methods. In both there is danger of contamination from the 

 acid, which may contain ammonia. This occurred in one of 

 my spore determinations showing a high amount of nitrogen, 

 traced to a stray bottle of H2SO4 which had been left in the place 

 of my tested acid. 



"^"Will, H., Uber die Assimilation des atmospherischen Stickstoffs durch 

 Pilze. Jahrb. f. wiss. Bot. 44: 353-408. 1907. 



Will, H., Beitriige zur Kentniss derSprosspilze ohne Sporbildung, etc. Centrb. 

 f. Bact., etc. 34: 1-42, especially pp. 17-23. 1912. 



'^Nitinsky, Jacob, Uber die Beeinflussuiig der Entwickelung einiger Schim- 

 melpilze durch ihre Stoffwechselproducte. Jahrb. f. wiss Bot. 40: 1-93. 1904. 



1^ Czapek, I. c. 



2° Pennington, I. c. 



