26 
AMERICAN JOURNAL OF BOTANY 
[Vol. 8 
organic nitrogen supplied in beef extract and peptone, beerwort, or humus, 
and in the two remaining solutions both ammonium sulphate and sodium 
nitrate were present; in none of the media was the nitrogen supplied as 
nitrate only. The cultures were grown in large flasks plugged with cotton, 
no other provision being made for aeration. No growth resulted on the 
media free from combined nitrogen; on those to which organic nitrogen 
sources were added, a vigorous development of the algae resulted in most 
cases but no increases in nitrogen content were obtained. On the media 
containing inorganic" nitrogen a good growth was obtained only in one 
experiment with cultures of several species of Stichococcus; in the remaining 
experiments with these media there was no growth because of the unfavor- 
able reaction of the solution. No increases in nitrogen content were ob- 
served, however, in any case. The results of these authors compare very 
favorably with those presented above in the tables, no fixation having been 
secured when ammonium sulphate or organic compounds served as nitrogen 
sources. 
In 1903, Charpentier substantiated Kossowitsch's results with Cysto- 
coccus. He grew the single species on a bean-glucose-gelatin medium; at 
the end of 20 days no change in the nitrogen content of the cultures was 
observed. However, these experiments were neither extensive enough, nor 
were they continued for sufficient length of time, to be conclusive. The 
failure to secure fixation may be attributed in this case also to the absence 
of proper nitrogen sources in the medium. 
As already pointed out in the first part of this discussion, the presence 
of nitrate nitrogen seems to be essential for the process of fixation. In this 
connection it is interesting to note that the presence of nitrates has been 
found beneficial for the growth of, and fixation of nitrogen by, Azotobacter 
forms and the legume bacteria. It has been repeatedly observed that the 
presence of very slight amounts of nitrates stimulates the development of 
the nodule bacteria. Thus, Fred and Graul (1916) report that in the pres- 
ence of small amounts of ammonium nitrate nodules were produced in 
great numbers on alfalfa and vetch by pure cultures of Bacillus radicicola; 
numerous nodules were formed also in the presence of small amounts of 
calcium nitrate, but with added amounts the degree of infection declined. 
In a recent paper. Hills (1918) reports that the presence of small amounts of 
nitrates exerted an enormous influence on the growth and reproduction of 
Azotobacter in sterile soil, the amount of fixation in such cultures being 
also increased in the presence of nitrates. In the case of Bacillus radicicola 
the nitrates stimulated growth and reproduction in sterilized soil, and on 
agar films the amount of fixation was greater when nitrates were added. 
The author concludes that this increased fixation may be the result of the 
increased growth of the organisms where nitrates were supplied. Though 
such observations as these cannot be claimed to have any direct application 
to the case of green plants, they are at least suggestive in view of the fact 
