1914) 
SCHRAMM—GRASS-GREEN ALG AND ELEMENTARY NITROGEN 173 
fore, to study representatives from this genus as well as other 
members of the group Cyanophycee. 
In the progress of the work about to be reported, it soon be- 
came obvious that the development of pure culture methods 
would constitute a very considerable portion of the investigation, 
and it was deemed advisable to limit the nitrogen phase of the 
problem to the fixation of atmospheric nitrogen in the complete 
absence of combined nitrogen, leaving for a subsequent report 
the problem of elementary-nitrogen fixation in the presence of 
combined nitrogen. Тһе work concerning pure culture methods 
will be found reported elsewhere (34). 
EXPERIMENTAL 
MATERIALS AND APPARATUS 
Most of the alge isolated were soil-inhabiting species. Pre- 
liminary experiments showed that in nearly every case better 
growth was obtained on solid media than in liquid ones. For 
this reason it was decided to conduct all experiments concerning 
the fixation of free nitrogen in the complete absence of combined 
nitrogen on а solid medium. Agar could not be sufficiently 
freed from all traces of combined nitrogen, and the difficulties 
involved in preparing large quantities of silicic acid jelly suffi- 
ciently pure were so great that a No. 21 ground quartz was 
finally decided upon. 
Preparation of the Sand.—The sand, after being thoroughly 
washed, was boiled in concentrated hydrochlorie acid for two 
hours, subsequently washed free from chlorides with distilled 
water, and then heated almost to dull redness for from four to 
five hours. Тһе sand was then boiled a second time in chemi- 
cally pure concentrated hydrochlorie acid and again washed 
with distilled water until chlorides could no longer be detected. 
When this stage had been reached the washing with distilled 
water was continued a dozen times more, after which the sand 
was drained as thoroughly as possible and the washing completed 
with from five to ten changes of nitrogen-free water. After 
drying the sand in a clean evaporating dish, a sample was boiled 
in nitrogen-free water and the liquid tested for ammonia, nitrites 
and nitrates, but only uniformly negative results were obtained. 
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