[Von 1 
170 ANNALS OF THE MISSOURI BOTANICAL GARDEN 
that under these conditions growth would be difficult. Because 
of the vast amount of energy necessary for free-nitrogen fixation, 
as illustrated by Clostridium Pasteurianum, the author suggests 
that there is a strong probability that Cystococcus is capable of 
assimilating free nitrogen only when the expenditure of energy 
in carbon assimilation is reduced to a minimum,—that is to 
say, when abundant available organic materials are furnished. 
He further emphasizes the necessity of employing combined 
nitrogen in a less readily available form than nitrates, suggesting 
organic nitrogenous compounds. On media composed of a 
decoction of beans to which were added 1 per cent and 2 per cent 
of dextrose and gelatin, respectively, Cystococcus was grown 
and the entire culture analyzed for total nitrogen. Although 
care was taken to have an abundance of available organic 
material (dextrose) present, Charpentier found that in no case 
was there any indication of free-nitrogen fixation. He further 
found that ammonia, asparagin, and peptone were each able to 
serve as the sole source of nitrogen. 
The association of blue-green algæ and soil bacteria is again 
referred to as an effective agent in free-nitrogen fixation by 
Bouilhac and Giustiniani (5, 6). Buckwheat, white mustard, 
corn, and cress were planted in clean sand moistened with a 
mineral nutrient solution free from combined nitrogen, and the 
substrata inoculated with Nostoc punctiforme and Anabæna sp. 
covered with bacteria. The phanerogams grew to maturity and 
analysis showed a marked fixation of free atmospheric nitrogen. 
Of particular interest are the observations of Heinze (14), 
who, however, fails to state whether or not the Chlorella ex- 
perimented with was in pure culture. He found that no appre- 
ciable growth took place in cultural solutions free from combined 
nitrogen, but that in the presence of the latter a rich growth ap- 
peared, unaccompanied, however, by a definite fixation of nitro- 
gen. More important are his experiments with Nostoc in impure 
condition, a good growth of the form being obtained in a min- 
eral nutrient solution free from combined nitrogen and sugar. 
These cultures, as well as others on soil inoculated with a similar 
Nostoc culture contaminated with bacteria and fungi, showed 
a definite amount of free-nitrogen fixation. Heinze was unable 
to find Azotobacter present, and this, together with the observa- 
