[Vor. 1 
164 ANNALS OF THE MISSOURI BOTANICAL GARDEN 
Incidental to his work on the respiratory quotient in alge, 
Sehloesing (30) reported that in а culture containing prin- 
cipally Protococcus vulgaris Ag., and smaller quantities of 
Chlorococcum infusionum Menegh., Ulothrix subtilis Kütz., and 
Scenedesmus quadricauda Bréb., there was at the end of two 
months no diminution of nitrogen in the supernatant atmos- 
phere. This fact led the author to place these alge among 
those forms which do not fix free atmospheric nitrogen. 
As will have been observed, the work reported upon in the 
contributions cited was done with impure cultures. While in 
some cases but a single species was used, bacteria were present 
in all cases. Although in many instances this is not expressly 
stated, the author’s experience convinces him that the technique 
employed by these earlier workers made the contamination of 
their cultures with bacteria very probable. It is evident, 
therefore, that in the work done thus far it is impossible to 
state with certainty whether the results obtained are due to the 
activity of the alge, or to the bacteria, or to both. 
The first work done on the fixation of free nitrogen by alge 
in which pure cultures were used was that of Kossowitsch (18), 
in 1894. The only form isolated in pure culture by this investi- 
gator was one which he states resembled both Cystococcus 
(Nägeli) and Chlorella vulgaris Bey. He leaves its identity 
uncertain but designates it, for convenience, Cystococcus. Pre- 
liminary experiments with impure cultures of this alga had 
demonstrated that asparagin and ammonium tartrate could 
not serve as the source of nitrogen and that growth took place 
only when nitrates were supplied. In the experiments with pure 
cultures, flasks containing 70 grams of clean sand moistened 
with a mineral nutrient solution containing a known amount 
of calcium nitrate were inoculated with a carefully tested pure 
culture of Cystococcus and allowed to remain four months. 
To a number of the cultures dextrose was added, and to others, 
in addition to this sugar, pea-tubercle bacteria. At the con- 
clusion of the experiments the cultures were carefully tested 
for purity. Analysis in every case showed an absence of free- 
nitrogen fixation, and demonstrated clearly for the first time 
that an alga, Cystococcus, under the conditions realized in the 
experiment, did not fix free atmospheric nitrogen. That the 
