Jan., 1921] 
WANN — FIXATION OF FREE NITROGEN 
5 
each flask. The flasks were then immediately weighed in the order in which 
the agar was introduced. The mouths of the flasks remained plugged with 
cotton except during the actual processes of introducing the agar and of 
w^eighing. Although water vapor condensed on the walls and necks of 
flasks as the agar cooled, the use, with a number of flasks, of rubber stoppers 
instead of cotton plugs, demonstrated that there was no detectable loss of 
water vapor through the cotton plug during the interval between the intro- 
duction of the agar solution and the weighing. Thus the actual weight of 
medium in each flask was known, and, as will be seen from the tables, 
differences resulted of one or two grams in the weight of approximately 
equal volumes between the first and last flask of a series to receive the 
medium, due to the cooling of the agar during the processes involved. 
After the second weighing each flask was provided with a two-hole 
rubber stopper carrying intake and outlet glass tubes, the outer arms of these 
tubes being adjusted so as to be readily connected in series by means of 
rubber tubing. Long cotton plugs were loosely adjusted in the bore of each 
outer arm. The flasks were sterilized at 15 pounds' pressure for 20 minutes, 
the stoppers resting lightly in the mouths of the flasks but being tightly 
adjusted upon removal from the autoclav, the hands being moistened with 
alcohol for this operation. The flasks were allowed to cool in a dust-proof 
case. 
Inoculation. — The inoculations were made in the laboratory under a 
glass dust shield open on one side only. The inoculum consisted of a 
suspension of the algal cells in a test tube of sterilized nutrient solution minus 
combined nitrogen. Special cultures on hard (2 percent) agar were pre- 
pared, so that in making the suspension no agar was introduced with the 
cells. The tube was thoroughly shaken to secure a uniform suspension of the 
inoculum, of which ten drops were added to each flask in the 191 7-1 8 
experiment and one cubic centimeter was similarly added in 1919. In the 
former experiment four species were used and two flasks in each series 
were inoculated with the same species, three flasks of each medium remaining 
uninoculated as checks. In 1919 seven species were used, three flasks of 
each series being inoculated with each species with the exception of species 
no. 3 and no. 7, in which cases only two flasks of any one medium were 
inoculated; three flasks of each series remained uninodulated as checks. 
After the rubber stoppers were tightly fitted in the flasks, melted 
paraffin was run in around the flared neck, and the stopper and a portion 
of the neck of each flask were covered with sterilized cotton. During the 
two experiments eighteen contaminations occurred out of a total of 340 
flasks. 
Aeration. — The intake and delivery tubes of the flasks of each series 
were connected with rubber tubing for the purpose of aeration. In making 
connections the free ends of the glass tubes were painted with 95 percent 
alcohol, which was used also in washing out the bore of the rubber tubing. 
