26 
Colorado Experiment Station. 
Attempts to secure pure cultures of blue-green algae (Cyan- 
ophyceae) have been attended with failure and hence there are no 
reliable experiments which prove that these possess the ability to 
assimilate free nitrogen. Our further studies will be directed to 
clearing up the relations of the blue-green algae to Azotobactcr 
cliroococcum and nitrogen fixation. It is a significant fact that 
the blue-greens are by far the most abundant algae in all Colorado 
soils examined. 
METHODS. 
In each case the samples included the surface 3 to 4 inches of 
soil. Any debris on the surface was first removed. The samples 
were taken during October, 1911, and brought from the field to the 
laboratory in sterilized, double, sugar sacks. In the laboratory, the 
soil was transferred to sterilized Mason jars. 
Florence flasks, 500 c. c. capacity, were filled to their greatest 
diameter with ground quartz which was previously washed fnee 
from all suspended matter. These were sterilized in an autoclave 
for 30 minutes at 120° C., in a moist condition. Each flask was 
plugged with sterile cotton. After removing flasks from the au¬ 
toclave they were so placed as to get a smooth, horizontal surface 
of the substratum. 
For inoculation 20 g. of each soil sample were shaken up 
for 5 minutes in 50 c. c. of sterile, distilled water. An amount of 
suspension material corresponding to 10 g. of soil, i. e- 25 c. c., 
was drawn off with a sterile pipette and distributed as evenly as 
possible over the ground quartz surface. 
With the above precautions, contamination was impossible; 
the abundant algal growth which appeared in all but two flasks 
assuredly represents only those forms existing in the '.soils used 
for inoculation. Of course, it is quite well known that sterile, dis¬ 
tilled water is essential; tap water may carry both vegetative and 
reproductive parts of algae. 
The flasks, 22 in number, containing as many different soil 
samples, were placed in the greenhouse in a sunny place. Late* 
they were removed to a shady situation in the botanical labratory 
where they grew fully as well as in the greenhouse. Each flask 
was tipped to one side so as to offer both a moist sand and a free 
water surface for the algae to grow on. 
A number of species of algae which appeared in the flask cul¬ 
tures were transferred to a 1% agar medium in which soil extract 
was used as the nutrient solution. The soil extract was prepared 
by filtering soil in Pasteur-Chamberland, unglazed porcelain fil¬ 
ters. In preparing Petri dish cultures, a small bit of algal material 
was removed from the flasks and shaken up vigorously in a test 
tube with about 2 c- c. of distilled water. Platinum wire loops from 
this were transferred to tubes of liquid 1% agar at 42 0 C. These 
were shaken and then poured into Petri dishes. Algal growth never 
