264 
Journal of Agricultural Research 
Vol. XXIV, No. 3 
that a prompt and heavy growth of Azotobacter can be secured by a 
vigorous aeration of a liquid culture in a manner similar to that used 
in stimulating yeast grow^. 
The experimental data which follow are merely a report of progress 
in an investigation having for its object the utilization of waste prod¬ 
ucts by Azotobacter for protein synthesis. 
METHOD OF CULTIVATION 
A rapid and vigorous growth of Azotobacter was produced by passing 
a current of air continually through a culture solution. The method used 
was identical with that described in a previous publication (14). 
Aeration was accomplished by attaching the culture flask equipped 
with Folin's aerating tubes to a vacuum system. Contamination and 
evaporation of the culture were reduced to a minimum by filtering the 
air through a sterile cotton filter and two or three flasks of sterile water. 
The air was thus filtered through cotton and rinsed in water before 
entering the culture flasks. 
Severn flasks of culture media could be easily aerated at the same 
time. Usually a cotton filter and a wash bettle were placed between 
the culture flasks. Little trouble occurred from contamination. A 
good grade of rubber tubing and tight fitting connections are a necessity, 
however. Flasks containing from 200 cc. to 2,500 cc. of medium were 
used. The quantity of air passed through the cultures was not meas¬ 
ured, but a vigorous bubbling of the liquid was maintained continuously. 
AZOTOBACTER CULTURES 
Cultures of Azotobacter were isolated from samples of soil received 
from various parts of Kansas, Colorado, California, Iowa, and Mississippi. 
Flasks containing dextrose-Ashby or mannite-Ashby medium were 
inoculated with the soil samples. Upon the formation of the charac¬ 
teristic surface growth, Ashby agar plates were streaked. Dextrose- 
Ashby agar slants were inoculated from well-isolated colonies and re¬ 
peated streakings of the cultures upon Ashby agar plates were made. 
A large number of the cultures were streaked consecutively from i to 
12 generations. The utmost care was exercisfed in attempting to obtain 
and maintain pure cultures. 
The inoculum used for seeding the medium was prepared by adding 
a portion of the emulsion from a young dextrose-Ashby culture to a 
flask containing dextrose-Ashby broth. This was aerated for two to 
four days. The quantity of this starter usually used for inoculating 
the experimental medium was 0.5 per cent to i.o per cent of the medium 
seeded. In all cases a morphological examination of the starter was 
made as a test for piuity before use. The temperature for incubation 
was 30° C. 
MEDIUM 
The .medium used in the following experiments, unless otherwise 
noted, was made from the following formula: 
Gm. 
Tap water.. 1,000.0 
K^HP O4. 5 
Mg S04. .2 
NaCl. 2 
Dextrose. 10. o 
