666 
Journal of Agricultural Research 
Vd. XXin, No. 8 
incapable of supporting rapid cell development. Such cultures have 
invariably shown a vigorous and heavy growth in 24 to 48 hours, a 
growth comparable to that exhibited by the common metatrophic forms 
growing on a nutrient medium. This rapid growth of Azotobacter on 
such agar slopes is attributed to maximum aeration. 
The purpose of the present investigation was to develop a method for 
inducing a prompt growth of Azotobacter in liquid media. The im¬ 
portance of aeration in this respect is indicated by the following data. 
METHOD OF AERATION 
Hoffman (j) suggested the sand slope cultural method for supplying a 
maximum surface exposure for aeration. Allen (7) used the mechani¬ 
cal agitation method employed by Bonazzi (6) in his nitrification experi¬ 
ments and reported variable results with this method of aeration. He 
attributed the greatest influence to the chemical composition of the 
media. 
The activity of organisms in many fermentation industries has been 
materially increased by aeration. Probably in most cases the interest 
has been focused upon the fermentation processes rather than on any 
direct interest in cell multiplication. However, within the last few years 
aeration has been employed commercially for the dirct purpose of increas¬ 
ing the growth and thereby the yield of microorganisms. The cultiva¬ 
tion of baker's yeast and the production of yeast protein in Germany by 
the fermentation of factory wastes is dependent upon this principle. 
Aeration was accomplished in the following experiments by bubbling 
air vigorously and continually through the culture medium. The air 
was passed through the culture by attaching the culture flask equipped 
with Folin’s aerating tubes to a vacuum System. Contamination and 
evaporation of the culture was reduced to a minimum by filtering the 
air through a sterile cotton filter and two or three sterile flasks of water. 
The air was thus filtered through cotton and rinsed in water before enter¬ 
ing the culture flasks. 
Several flasks of culture media could be easily aerated at the same 
time. Usually a cotton filter and wash bottle were placed between each 
two culture flasks. Little trouble occurred from contamination. A 
good grade of rubber tubing and tight-fitting connections are necessary, 
however. Flasks containing from 200 cc. to 250 cc. of media were used. 
The amount of air passed through the cultures was not measured, 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 Missis¬ 
sippi. Flasks containing dextrose or mannite Ashby media were inocu¬ 
lated with the soil samples. Upon the formation of the characteristic 
surface growth Ashby agar plates were streaked. Dextrose-Ashby-agar 
slants were inoculated from well-isolated colonies, and repeated streak¬ 
ings of the cultures upon Ashby agar plates were made. A large number 
of the cultures were streaked consecutively from 1 to 12 generations. 
The utmost care was exercised in attempting to obtain and maintain 
pure cultures. Much difficulty was experienced in satisfying one's self 
of the absolute purity of a culture. This is accounted for by several 
justifiable reasons. 
