9o8 
Journal of Agricultural Research voi. xxiv, no. h 
were obtained from a number of different States. These were secured, 
in most instances, through the various experiment stations. The 
collector was requested to send samples, where possible, from alkaline 
or limed, and acid or unlimed, adjacent soils. 
METHODS 
Soon after the soils reached the laboratory they were well mixed and 
four 300 cc. Erlenmeyer flasks containing 50 cc. of mannite cultural 
solution were inoculated from each soil. Ten cubic centimeters of a 
suspension prepared by shaking i part of soil with 2 parts of sterile 
water was used as an inoculum. The suspension was allowed to stand 
a few minutes to let the heavier soil particles settle out. Two of the 
cultures were immediately sterilized in the autoclave to act as controls 
on total nitrogen determinations. 
The culture medium employed had the following composition: 
Magnesium sulphate. 
Di-basic potassium phosphate 
Sodium chlorid. 
Ferric chlorid. 
Calcium chlorid.. 
Mannite.. 
Distilled water . 
0.2 gm. 
0.2 gm. 
0.5 gm. 
Trace. 
Trace. 
2o.ogm. 
1,000.0 cc. 
This medium was rendered slightly alkaline to phenolphthalein with 
sodium hydroxid. In all experiments, except those reported in Table 
V, a small quantity of sterile calcium carbonate was added to each cul¬ 
ture flask before inoculating. In all cases the cultures were incubated 
at room temperature for 3 weeks, after which total nitrogen determina¬ 
tions Were made according to the modification of the Kjeldahl method 
suggested by Tatshaw (10). The quantities of nitrogen reported repre¬ 
sent the average of duplicate cultures after deducting the average of 
duplicate controls. 
During the incubation period frequent examinations were made both 
macroscopically and microscopically, to ascertain the character of the 
growth. When “no film’' is reported, no growth resembling Azoto- 
bacter took place during the first two weeks of incubation. After 
approximately 2 weeks of incubation a heavy fungus growth usually 
appeared, especially where no Azotobacter growth, or a nontypical 
Azotobacter film developed. After the development of a fungus film 
the growth became so complex that it was difficult to detect Azoto¬ 
bacter either macroscopically or microscopically. Such was the ap¬ 
pearance of those cultures in which Azotobacter is reported as questiona¬ 
ble. It is believed that the results would have been more consistent 
and striking if incubation had been reduced to 2 weeks. This would 
have avoided, to a large extent, the complications arising from the 
growth of fungi. 
The microscopic examinations were made by placing on a slide a loop 
of that part of the surface growth which appeared most characteristic 
of Azotobacter, covering witii cover glass, and examining with the 1/6 
objective. If typical Azotobacter were present in appreciable numbers, 
the picture was so striking as to be almost unmistakable. If Azotobacter 
are not present in a soil in sufficient numbers and vigor to develop a visi¬ 
ble film or to produce sufficient growth to be observed microscopically by 
the methods employed, it is questionable whether they are of any sig¬ 
nificance in the nitrogen economy of a soil. 
