Feb. 24,1923 
Stimulating the Growth of Azotobacter 
667 
The purity of the culture is dependent practically entirely upon mor¬ 
phological considerations. The evident complex life cycle of Azotobacter, 
with its varied and fluctuating forms, makes a morphological differentia¬ 
tion indefinite, especially since our present knowledge pertaining to this 
life cycle, and its relative importance to the physiological activities of 
the cell is limited. . 
In all, 16 cultures of Azotobacter were used in these experiments. 
No attempt has been made to classify them as to species. In general, 
cultures 232, 6A, 12B, 9B, 8B, iB, 10B, and 19 were all typically Azoto¬ 
bacter organisms producing a brown to blade pigment. Culture 3A, 
213, 2B, 5B, 11B, 9A, 222, and 3B were likewise typical, but pigment 
production by them was absent or doubtful. Culture 6A and 9B were 
isolated from Mississippi soil, 10B from California soil, 5B from Colorado 
soil, and 3A from Iowa soil. The remaining cultures were isolated from 
soil obtained from various parts of Kansas. 
The inoculum used for seeding media was prepared by adding a portion 
of the emulsion from a young dextrose-Ashby-agar culture to a flask of 
dextrose-Ashby broth. This was aerated for two to four days. The 
amount of this starter usually used for inoculating the experimental 
medium was 0.05 per cent to 1 per cent of the medium seeded. In all 
cases a morphological analysis of the starter was made as a test for 
purity before using. The temperature /or incubation was 30° C. 
MEDIUM 
The medium used in the following experiments, unless otherwise noted, 
WaS: Grams. 
Tap water. 1,000.0 
Potassium phosphate (K 2 HP 0 4 ). .,5 
Magnesium sulphate (MgS 0 4 ). .2 
Sodium chlorid (NaCl). .2 
Dextrose. 10. o 
This solution was neutralized, filtered if necessary, and the required 
amounts placed in flasks and sterilized in the autoclav at 20 pounds 
pressure for 30 minutes. A pinch of calcium carbonate (CaCO s ) was 
added to each flask before sterilization. 
CHEMICAL ANALYSIS 
Total nitrogen determinations and sugar analyses of the Azotobacter 
cultures were made at frequent intervals. In all cases the total nitrogen 
was determined by the usual standard methods. Unless otherwise noted, 
50-cc. portions of the media were used in duplicate. The figures referred 
to in the tables denote the average of the duplicate analyses. Nitrogen 
is recorded in all cases unless stated as net gain in milligrams of nitrogen 
per 100 cc. of media. This, in other words, refers to the amount of 
nitrogen fixed per gram of dextrose, as 100 cc. of media contains this 
amount of sugar. 
Sugar determinations were made according to the method proposed by 
Shaffer (5). The copper resulting from the reduced Fehlings was deter¬ 
mined by colorimeter readings. Duplicate readings were made each time, 
and the average of these was recorded. As a general rule, 50 cc. of the 
media were used. The protein was precipitated and removed, the fil¬ 
trate was diluted to 100 cc., and 20 cc. of this filtrate were used for 
reduction. The figures cited, unless so stated, refer to grams of dextrose 
per 100 cc. of medium. 
