Mar. io, 1923 Growth-Promoting Substance by Azotobacter 827 
improvement in the medium used and in the method of cultivation were 
desired for further investigation. This was accomplished by devising a 
means for cultivating the Azotobacter rapidly in a large volume of liquid 
culture medium. 
A description of the method of procedure and experimental data 
follows. 
MEDIUM 
To insure the absence of any known food accessory compound, 
chemically pure chemicals were employed in the preparation of the 
synthetic medium. All media were made up with a reaction of P H 7.0 
to 7.4 and were thoroughly sterilized at 20 pounds pressure for 30 
minutes. 
The composition of the medium was as follows: 
Distilled water. i, 000. 00 cc. 
Potassium acid phosphate. . 50 gm. 
Magnesium sulphate. .20 gm. 
Sodium chlorid. . 20 gm. 
Dextrose. 10. 00 gm. 
STARTERS 
Starters of pure Azotobacter cultures were made by inoculating flasks 
containing about 300 cc. of the dextrose media with a portion of the 
emulsion from a young dextrose Ashby slant culture. This was thor¬ 
oughly aerated for two days at a temperature of 30° C. Several strains 
of Azotobacter cultures have been used in these experiments. All 
cultures were isolated in this laboratory from different samples of soil. 
No attempt has been made to differentiate the cultures as to species. 
Pigment production was common in all cultures used. 
It was aimed to use only pure cultures of Azotobacter, and repeated 
streakings upon dextrose Ashby agar plates were made to insure freedom 
from contamination. Frequent morphological examinations were made 
to insure purity of the culutres. 
method of cuetivation 
A rapid multiplication of Azotobacter cells was encouraged by vigorous 
aeration of the liquid cultures. Bottles containing 2,000 cc. of sterile 
media were inoculated with 10 to 20 cc. of the Azotobacter starter. These 
were placed in the incubator, temperature of 30° C., and aerated by bub¬ 
bling air vigorously and continually through the medium. The air was 
passed through the cultures by attaching the bottle equipped with 
Folin’s aerating tubes to a vacuum system. Contamination and evapo¬ 
ration of the cultures were reduced to a minimum by filtering the air, 
first through a sterile cotton filter and then by washing in two or three 
flasks of sterile water. The air was thus filtered through cotton and 
rinsed in water before entering the culture media. From 12 to 24 of 
these bottles were aerated at the same time. A vigorous growth was 
produced within two to four days. If the culture is pure and no con¬ 
tamination occurs, the reaction of the media should remain close to the 
reaction of the media before inoculation. 
It is not believed that the inoculum supplies accessory food factors for 
stimulating the growth of Azotobacter (in this synthetic medium). 
Ten flasks containing 200 cc. of the media were inoculated, respectively, 
