676 
Journal of Agricultural Research 
Vol. XXII*, No. B 
Table VI .—Rearrangement of data from■ Table III 
1 dsy. 
! 2 days. 
1 
3 days. 
4 days. 
5 days. 
6 days. 
Total grams dextrose consumed 
o. 43 
0. 83 
°. 95 
0. 965 
O. 965 
O. 97 
Total milligrams nitrogen fixed. 
2. 90 
4. 40 
6. 10 
6. 40 
7J0 
Grams dextrose consumed per 
day. 
• 43 
. 40 
. 12 
. 015 
0 
. 005 
Milligrams nitrogen fixed per 
day .... 
2. 90 
I- 50 
1.70 
•30 
I. 40 
Rate azofication per gram dex¬ 
trose per day. 
6. 51 
tr. 30 
6. 42 
6. 56 
8. 04 
EFFECT OF CaC 0 3 
Calcium carbonate appears to be a universal constituent of the media 
used for the cultivation of Azotobacter. Lipman ( 2 ) asserts that calcium 
carbonate stimulates the growth of Azotobacter by either directly 
furnishing calcium or indirectly by making available more phosphorus, 
sulphur, and magnesium. Other investigators attribute its beneficial 
influence to the maintenance of a neutral reaction. Allen (7) in his 
studies on the composition of media for Azotobacter found CaC 0 3 to be 
essential. 
For further investigation with the cultivation of Azotobacter a sub¬ 
stance fulfilling all the requirements of a good medium was desired— 
namely, one that provides the proper food constituents, proper reaction, 
and is clear and free from sediment. The presence of CaC 0 3 inter¬ 
fered with the latter qualification. Preliminary experiments with a 
dextrose solution similar to the medium already mentioned, with and 
without CaC0 3 , had furnished evidence supporting the doubtful value 
of CaC 0 3 for the growth of Azotobacter in pure culture. 
Flasks containing 250 cc. of media with and without CaC 0 8 were 
inoculated with pure cultures of Azotobacter and aerated for six days. 
One flask of media was removed each day, and duplicate determinations 
of total nitrogen were made. The results are given in Table VII. 
The averages of the analyses for two and four days indicate a slight 
increase in the amount of nitrogen fixed in the CaC 0 3 medium. However 
on the sixth day the non-CaC0 3 medium shows a slight increase over the 
CaC 0 3 solution. The slight increase of nitrogen fixed in one medium 
over the other is attributed to experimental error rather than to any 
marked effect of the media. These results are comparable with numer¬ 
ous others obtained in this laboratory. The conclusions are that with 
pure cultures undergoing a vigorous aeration the presence of CaC 0 3 in 
the media is not essential for the prompt growth of the Azotobacter. 
It should be stated that the medium used in these experiments and in 
all other work where CaC 0 3 is not added was always corrected to a 
reaction near a P H of 7.0 to 7.4. The reaction of such a medium inocu¬ 
lated with pure cultures of Azotobacter and aerated will vary but little. 
