Bacteriological Studies of the Fixation of Nitrogen. 
9 
General Methods. 
For determining the nitrogen fixing power of the different soils 
in solutions, we have used the mannite solution recommended by 
Lipman 1 except that the tri-basic potassium phosphate, (KsPCh), has 
been substituted for the di-basic, (K2HPO4). 
Mannite Solution for Nitrogen Fixation. 
Tap water 
Mannite 
k 3 po 4 
Mg S0 4 
Ca Cl 2 
10 per cent solution Fe Cl 
-1000.00 c. c. 
15.00 grams 
. 5 grams 
. 2 grams 
.02 grams 
1 drbp 
This was made neutral to phenolphthalein with normal NaOH. 
100 c.c. of this solution containing 1.5 grams of mannite were em¬ 
ployed for each soil examined. It was placed in 500 c. c. Erlenmeyer 
flasks and sterilized in the autoclave for five minutes at 120 degrees C. 
These solutions were inoculated with 20 c.c. of the given soil infusion 
corresponding to 10 grams of the soil. The infusion was prepared by 
mixing 150 grams of soil with 300 c.c. of sterile physiological salt so¬ 
lution (.75% Na Cl), shaking the mixture for five minutes and then- 
allowing it to stand for thirty minutes for the coarser particles to' sub¬ 
side, after which the inoculating suspension was withdrawn with a 
sterile pipette. Four flask cultures were prepared from each soil, 
two of which were analyzed for total nitrogen immediately, and the 
remaining two after thirty days’ incubation at 28 degrees C. 
For isolating and growing our stock cultures we have employed 
a mannite agar of the same composition as the mannite solution with 
the addition of 15 grams of agar per 1000 c.c. of solution. 
In isolating Azotobacter from the crude soil cultures we have de¬ 
pended solely upon repeated plating for obtaining cultures in a pure 
condition. We were troubled a great deal with a small bacillus which 
was almost invariably associated with the Azotobacter colonies in the 
original plates but by replating through three dilutions, sometimes 
three different times, we were able to obtain pure cultures. The inter¬ 
mediate glycerine solution recommended by Lipman did not prove 
satisfactory in our hands. 
Our stock cultures of Azotobacter were isolated in March, 1910, 
and have been transferred every fourteen days since that time on man¬ 
nite agar. 
For determining the total nitrogen in the cultures and in the soils, 
we have used the Gunning Method modified to include nitrates as de¬ 
scribed in the Official Methods of Analysis,. 2 page 8. 
1. Rept. of Soil Chemist and Bacteriologist, New Jersey Exp. Sta. 1908, p. 137 
2 . Bulletin No. 107 (Revised) Bureau of Chemistry, U. S. Dept. Agr. 1908. 
