Bacteriological Studies of the Fixation of Nitrogen. ii 
character, a mixture of soil and crystals. Below this, the soil grew 
wet very rapidly and at sixteen inches was practically mud. There 
was no free water at three feet and in a nearby excavation which had 
been made for a cellar, over three feet deep, there was no water, yet 
the soil which varied from a sandy loam to a calcarious clay was de¬ 
cidedly wet and sticky sixteen inches below the surface. It was nec¬ 
essary to dig to a depth of six feet to reach the flow of ground water. 
Sample No. i was composed of the surface crust of which 
12.523 per cent was soluble in water. 19.822 per cent of this, or 
1.482 per cent of the air dried soil consisted of nitrates. The flask 
culture did not develop a typical brown Azotobacter membrane, but 
rather a whitish yellow scum. This was composed mostly of rod 
shaped organisms with scattering Azotobacter like forms which dis¬ 
appeared with age. A marked gaseous fermentation of the culture 
solution took place, accompanied by formation of acetic acid. In 
thirty days, this soil fixed 1.05075 m. g. of nitrogen. 
Sample No. 2 came from the mealy layer beneath the crust; 8.44 
per cent of this was water soluble, 15.421 per cent of which or 1.301 
per cent of the air dried soil was nitrates. A very delicate, white 
scum with almost no growth in the body of the liquid was all that 
was obtained in the culture. There was some fermentation and slight 
acid production with the odor of butyric ether. In thirty days, the 
increase in nitrogen was so small as to be practically negligible, being 
only .5604 m.g. 
Sample No. 3 consisted of the twelfth to fourteenth inch in¬ 
clusive where the ground was wet. Unfortunately I do not have the 
analysis of this portion of the soil, but in a nearby orchard the fourth 
to fifteenth inch inclusive contained .676 per cent nitrates. In all 
probability, my sample carried less than this since it contained a smaller 
amount of the rich surface material. With this, I secured the Azoto¬ 
bacter forms in a limited number, comparatively speaking, along with 
the ordinary rods which made up the patchy film and flocculent growth 
of the culture. Some fermentation and slight acid production with a 
cheesy odor were observed. An increase of 3.43245 m. g. of nitrogen 
was obtained after thirty days. 
Sample No. 4 came from near a tree along the edge of the affected 
area. The soil appeared normal and the few trees close by seemed in a 
healthy condition. The surface two inches were removed and the 
second to sixth inch inclusive were collected. A typical chocolate 
brown, wrinkled Azotobacter membrane was obtained with this mater¬ 
ial in five days. This form was very abundant and dominated in the 
culture which was slightly acid and possessed an earthy odor. After 
thirty days, the determination of nitrogen showed an increase of 
12.4689 m. g. 
A comparison of the results obtained with these four samples sug¬ 
gests that the nitrates were so abundant in the first two that the 
Azotobacter had either been destroyed or so weakened in virulence that 
