The Fixation oe Nitrogen. 87 
samples of the soil were brought to a thoroughly air-dried con¬ 
dition and the total, and also the nitric nitrogen determined. Four 
of the large dishes with their charges of soil were placed in an 
incubator and were kept at a temperature of about 29 0 C. for 27 
days, when a sample was removed from each dish for examina¬ 
tion. The fifth dish was covered and placed in an unused room. 
Two of the four dishes placed in the incubator were inoculated 
by the addition of 25 grams of another soil in which the total and 
nitric nitrogen was determined, because a culture test made from 
the sample of soil taken for this experiment gave unsatisfactory 
results, as no proper membrane was formed, only a few gelatinous 
masses at the edge of the culture medium. I feared that I would 
have to collect other samples of soil and repeat the experiment, as 
I had already had to do once, for the first sample of soil that I 
gathered for this purpose, though it was very rich in nitrates, 
seemed to contain no living bacteria which Professor Sackett, on 
whose judgment I relied, was willing to accept unqualifiedly as 
virile azotobacter. This precaution, however, proved to be un¬ 
necessary, for a sample of soil taken from the fifth dish, on the 
thirteenth day of the experiment, gave a very heavy membrane 
in four days. The soil in this dish had already begun to show a 
change of color on its surface at the time this sample was taken. 
The membrane in the culture flask began to show brown points by 
the eight or ninth day. This fifth dish had been kept in a south 
room where for part of the day it received the sunshine, modified 
by a screen of paraffined paper. We have five dishes, each con¬ 
taining originally 1,250 grams of soil with eighteen percent of 
moisture. On the twenty-seventh day I removed from each dish 
170 grams of the moist earth, brought it to a thoroughly air-dried 
condition and made nitrogen determinations in triplicate on each 
sample. I have mentioned five samples, but I added another, i. e., 
a sample of the original soil which had been put into a show bottle 
while moist and allowed to stand in the room. If moisture, air and 
an equitable temperature be the requirements for the fixation of 
nitrogen I could see no reason why I should not expect an increase 
in this sample as well as in those placed in the incubator, perhaps 
not so great an increase, but still an increase. Samples A, B. C 
and D were put into the incubator, sample E was moistened and 
kept at the room temperature and F was the original sample in 
its moist condition. 
The original soil, thoroughly air-dried, gave 0.105875, 0.11000 
and 0.105875 percent of nitrogen, average for total nitrogen 
0.10725 and o. 0055 percent of nitrogen as nitrates, equal to 3.263 
percent of the total. 
