10 
COLORADO EXPERIMENT STATION 
bonate were mixed with this by adding the respective solutions drop 
by drop to a portion of the ioo grams, and then more of the soil was 
added and more solution, and so on until io c. cm. of solution corre¬ 
sponding to loom. g. of nitrogen had been used with the whole sample. 
For the organic nitrogen, dried blood (i) corresponding to ioo 
m. g. total nitrogen was added to like portions of soil and was thorough¬ 
ly incorporated by stirring the mixture for five minutes with a sterile 
glass rod. Five cultures were prepared from each of the Colorado soils. 
Three received the ammonia salts, one the dried blood, and one to which 
no form of nitrogen was added, was kept as a control. The ammonium 
chlorid series was omitted from the foreign soils. 
Inoculation of Soils .—Each culture mixture was inoculated with 
io c. cm. of soil infusion prepared from fresh soil of the same source 
as that already in the dish and corresponded to 5 grams of the undried 
material. These infusions were made by shaking 100 g. of undried soil 
with 200 c. cm .of sterile distilled water for five minutes. They were 
allowed to stand thirty minutes for the coarser particles to settle after 
which 10 c. cm. of the turbid suspension were drawn off with a sterile 
pipette and distributed uniformly over the surface of the soil in the 
culture dish. In order that the dried blood and control series might 
have the same moisture content as the other three, sufficient sterile 
distilled water was added to make the moisture approximately 22 per 
cent. Additional water was used for the dried blood at the rate of 1.5 c. 
cm. for each gram of material. 
The weight of each culture was determined at the beginning of 
the experiment and the loss of water by evaporation was restored every 
ten days with sterile, distilled water. 
Incubation of Samples .—All of the cultures were kept in the incu¬ 
bator at a temperature of 28 - 30 degrees C. throughout the experi¬ 
mental period of six weeks. At the end of this time quantitative de¬ 
terminations were made for nitrites and nitrates. 
Chemical Methods .—10 grams of each sample, air dried and pulver¬ 
ized, were used in the chemical determinations. This was extracted 
with hot water and washed free from chlorides and nitrates on a 
Buchner funnel containing asbestos. The resulting extracts were ren¬ 
dered colorless by means of carbon black. That portion of the extract 
which was used for the determination of nitrites and nitrates was freed 
from chlorides by the use of silver sulphate. The loss of nitrous and 
nitric acid during the evaporation and concentration of the extracts 
on the water bath was prevented by the addition of milk of lime in 
excess. 
The nitrates were determined by the phenoldisulfonic acid method; 
the nitrites by Ilsovay’s modification of Griess’s test (sulfanilic acid 
and naphthylamine acetate) ; the chlorids, volumetrically, by titration 
with silver nitrate. 
(1) The dried blood contained 13.0503 per cent, total nitrogen. 
