14 
Colorado Experiment Station. 
to me quite desirable that we should have a specimen of soil from some 
locality where this nitrate trouble had not been heard of. It looked 
only reasonable that if the nitrates were killing apple trees, they were 
probably also destroying the microbic soil flora, at;d I wished to have 
a sample of what might be considered a normal soil as a check. For 
this purpose we obtained material from an alfalfa field where there was 
no history of any trouble. The ground was light, well drained, and 
judging from the size of the plants, it had been in alfalfa for a number 
of years. The surface two inches were discarded and the next four 
inches were taken for the sample. 
In ten days a heavy, gelatinous, white Azotobacter membrane 
formed in the culture flask. Azotobacter was the dominant form, 
but other small rods were also present. There was some fermentation 
and an odor of rotten cabbage was developed. After thirty days the 
membrane was brown in color and had the physical appearance of cold 
grease which has hardened on top of a beef infusion and later has been 
disturbed and broken. This soil gave an increase in nitrogen in 
thirty days amounting to 10.15925 m. g. After obtaining this result 
and a similar one with No. 4, I felt pretty well satisfied that the true 
nitrogen fixing power of the soil could not be judged from a sample 
which was taken from either the dark crust of a nitre spot or an area 
where all vegetation had been dead for some time and where a chemical 
analysis showed the nitrates to be extremely high. I felt very confi¬ 
dent from our work thus far that some of our soils, at least, were 
stocked abundantly with Azotobacter chroococcum, but it was very evi¬ 
dent that this genus had either been destroyed or become greatly attenu¬ 
ated where the nitrates were excessive. I am inclined to accept the form¬ 
er view by way of an explanation since I have plated out crude cultures 
repeatedly which were made from very bad soils and have failed to 
obtain anything which resembled Azotobacter. Again I have had no 
difficulty at all in isolating pure cultures of Azotobacter from crude 
cultures prepared from soils which were high in nitrates, but not ex¬ 
cessively so, and which a month later developed fatal quantities. After 
my experience with these two samples, I decided to take the soil for 
future work from areas where the nitre was just beginning to manifest 
itself on the vegetation and toward which the wave of nitrate destruc¬ 
tion was advancing. I am glad to say that I was not disappointed in 
adopting this new way of sampling as the following experiments will 
testify. 
Sample No. 10. 
In April, 1910, Dr. Headden called my attention to the unmistak¬ 
able brown stain on the irrigating furrows of a young orchard belong¬ 
ing to the Experiment Station. This was the first indication of the 
trouble that had been observed in this immediate vicinity. The color 
was confined to the furrows, there was no mealy condition of the soil 
and the trees were in perfect health. The soil was a clay loam, well 
drained, with gravel and ground water at 18 to 20 feet. Up to the pres- 
