21 
A Bacterial Disease oe Alfalfa 
12. Relation to Freezing. 
1/100 c.c. and 1/10,000 c.c. portions of a 24 hour broth cul¬ 
ture grown at 28° C., were plated in nutrient agar as controls. 
This culture was then frozen in a mixture of snow and salt, and 
kept in this condition for 24 hours, at the end of which time it 
was thawed out by placing it in an incubator at 2 8° C. As soon 
as melted, dilution plates were made using the same amounts as 
before freezing. After four days, colony counts were made 
which showed the culture to have contained 470,000 germs per 
c.c. before freezing and 5,100 after freezing, or 98.9 % had been 
killed. 
13. Relation to Light. 
DIRECT SUNLIGHT.—Three tubes of liquefied agar were sown 
thinly with different amounts of a 24 hour broth culture and 
poured into sterile Petri dishes. As soon as the agar had solidi¬ 
fied, one half of each plate was covered with one thickness of 
heavy, black, glazed paper. They were then placed on a bed of 
firmly packed snow in a large crystalizing dish and the whole 
was exposed for fifteen minutes to the bright sunshine of Fort 
Collins, Colorado (altitude 4,981 feet above sea level), Nov. 20, 
1909, 11:30-11:45 A. M. 
100 % of the germs were killed in the exposed portions of 
the plates, while 2 5, 9, and 5 colonies, respectively, developed 
in the protected parts. 
DIFFUSED LIGHT.—Our cultures have been kept in diffused 
light in a culture room supplied with light from a north window, 
which was perhaps eight feet from the table on which the cul¬ 
tures were kept. We have never noticed any detrimental effect 
from this light upon either the vigor or the virulence of the 
cultures. Other cultures have been kept next to the window for 
a week at a time, and when subcultures were made from these, 
no difficulty has been experienced in getting good growths. 
14. Production of Hydrogen Sulphide. 
Nutrient broth in tubes was inoculated with a 4 8 hour agar 
culture, and at the same time narrow strips of filter paper, 
moistened with lead acetate, were suspended in the upper part 
of the tubes and held in place by the cotton plugs. These strips 
of paper were remoistened with lead acetate every twenty-four 
hours for a period of ten days but there was no blackening to 
indicate the production of hydrogen sulphide. 
A second method of demonstrating H 2 S was to make stab 
cultures in nutrient gelatin to which .5% of iron potassium 
tartrate had been added. If hydrogen sulphide had been pro¬ 
duced, the line of puncture should have been marked by a well 
defined black line due to the formation of iron sulphide. Tubes 
of this medium inocualted with Ps. medicaginis, n. sp. failed to 
show the production of any H 2 S. 
15. Production of Ferments. 
Determinations have been made for the presence of diastase, 
invertase, zymase, rennet, pepsin and glucase. Tests were made 
for these enzymes after five, seven and thirty days, and th3 
only one which we have been able to demonstrate was in¬ 
vertase. A trace of this was evident on the seventh day, and 
was abundant at the end of thirty days. A 48 hour agar culture 
has been employed in all of the inoculations. 
DIASTASE.—100 c.c. of sugar free beef broth containing.1 % 
soluble starch were inoculated with the organism under study. 
After five, seven and thirty days, portions were removed from 
the flask with a sterile pipette and tested for starch with iodine 
