16 The Colorado Experiment Station 
26. Sodium Chloride in Bouillon. 
The effect of sodium chloride upon growth has been determined 
by inoculating tubes of nutrient broth ( + 15° Fuller’s Scale), 
containing different amount of chemically pure sodium chloride, 
with two loopfuls of a 24 hour broth culture. A preliminary 
test in which the broth contained from 0 to 12 per cent of salt, 
each tube differing by 1%, showed that the toxic strength lay 
somewhere between 3 and 4 per cent. In the next determina¬ 
tions, the tubes contained 3.0, 3.25, 3.5, 3.75 and 4 per cent of 
NaCl respectively. This same series was repeated three times 
with the result that growth took place in the presence of 3.75 
per cent of NaCl, but 4 j)er cent was sufficient to inhibit it. 
27. Nitrogen. 
Nitrogen has been obtained from peptone, asparagin, glycerine, 
and beef broth, but not from ammonium tartrate. No determina¬ 
tions have been made with other ammonium salts, urea or free 
nitrogen. An abundant growth has been obtained on a synthetic 
agar low in nitrogen, such as is used for growing the nodule 
forming bacteria; this would seem to indicate the possibility of 
the organism being able to utilize atmospheric nitrogen. Deter¬ 
minations of this particular feature are in progress at the pres¬ 
ent time. 
28. Best Media for Bong Continued Growth. 
Standard nutrient agar with a reaction of +15° Fuller’s scale 
has proved entirely satisfactory. 
29. Quick Tests for Differential Purposes. 
Fluorescent green color produced in the nutrient agar streak; 
no growth at 37 y 2 ° C.; non liquefaction of gelatin; surface 
pellicle on nutrient broth; luxurient and characteristic growth 
on synthetic agar low in nitrogen (q. v.). 
III. PHYSICAL AND BIOCHEMICAL FEATURES 
1. Gas Production. 
Four fermentation tubes, each, of dextrose, saccharose, lactose, 
maltose, glycerine and mannite broth were inoculated with a 
4 8 hour culture; two of each were kept at 2 8° C. and two at 
37 y 2 ° C. Those held at 28° C. gave growth in the open arm 
after 24 hours, but none in the closed arm and no gas; those at 
37%° C. showed no growth whatever. The turbidity in the 
open arm of those kept at 28° C. increased with age, extending 
over half way into the U, but at the end of ten days there was 
no growth in the closed arm and no gas. There was a well de¬ 
fined line between the growth in the bulb and the clear liquid 
in the closed arm, which seemed to point strongly toward the 
aerobic nature of the organism. 
2. Production of Acid and Alkali. 
60 c.c. portions of dextrose broth, saccharose broth, lactose 
broth, maltose broth, glycerine broth and mannite broth in 200 
c.c. Erlenmeyer flasks were inoculated with two loopfuls of a 
2 4 hour broth culture of Ps. medicaginis, n. sp. Five cubic 
centimeters weie taken from each flask for titration everv 
twenty-four hours for a period of ten days. This was diluted 
with 4 5 c.c. of distilled water in a porcelain evaporating dish 
and titrated cold with N/10 NaOH or N/10 HC1 as the case re¬ 
quired. Phenolphthalein was used as the indicator. An ex¬ 
amination of Table No. 1 will show that in no instance has there 
been any acid produced, while on the other hand, after the third 
day, there has been a gradual production of alkali until after 
