478 
Journal of Agricultural Research 
Vol. XXV, No. 12 
per cent potassium nitrate had been added. Satisfactory growth oc¬ 
curred, but no indication of nitrites was secured when the test was applied 
at the end of 7, 14, and 21 days. 
Toleration of sodium chlorid. —Tubes of neutral beef extract- 
peptone bouillon containing 0.5, 1, 1.5, 2, 3, 4, and 5 per cent of pure 
sodium chjorid were used in this test. Best growth occurred in the 
presence of 0.5 per cent sodium chlorid. Higher concentrations of salt 
were progressively inhibitive, since the growth in 1 and 1.5 per cent was 
less than in 0.5 per cent, and that in 2 per cent very slight and with no 
visible clouding in higher concentrations. 6 
Gas production. —These tests were conducted by using fermentation 
tubes filled with solutions prepared as follows: A 2 per cent peptone 
solution was used as the base for six solutions made by adding 2 per cent 
of the following carbon compounds—glycerin, mannite, lactose, maltose, 
dextrose, and saccharose. These were prepared in sets of 14 each, 
and were then sterilized and incubated to determine their sterility before 
inoculation. Eight were then inoculated with Wisconsin type strain 
J9i6-II, three with strain 1919-1, and three with 1920-III. They 
were not disturbed during the period covered by the test. No gas was 
produced in any case, and growth was sharply limited to the open arm 
m all media with each of the strains. 
The media for another series were prepared by using a bouillon con¬ 
sisting of 1 per cent Difco peptone, 0.3 per cent Liebig's beef extract, and 
0.5 per cent sodium chlorid, as a stock solution. The same carbon com¬ 
pounds were employed and they were prepared separately in 20 per cent 
solutions in distilled water. These solutions were sterilized at 10 pounds 
pressure for 10 minutes. The sugars were then added to the bouillon 
under aseptic conditions and the solutions poured into sterile fermenta¬ 
tion tubes. After incubation for 48 hours to determine their freedom 
from contamination, they were inoculated with strain 1920-III and a 
single strain from each of three species of clover. Five tubes were used 
in each set of sugars with each strain. No ga£ was developed, and no 
growth took place in the closed arm, results entirely in accord with the 
previous test. 
CARBON METABOLISM 7 
Agar with sugars. —Agar was employed in the qualitative studies to 
determine the production of acid from the several common carbon com¬ 
pounds, It was prepared by adding to flasked and sterilized bacto-agar, 
cooled to 6o° C. and adjusted colorimetrically to P H 7.4, sufficient of the 
stock solutions of the carbon compounds to make 1 per cent of the sugar 
to be tested. Phenol red was added as an indicator. Before the agar 
had solidified it was poured into sterile test tubes for use in stab cultures. 
Dextrose, saccharose, lactose, maltose, and glycerin were tested in this 
manner. After the tubes of media had been incubated sufficiently long 
to determine freedom from contamination, they were inoculated in sets 
of five cultures on each sugar with Wisconsin strain 1920-III, and with 
a strain from red clover, one from white, and one from alsike from Raleigh, 
N. C. The red color disappeared with all strains in dextrose agar and in 
saccharose agar within five to seven days, indicating acid production, 
6 At Washington in beef infusion bouillon+13 Fuller’s scale, Ph 6.7, with sodium chlorid added, the Wis¬ 
consin as well as the Virginia organisms gave growth in concentrations up to 4 per cent sodium chlorid. 
7 These statements concerning carbon metabolism are based on studies made by F. A, Wolf at Raleigh, 
N. C. The results with all carbohydrates tested in Wisconsin were in agreement with these. 
