28 
TETANUS TOXIN IN GLYCERIN. 
A series of tests was then made to determine the effect of glycerin 
upon tetanus toxin. 
In each instance a quantity of the glycerin containing 0.0025 c. c. of 
the toxin was inoculated into the flank of a mouse and observed for 
symptoms. This quantity was over ten times the M. L. D. 
It was found that the toxin exposed to the glycerin gradually lost 
its virulence after sixty days at the body temperature, but that it was 
still active after one hundred and eighty days in the ice chest or at 
room temperature. 
If vaccine virus, therefore, was contaminated with such toxin the 
glycerin would have no influence upon it. 
VIABILITY OF MIXED CULTURE OF TETANUS IN VARIOUS PERCENTAGES 
OF GLYCERIN. 
This culture was the same as was used for testing the viability of the 
mixed culture of tetanus on dry points and in glycerinated virus. ^ It 
was planted into 100 c. c. of bouillon on June 25 and kept at 37^ for 
two weeks in a Novy jar. It contained a coccus, a slender motile rod 
of good length, which contained a central oval spore, in addition to a 
rich growth of tetanus organisms. 
On July 14 the 400 c. c. were subjected to a Pasteur Alter to remove 
the toxins. The residue was washed several times with distilled water 
to remove an}^ trace of toxins from the tetanus organisms. The excess 
of water in which the tetanus remained suspended was evaporated in a 
vacuum containing a vessel of calcium chloride until it reached a bulk 
of about 2 c. c. 
Three series of test tubes were prepared. Each series represented 
ten difl'erent percentages of pure glycerin in water, namely, 10, 20, 30 
per cent, etc. , to 100 per cent. Into each of the thirty tubes compris- 
ing the three series we put an equal amount of the watery suspension 
of mixed tetanus organisms on July 16 and paraffined the cotton plugs. 
The three series were then put at different temperatures — one at room 
temperature, about 20° C. ; another in the incubator at 37°, and the 
other in the ice chest at 10° to 12° C. Before placing the series at 
different temperatures, however, they were tested on mice. 
As a control, inoculations into mice were made from three tubes 
selected at random, which were the 10 per cent incubator, 30 per cent 
ice chest, and 60 per cent room temperature. The three series were 
then placed in their appropriate temperatures and tested on mice at 
intervals. 
It was found that mixed cultures of tetanus in glycerin at the body 
«See Bulletin No. 12. — The bacteriological impurities of vaccine virus; an experi- 
mental study. By M. J. Rosenau. 
