46 
for two weeks. The resulting growth was then tested on mice to deter- | 
mine its virulence. Each of two mice were given 0. 00002 of the culture, j 
Both animals took the disease. The one died in eight da^^s; the other 
recovered. One month later, viz, May 28, two more mice were inoc- j 
ulated with an equal amount of the same culture, and both died. This i 
amount was, therefore, considered to represent approximate!}’ the 
minimal fatal dose, and was used to contaminate the vaccine virus. 
The glycerinated virus of two manufacturers was bought in the : 
open market. With the mixed tetanus culture we prepared an arti- 
licial contamination of both these viruses and sealed them hermetically 
in small capillary tubes. By means of dilution each capillary tube 
was made to contain just 0.00002 c. c. of the tetanus culture in addi- 
tion to a large-sized drop of the glycerinated virus. 
These tubes were kept at 20^-22° C. in a dark place and tested from 
time to time. 
Glycerinated virus {manufacturer Xo. ^4) contaminated with 0.00002 c. c. of a mixed culture 
of tetanus. 
Date 
pre- 
pared. 
Inocu- 
lated 
into 
mice. 
Xum- 
Sjunptoms 
Result. 
ber 
A 1st 
days. 
2d 
day. 
3d 
day. 
4th 
day. 
5th 
day. 
6th 
day. 
7th 
day. 
8th 
day. 
9th 
day. 
10th 
day. 
Apr. 30 
Apr. 30 
0 P 
P 
P 
P 
P 
P 
P 
P 
P 
P 
Po.sitiye: recoyered. 
Do.. 
May 28 
28 X 
Xegatiye. 
Do.. 
June 17 
48 X 
Do. 
Do.. 
June 27 
58 X 
Do. 
w e see from this table that one montlrs action of the glycerinated 
virus reduced the virulence of the tetanus which it contained to a ! 
point where it no longer produced any symptoms on mice. 
A comparison of the table above with the control which immediately / 
precedes it indicates strikingly the attenuating power of the glvcerin- 
ated virus in this instance. Observe that on Mav 28 the glvcerinated 
tube produced no symptoms of tetanus, while the two control tests 
made on the same date both proved typical tetanus. Whether the 
virus had killed the tetanus or merely diminished its virulence was 
determined by planting on July 8 six of the tubes each into a separate 
cubic centimeter of bouillon and growing it under anaerobic conditions i 
for one week. At the end of this time we tested the growth 'in mice j 
and were surprised to find that three of the tubes when injected into j 
mice caused no sickness whatever, while the other three tubes pro- ; 
duced typical symptoms of tetanus in mice. . | 
We conclude that the glycerinated virus killed the tetanus entirely 
in some of the tubes in two months and that if it had been allowed to 
act longer on the other tubes they, too, probably would have contained 
no living tetanus. 
