10 
BULLETIN 340, U. S. DEPARTMENT OF AGRICULTURE. 
of guinea pigs were inoculated intraperitoneally with varying 
amounts of serum, and 48 hours later were injected with 0.25 c. c. 
of a 24-hour bouillon subculture of an attenuated strain known as 
" Davis D." This culture had been attenuated by growing it at a 
temperature of 42°-43° C. for a period of 20 days. Previous tests 
of this culture showed that it was uniformly pathogenic for guinea 
pigs, killing them in two to three days, but it failed to kill rabbits. 
The results of this test are contained in the following table : 
Standardization tests of anthrax serum (serum injected intraperitoneally; 
virus 24 hours later sudcutaneously) . 
SERUM 48. 
Guinea pig No. 
Amount of 
serum. 
Amount of 
vims. 
Result. 
1 
1.0 c. c. 
1.5 c. c. 
2.0c.c. 
2.5 c. c. 
3.0 c. c. 
3.5 c. c. 
0.25c.c. 
0. 25 c. c. 
0.25c.c. 
0.25c.c. 
0.25 c. c. 
0. 25 c. c. 
2 
Died on third day. 
3. . 
4 
Do. 
5 
Do. 
6.. 
Do. 
SERUM 96. 
1 
1.0 c. c. 
1.5 c. c. 
2.0c.c. 
2. 5 c. c. 
3. Oc.c. 
3.5 c. c. 
0.25c.c. 
0. 25 c. c. 
0.25 c. c. 
0. 25 c. c. 
0. 25 c. c. 
0.25 c. c. 
Remained alive. 
2 
Do. 
3 
Died on third day. 
Remained alive. 
4 
5 
Do. 
6... 
Dor 
NORMAL HORSE SERUM. 
1.0 c. c. 
0.25c.c. 
1.5c. c. 
0. 25 c. c. 
2.0 c. c. 
0.25c.c. 
2.5 c. c. 
0.25 c. c. 
3. Oc.c. 
0. 25 c. c. 
3.5 c. c. 
0.25C.C. 
Died on fourth day. 
Died on third day. 
Died on fourth day. 
Remained alive. 
Died on fourth day. 
Died on third day. 
In view of these results it was decided to use the " Davis D " cul- 
ture in the preparation of our spore vaccine, to be used simultane- 
ously with the serum. 
Extensive tests to determine whether or not the immune serums 
possessed a bactericidal property proved negative. 
PREPARATION OF SPORE VACCINE. 
The four cultures used for the hyperimmunization of the horses 
were attenuated at a temperature of 42.5° C. for varying periods. 
From time to time they were tested for their pathogenicity by inocu- 
lation into mice, guinea pigs, and rabbits. The cultures, which were 
removed from the incubator after 20 days of attenuation, proved sat- 
isfactory for the purpose, inasmuch as the test inoculation demon- 
