8 BULLETIN 340, U. S. DEPARTMENT OF AGRICULTURE. 
serum has an extraordinary potency. It should be noted that it 
does not follow that those rabbits which receive the smallest serum 
doses should die, since not infrequently they may remain alive when 
the rabbits receiving larger doses succumb. 
This method of standardization has not been proved as accurate 
and reliable as the test recommended by Ascoli, and which has been 
emploj^ed in the experimental . work with serum prepared in con- 
nection with our experiments. In this test a 24-hour-old attenuated 
bouillon culture is used, which is of such virulence that when intro- 
duced subcutaneously in a 0.25 c. c. dose into 350-gram guinea pigs 
it will kill them in from two to three days. These test cultures must 
be previously standardized in such a way that they will kill guinea 
pigs which 24 hours previously have been injected intraperitoneally 
with 2 c, c. of normal serum. Guinea pigs treated in the same man- 
ner and with the same dose of titrated standardized immune blood 
serum must remain alive. 
The testing of the serum is carried out on six guinea pigs, each 
receiving intraperitoneally 2 c. c. of the serum to be tested, and 24 
hours later the established dose of the test culture is injected sub- 
cutaneously in the axillary region. The serum is considered satis- 
factory for immunization purposes if at least four of the guinea pigs 
remain alive over six days while the control animals die within 
three or four days. For protective and curative purposes in man, 
only such serum should be selected which, by carrying out the same 
conditions of the test, protect the guinea pig in 0.5 to 1 c. c. doses. 
EXPERIMENTAL DATA. 
HYPERIMMUNIZATION OF HORSES. 
On September 8, 1914, two horses, Xos. 48 and 96, were vaccinated 
against anthrax according to Pasteur's method. On September 29 
these two horses were given approximately 0.01 of a loopful of 
virulent anthrax bacilli subcutaneously. Horse No. 48 showed no 
apparent reaction following the injection. Horse Xo. 96, however, 
developed local anthrax at the point of inoculation. The swelling 
became enlarged and there was a considerable area of edema below 
the same. This condition persisted for approximately a week, and 
finally disappeared. The animal, however, showed no appreciable 
rise in temperature during this period. 
The following table gives in detail the process of hyperimmuni- 
zation : 
