Jan. 8, 2917 
Immunity Studies on Anthrax Serum 
53 
methods so far employed being quite indefinite and the results variable. 
The writers have tried several methods of standardization. Sobem- 
heim’s method (10), consisting of the intravenous injection of graduated 
doses of the serum into a series of rabbits, followed immediately with 
subcutaneous injections of 0.001 loopful of a suspension of virulent 
anthrax bacilli in saline solution was the first method tried. This, how¬ 
ever, did not prove as satisfactory as Ascoli’s method. In this test a 
24-hour-old attenuated bouillon culture is used, which is of such virulence 
that when introduced subcutaneously in a 0.25 c. c. dose will kill guinea 
pigs weighing 350 gm. each in from two to three days. 
These 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 manner 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 2 c. c. of the serum intraperitoneally, 
followed in 24 hours with a subcutaneous injection of the established 
dose of the test culture. The serum is considered satisfactory for im¬ 
munization purposes if at least four of the guinea pigs remain alive over 
six days while the control animals die in three or four days. This test 
has been modified by using graduated amounts of the serum under test. 
The results obtained with the above methods are greatly influenced 
by the variance in individual susceptibility of the test animals, the 
character of the virus employed, etc. For this reason the writers have 
undertaken a series of experiments with the complement-fixation test, 
with a view to ascertaining its value in the standardization of anthrax 
serum, and while our work on this phase of the subject is only in the 
experimental stage and incomplete, the results so far are quite gratify¬ 
ing, and point to the possibility of employing this test as a means of 
more accurate standardization. So far the serum from two horses 
and a mule used by the Bureau of Animal Industry in the preparation 
of anthrax serum, as well as serum from horses in various stages of 
hyperimmunization obtained from various biological firms of the country, 
have been employed in this work. At the present time the serums of 
four horses under the course of hyperimmunization are being studied. 
Several antigens have been tried, the best results being obtained with 
a bouillon culture of a slightly virulent strain of Bacillus anthracis 
(virulent for white mice and occasionally for small guinea pigs). Such 
a culture is grown at incubator temperature from three to five days and 
then placed in the refrigerator for two or three weeks or even longer. 
It is then heated at 6o° C. for one-half hour and titrated against a known 
potent anthrax serum used as a standard. 
In standardizing the serum the usual technic employed in the comple¬ 
ment-fixation test applies, with the exception that varying quantities 
of the serum under test are used. So far in the work the writers have 
