258 
Journal of Agricultural Research 
Vol. XIV, No. 6 
the organism. A number of cultures should be made and care exercised 
to select for inoculation only a pure culture of the blackleg organism. 
It has been the procedure of the writer to plan the work so that the 
culture would be ready for inoculation on the day the medium for toxin 
production was sterilized for the third time. The medium is allowed to 
cool down to approximately 40° C., and then several cubic centimeters 
of the culture are inoculated in the bottom of each of the flasks with a 
sterile pipette and the flasks placed in the incubator at 37.5 0 . If the 
inoculation is not made immediately following the third sterilization, the 
medium should be heated to 6o° to drive off the oxygen and should be 
inoculated after it has cooled down to approximately 40°. Incubation 
for 10 to 12 days appears to be the approximate time for satisfactory 
toxin production. 
The product is then removed from the incubator and filtered through 
several thicknesses of cheesecloth, next through a thin layer of asbestos 
wool, and then twice through Berkefeld filters of “N” porosity. 
It is preserved with 0.5 per cent chloroform and stored in amber-glass 
bottles, which should be well filled, so as to leave as little air space as 
possible. 
The product is tested culturally, and sublethal doses are administered 
to guinea pigs to determine whether or not it has been rendered free of 
organisms. « 
The potency of the material is determined through animal inoculation 
tests. In connection with the test for potency, attention has also been 
given to the degree of toxicity, because of the apparent relation of one 
to the other. This phase of the question will be given further con¬ 
sideration in a subsequent chapter of this article. 
POTENCY TESTS OF THE TOXIC CULTURE FILTRATE 
As in the case of the natural aggressin, numerous tests have been car¬ 
ried out with the culture filtrate on calves and guinea pigs. There is one 
important factor which has been uniformly noted in all the tests with 
the culture filtrate, and that is that there appears to be a direct ratio 
between the toxicity and potency of the product. In all potency tests 
thus far undertaken by the writer, no immunizing properties could be 
demonstrated in nontoxic culture filtrates. It is contemplated that if 
this relation of toxicity to potency is definitely proved, it will be a valu¬ 
able factor in connection with standardization of the product. 
Tables IV-VIII give results representative of the tests to which the 
filtrate has been submitted. 
