26 o 
Journal of Agricultural Research 
Vol. XIV, No. 6 
vary greatly with the different amounts weighed. Depending on how 
well the material is emulsified, a greater or smaller number of the organ¬ 
isms would be left behind in the small particles of tissue which are filtered 
out. Foreign organisms frequently present in such affected tissue also 
are complicating factors when injected into the guinea pigs. 
The writer has prepared a type of test virus which has given very good 
results in the guinea-pig tests and possesses a number of advantages 
over the emulsion of affected tissue. It is prepared in the following 
manner: Guinea pigs are inoculated intramuscularly with an emulsion 
of virulent blackleg tissue and usually die of the disease in from 24 to 48 
hours. Cultures are then made from the guinea-pig carcasses into 
fermentation tubes containing dextrose bouillon. The culture medium 
is heated for approximately 10 minutes in the Arnold sterilizer just prior 
to inoculation in order to drive off the oxygen. It is allowed to cool 
down to about 45 0 C., inoculated, placed in vacuum jars, and incubated 
24 hours at 37.5 °. At the expiration of the incubation period the jars 
are removed from the incubator and all fermentation tubes showing 
evidence of good growth removed and examined for purity. The cultures 
are then thoroughly mixed in a crystallizing dish with sufficient lactose 
to make a soft paste, and this placed in a vacuum desiccator containing 
sulphuric acid. Care should be taken to protect the material from direct 
light by covering the desiccator with towels or by keeping it in a dark 
place. The material dries very rapidly. It is then removed and pulver¬ 
ized to a very fine powder in a sterile mortar and stored in wide-mouth 
amber-glass bottles at refrigerator temperature. When ready for use a 
definite amount of the powder is weighed out and taken up in a measured 
amount of distilled water. 
The approximate minimal lethal doses of this virus for guinea pigs 
weighing 350 gm. can be established and this increased 10 times for a 
test dose in potency tests of blackleg products. 
Virus thus prepared contains no foreign organisms, eliminating the 
possibility of complications in the animals inoculated with it; it is readily 
absorbed, and can be fairly accurately standardized. In this form the 
virus retains its virulence for a considerable time. 
Table VI .—Results of subcutaneous vaccination of calves with toxic culture filtrate 
followed after 14 days with intramuscular inoculation of blackleg virus 
Calf No. 
Amount 
of culture 
filtrate. 
Amount 
of 
virus.® 
Result. 
I . 
Cc. 
£ . 
Cc. 
10 
IO 
IO 
IO 
IO 
10 
Slight swelling. Remained alive. 
Very slight swelling. Remained alive. 
Dead of blackleg within 48 hours. 
Do. 
Do. 
Do. 
2 . 
5. 
7 . 
Control. 
O 
A . 
.do. 
C. 
.do. 
6...... 
.do. 
a The virus employed was an emulsion of affected blackleg muscle tissue. 
