Aug. 5,1918 Immunization Methods against Symptomatic Anthrax 257 
plus sterile bovine serum, dextrose-liver bouillon, dextrose-liver bouillon 
plus cubes of beef, dextrose-veal bouillon plus calcium lactate, dextrose- 
liver bouillon plus calcium lactate. 
The medium with which best results were obtained is a modification 
of Martin's peptone bouillon, and is prepared as follows: 
Fresh pig stomachs with their contents are obtained, and after trim¬ 
ming away the fat, are opened and the contents expelled. They are then 
lightly rinsed in water, care being taken not to wash away the gastric 
mucosa. The material is then cut in pieces of suitable size for a meat¬ 
chopping machine and finely ground. To every 200 gm. of this finely 
ground stomach tissue are added 1 liter of water at 50° C. and 20 cc. of 
hydrochloric acid, C. P. This mixture should be made in glass flasks, 
and from here on up to the time the material is neutralized it should not 
come in contact with metal. The mixture is placed in an incubator 
maintained at approximately 50° and allowed to remain there for 20 to 
24 hours. It is then filtered through several thicknesses of cheesecloth, 
heated to 8o° to stop peptonization, allowed to cool down to 70°, and 
neutralized to litmus. Flocculation occurs at this point, and the material 
is then filtered through cotton. Sterilization is accomplished by auto¬ 
claving at 15 pounds' pressure for 20 minutes. 
A piece of fresh beef is then obtained, and with as much precaution 
as possible to prevent undue contamination, a thin layer is removed, 
taking all of the exposed surface of the beef. 1 As much of the remainder 
as will be required is cut in small pieces and put through a meat chopper 
which has been previously boiled; 450 gm. of this ground beef and 10 gm. 
of dextrose are then added to every 1,000 cc. of the peptone solution. 
The flasks containing the medium are filled close to the cotton stopper 
in order to eliminate all the air space possible. The medium is then 
allowed to remain at refrigerator temperature for several hours, at the 
end of which time it is titrated against phenolphthalein and the reaction 
adjusted to + 0.5, and is then sterilized by heating and maintaining it 
at a temperature of 65° to 70° C. for one hour on three successive days. 
It is then ready for inoculation. 
The inoculation may be made either with a freshly isolated, virulent 
culture of the bacillus of symptomatic anthrax or with fresh affected 
muscle tissue known to contain only the blackleg organism. The latter 
method is not as easy of accomplishment as the first. 
The culture that gives good results is one 24 to 48 hours old, recov¬ 
ered from a guinea pig which has been inoculated with virulent blackleg 
material. The cultures may be taken from the affected musculature, 
peritoneal fluid, or heart blood after the animal has been dead a few 
hours. Dextrose, beef, or liver bouillon may be employed in recovering 
1 This rejected material can be utilized in the preparation of ordinary beef bouillon. 
