PROTECTIVE INOCULATIONS. 359 
occurred in a dialyzer placed in running water. But by shaking up 
the dry powder in chloroform the heavy salt sank to the bottom and 
the purified antitoxin floated on the surface and could be recovered by 
skimming it off. The powder thus obtained consisted of a mixture of 
various substances, including the antitoxin, and when obtained from 
milk having an antitoxic value of 90,000 it was found to have a value 
of 25,000,000 immunization units. By further purification a still 
higher value was obtained (55,000,000). In experiments on mice a 
dose ten thousand times as great as was necessary to produce immu- 
nity proved to exercise a curative power—i.e., a dose of 0.02 gramme 
for a mouse weighing twenty grammes saved it from being killed by 
double the minimum fatal dose of the tetanus toxin, after tetanic 
symptoms had been developed. 
Reference has been made to the production of immunity by the 
use of cultures made in thymus bouillon. This was made known 
through the experiments of Brieger, Kitasato, and Wassermann 
(1892). The thymus bouillon is made from the thymus glands of 
calves, which are chopped fine in a hash machine and covered with 
an equal volume of distilled water. The mixture is stirred for some 
time and then placed in an ice chest for twelve hours; the liquid is 
then obtained by filtration through gauze with pressure—by means of 
a flesh-press machine. A turbid, slimy fluid is thus obtained, which 
is diluted with an equal volume of water and made slightly alkaline 
by the addition of soda solution. It is then sterilized at 100° CO. for 
fifteen minutes. As a result of this the liquid has a grayish-brown 
color, and some large flocculi in suspension, which are removed by 
passing it through fine linen. The fluid is then of a milky opal- 
escence. It is next placed in test tubes and again sterilized. The 
tetanus bacillus when cultivated in this medium does not form spores, 
and the toxic potency of the culture is very much reduced—1: 5,000 to 
1:3,000 of the toxic potency manifested by cultures of the same bacil- 
lus in ordinary media. Inoculations with cultures in thymus bouillon 
were found to kill mice in the dose of 0.5 cubic centimetre, while 
smaller amounts failed to kill and caused the animals to be immune. 
A culture in ordinary bouillon was fatal to mice in the dose of 0.001 
cubic centimetre. 
Experiments on rabbits (thirty-five) gave a uniformly successful 
result in immunizing these animals. Immunity was established in 
the course of two weeks, and the blood serum of these animals tested 
on mice showed an antitoxic value of 1,000. 
Reference has already been made to the earlier researches of the 
Italian investigators, Tizzoni and Cattani. These have been followed 
