PROTECTIVE INOCULATIONS. 303 
of the animal into the peritoneal cavity. Starting from this point I have 
worked out a method which permits the culture of the microbe in the animal 
organism in a state of purity during indefinite generations, the exaltation of 
it to a well-determined maximum of strength, and keeping it at the same 
degree of virulence for an unlimited period of time. 
‘This method is illustrated by three series of experiments which were the 
subject of our publications in the Comptes rendus de la Société de Biologie 
of Paris, and which are: 
“1, Giving the first animal a dose larger than the fatal dose, and killing 
this animal in a sufficiently short space of time to be able to find the more 
resisting microbes. 
‘*2. To expose the exudation taken from the peritoneal cavity to the air 
for several hours. 
‘*3. Then to transfer this exudation to the next animal, of large or small 
size, according to the concentration of the exudation. : 
‘‘In the hands of a number of other experimenters this method has given 
the same results and showed a pertect consistency. 
‘‘The properties of the virus which is obtained in this manner of cultiva- 
tion are as follows: Upon intraperitoneal inoculation it kills guinea-pigs 
regularly in the space of about eight hours, and the fatal dose for this animal 
is reduced to about twenty times less than that which it would have been 
necessary to take for the microbe with which I started. The same inocula- * 
tion kills rabbits and pigeons with a dose which would have been perfectly 
harmless at the beginning of the experiments. It kills guinea-pigs by intra- 
muscular inoculation. 
‘‘The subcutaneous inoculation brings about the formation of a large 
cedema, which tends toward sequestration of a whole part of the cutaneous 
tissues and to the formation of a wide open wound, which is cured in from 
two to three weeks. 
‘‘The basis of anticholeraic vaccination is founded on the virus obtained 
in the manner we have just described. 
“‘This virus, injected under the skin of a healthy animal, gives it, after 
several days, immunity from all choleraic contamination, in whatever man- 
ner this may arise; that is to say, if an animal that has been thus treated be 
taken, and an attempt made to infect it either by the digestive canal, by 
neutralization of the gastric juice and the injection of opium into the peri- 
toneum, or by the introduction of the microbe into the intestines by the 
method of Nicati and Rietsch, or by intramuscular inoculation, or finally, 
by intraperitoneal injection, the most terrible of all, it resists, whilst the con- 
trol animals succumb. 
“‘ Anticholeraic vaccination of animals in this manner is then definitely 
established. But the operation described cannot be, such as it is, applied to 
man. The wound following on the subcutaneous inoculation is terrible to 
look at, and, in all probability, extremely painful. Besides, although it 
does not in itself present any danger to the health of the individual, it exposes 
him to all the complications inseparable from an open wound. 
“This power of producing necrosis of the cutaneous tissues has been 
removed from the exalted vaccine by cultivating it at a temperature of 39° 
C., and in an atmosphere constantly aérated. nder these conditions the 
first generations of the cholera microbe would die rapidly, in an interval of 
two to three days, and therefore care must be taken to sow them again in 
new media immediately before death, and after a series of generations of this 
kind a culture is obtained which, if injected under the skin of animals, even 
in exaggerated doses, produces only a passing cedema, and prepares the 
organism in such a, manner that the injection of exalted virus, the definite 
vaccine, only produces a local reaction of the slightest description. 
