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 Societ 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 perlect 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 

 oedema, 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 aerated. Under 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 oedema, 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. 



