MODIFICATIONS OF BIOLOGICAL CHARACTERS. 131 
could be modified by special treatment in such a manner that it no 
longer produced a fatal form of the disease. He found that the viru- 
lence was greatest when cultures were made from fowls which had 
died from a chronic form of the disease, and that this virulence was 
not lost by successive cultivations in chicken bouillon, repeated at 
short intervals. But when an interval of more than two months 
was allowed to elapse without renewing the cultures, the virulence 
was diminished and fewer deaths occurred in fowls inoculated with 
such cultures. This diminution of virulence became more marked 
in proportion to the length of time during which a culture solution 
containing the microbe remained exposed to the action of the atmo- 
sphere, and at last all virulence was lost as a result of the death of 
the pathogenic microdrganism. When the virus was preserved in 
hermetically sealed tubes it did not undergo this modification, but re- 
tained its full virulence for many months. According to Pasteur, 
the various degrees of modification of virulence resulting from pro- 
longed exposure to the air may be preserved in successive cultures 
made at short intervals. Subsequent experiments with cultures of 
the anthrax bacillus gave similar results and enabled him to produce 
an “attenuated virus” for his protective inoculations. 
In the case of the anthrax bacillus it was found that the spores 
retain their full virulence for years, and that the production of an at- 
tenuated virus required the exclusion of these reproductive elements. 
Cultivations were consequently made at a temperature of £2° to 43° 
C., at which point this bacillus is incapable of producing spores. 
Cultivation at this temperature for eight days gave an attenuated 
virus suitable for use in protective inoculations. 
Attenuation by Heat.—Toussaint has shown that a similar modi- 
fication of virulence may be produced by exposure fora short time 
to a temperature a little below that which destroys the vitality of the 
pathogenic organism. This is best accomplished, according to Chau- 
veau, in the case of the bacillus of anthrax, by exposure for eighteen 
minutes to a temperature of 50° C. Exposure to this temperature for 
twenty minutes is said to completely destroy the vitality of the bacillus. 
Attenuation by Antiseptic Agents.—The writer, in 1880, ob- 
tained evidence that attenuation of virulence may result from ex- 
posure to the action of antiseptic agents. Ina series of experiments 
made to determine the comparative value of disinfectants, the blood 
of a rabbit recently dead from a form of septicaemia induced by the 
subcutaneous injection of my own saliva, and due to the presence of 
a micrococcus (Micrococcus pneumonize croupose), was subjected to 
the action of various chemical agents, and subsequently injected 
into a rabbit to test the destruction of virulence. In the published 
report of these experiments the following statement is made : 
