o 
in bouillon or agar, the bodies of animals dead of a preceding passage 
were fed to others. 
It was, therefore, plain that in the evolution of an epidemic caused 
by this microbe it was necessary to take account of the indisputable 
diminution of the virulence of the microbe, as well as the natural 
resistance of the survivors. 
Finally, passages of cultures in collodion sacks, inclosed in the peri- 
toneal cavities of rats, were tried, both in interrupted series and by 
alternating each sack culture with a culture in bouillon or on agar, but 
the end was invariably a notable diminution of virulence when admin- 
istered by the digestive tract. 
Danyz finally managed to increase the virulence of the organism, so 
that it was pathogenic for rats, by the following process : 
danyz’ S METHOD FOR INCREASING AND MAINTAINING THE VIRULENCE 
OF THE VIRUS. 
A culture of the bacillus was selected that was fatal for mice in four 
to five days, and grown in bouillon to accustom it to an anaerobic exist- 
ence. This was accomplished by growing the culture in fiasks as com- 
pletely filled as possible. The flasks were jilaced in the incubator until 
the culture developed, and then kept at ordinary temperature until a 
deposit formed and the bouillon became perfectly clear. This may 
take four or five days, and its object is, as above stated, to accustom the 
microbe to an anaerobic existence. 
From the flasks the culture was passed in a collodion sack, which is 
kept from twenty-four to thirty- six hours in the abdominal cavity of a 
rat, and then planted anew in ordinary bouillon and thence again into 
flasks. The culture was transferred from these last flasks to agar, and 
it is these cultures on agar that Danyz gave to mice to eat, after having 
diluted them with water and soaked bread and grain in the dilution. 
This series of operations was repeated several times, and at the fourth 
or fifth repetition a decided increase in virulence was noted. Mice, 
which died only at the end of four to seven days, now died in thirty-six 
to sixty hours after the ingestion. 
After this decided increase in the virulence for mice was obtained, 
the mice were replaced by white rats, commencing with young rats a 
mouth or six weeks old and, as the passages are continued, taking older 
rats. Proceeding thus and making collodion sack cultures in the 
abdominal cavity of the species of the animal which it was desired to 
infect, the culture was specialized, so that it was rendered sufiiciently 
virulent in ten passages. Operating in this manner, Danyz linally suc- 
ceeded in rendering regularly virulent for gray rats (?«. dec urn an us), 
then for black rats (m. ratus), and finally for white rats, a culture which 
was originallj’ but slightly virulent for the gray rat and entirely innocu- 
ous for the other two. 
