IMMUNIZATION WITH BACTERIOPHAGE SUSPENSIONS 521 



I may state here, once for all, that each time that the immunity 

 of one animal has been tested by the inoculation of a culture of the 

 bacterium of barbone this test has been controlled by the injection of 

 an equal dose into a control animal of the same weight, and never has 

 the control resisted. Furthermore, although there can be no possible 

 doubt concerning the cause of death, confirmation has always been 

 made by microscopic examination, by blood culture, and by the dem- 

 onstration of the lesions at autopsy. The temperature of the experi- 

 mental animals was taken regularly, morning and evening, and the 

 slightest reaction in the immunized animals could not have passed 

 unobserved. 



The race of bacteriophage employed for the preparation of the 

 suspensions destined for use in the immunization experiments had 

 been isolated from the feces of a buffalo which had passed unaffected 

 through the epizootic mentioned in the preceding chapter. This 

 bacteriophage possessed, when derived from the organism, a strong 

 virulence (+ ++) for the bacterium of barbone. After about ten 

 passages in vitro the virulence became extreme (+ + ++), and at this 

 time it was used. 



A fairly turbid bouillon culture of the bacterium of barbone about 

 12 hours old received one drop of the previously described active 

 (+ + + +) Barbone-bacteriophage. After about 12 hours the medium 

 became perfectly limpid. This culture was filtered through a Cham- 

 berland filter (L3) and distributed into ampoules, which were sealed. 

 I would call attention to the necessity of employing only suspensions 

 in which the dissolution of the bacteria has been complete. Such 

 suspensions ought, moreover, to be filtered because of the fact that a 

 secondary culture may develop in some of the tubes. 



The suspensions of Barbone-bacteriophage have been used after a 

 variable length of time, — from twenty days to five months after their 

 preparation. No difference has ever been observed in their mode of 

 action, whatever the time elapsed between the date of preparation 

 and the time of use. 



All of the experiments, except those deahng with the effect of the 

 age of the animal upon the development of immunity, have been 

 effected on steers of the indigenous race, in a perfect state of health, 

 aged from twelve to eighteen months, and of an average weight of 

 100 kgms.,* and on buffaloes aged from one to twelve years. The 

 bovine race and the buffalo are equally susceptible to barbone. In 



* The race in Indo-China is of small size. 



