1911.] Cultivating the Mycobacterium enteritidis, etc. 



531 



and if generally used in the infected areas of the countries in which the 

 disease is prevalent, and if followed by the slaughter of the diseased animals, 

 would soon tend to diminish if not to completely eradicate this condition. 



It must also be remembered that when animals suffering from this 

 disease are slaughtered, the flesh is not condemned as food unless the- 

 carcase shows marked emaciation, so that an early diagnosis would not 

 only help to prevent dissemination of the disease, but would allow of a better 

 price being obtained for the animal. 



From this it follows that the use of such a vaccine would be of direct 

 monetary value to farmers and stockowners, and if State legislation were 

 adopted, no Government compensation would be necessary. 



From the strains of Johne's bacillus which had been grown, we now 

 attempted to prepare a vaccine which would be both efficient and specific 

 as a diagnostic agent for the disease under discussion. In the first experi- 

 ments we used an alkaline peptone bouillon, containing 4 per cent, of 

 glycerine and 1 per cent, of dried human tubercle bacilli. This was placed 

 in Duclaux flasks and sterilised by steaming. These flasks were inoculated 

 with pure cultures of Johne's bacillus, and the main opening of each was 

 capped with gutta-percha tissue. The flasks were incubated at 39° to 40° C. 

 After the lapse of a month small yellowish-white grains of growth became 

 visible. These grew just above the sediment at the bottom of the flasks, 

 and gradually increased in size and number. No film formation was. 

 observed. After two months the flasks were steamed, their contents passed 

 through a Doulton white filter, and the filtrate so obtained placed into small 

 sterile flasks in quantities of 2£ and 5 c.c. The vaccine was not evaporated 

 to obtain a more concentrated solution, as we considered this unnecessary 

 for experimental work. 



A second batch of vaccine was prepared in a manner exactly similar to 

 the above, except that the dried human tubercle bacillus was replaced by 

 the dried timothy-grass bacillus. A third batch was prepared by growing 

 Johne's bacillus in a broth medium containing a glycerine saline extract of 

 the timothy-grass bacillus, so as to represent 1 per cent, of dried bacilli 

 and 4 per cent, of glycerine. The medium used for preparing the last 

 vaccine was filtered and autoclaved before inoculating, and in it the specific 

 bacillus grew fairly well as tiny masses which settled down to the bottom 

 of the flasks. Incidentally, this proved that Johne's bacillus will grow in an 

 albumen-free medium. 



A fourth batch was prepared from cultures of Johne's bacillus on the- 

 special timothy-grass bacillus egg medium, the growth being scraped off and 

 an emulsion made with vaccine No. 3 described above. 



