VACCINES : DIAGNOSTIC AND CURATIVE 121 



lized 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 con- 

 tents passed through a Doulton white filter, and the 

 filtrate so obtained placed in small sterile phials in 

 quantities of 2 J and 5 c.c. The vaccine was not evapo- 

 rated to obtain a more concentrated solution, as we con- 

 sidered this unnecessary for preliminary experiments. 



Vaccine No, 2. — A second batch of vaccine was pre- 

 pared in a manner exactly similar to the above, except 

 that the dried human tubercle bacillus was replaced 

 by the dried timothy-grass bacillus. 



Vaccine No. 3. — A third batch was prepared by 

 growing Johne's bacillus in a broth medium containing 

 a glycerine-saline extract of the timothy-grass bacillus, 

 the extract representing i per cent, of dried bacilli and 

 4 per cent-, of glycerine. The medium used for pre- 

 paring this vaccine was filtered and autoclaved before 

 being inoculated, and in it the specific bacillus grew 

 fairly well in tiny masses on the bottom of each flask. 



Vaccine No. 4. — A fourth batch was prepared from the 

 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. 



Vaccine No. 5. — This was made in a similar manner 

 to No. 4, except that the cultures of Johne's bacillus 

 were suspended in o*8 per cent, sodium chloride in 

 place of Vaccine No. 3. 



As controls to the above vaccines we used diagnostic 



