528 Messrs. Twort and Ingram. Isolating and [Nov. 7, 



the ileo-csecal valve ; the disease was in an early stage, and the thickening of 

 the intestine was quite moderate. Films were made from the ileum and the 

 ileo-caecal valve, but only a very few Johne's bacilli could be found, even after 

 searching for some considerable time. Small pieces of tissue were removed 

 aseptically in the manner previously described and inoculated on to six tubes 

 of Dorset's egg medium, and on to two tubes of special medium containing 

 \ per cent, of dried timothy-grass bacillus. All were capped with gutta- 

 percha tissue and incubated at 39° to 40° C. as with the previous cases. 



After 48 hours one of the tubes of special medium was found to be 

 contaminated and was discarded, After five weeks' incubation films were 

 made from the tubes and examined microscopically. Those taken from the 

 cultures on Dorset's egg medium showed no acid-fast bacilli, but that taken 

 from the remaining tube of special medium, made with dead timothy-grass 

 bacilli, showed some small clumps of acid-fast bacilli presenting the 

 characters of Johne's bacillus. Accordingly sub-cultures were made from 

 this tube on to fresh tubes of the same medium and on to fresh tubes of 

 Dorset's egg medium. All the tubes were capped and placed in the 

 incubator at 39° to 40° C. Films were now made at intervals of about 

 a week from the various tubes, and without describing all in detail it will be 

 sufficient to note that the bacillus found on the original tube of special 

 medium continued to grow on this and on all the sub-cultures made on to 

 the timothy-grass bacillus medium, and on media containing the human 

 tubercle bacillus, but that the sub-cultures on Dorset's egg medium remained 

 sterile. The bacillus isolated resembled in every way the bacilli isolated 

 from the four previous cases, and the cultural characters were also the same. 



From the above experiments it is clear that Johne's bacillus will grow on 

 media containing the dead timothy-grass bacillus, not only after it has been 

 cultivated in the laboratory for a considerable period, but also when taken 

 direct from the diseased gut of cattle. 



Having determined the various acid-fast bacilli most suitable for the 

 growth of Johne's bacillus, an attempt was made to extract the essential 

 substance from certain of these bacilli. The timothy-grass bacillus was 

 chosen, chiefly because it gave the best results in the above experiments, also 

 because it is harmless to man and grows quickly on simple media, thus 

 enabling a large quantity of growth to be obtained in a short time. 



Dr. W. Bulloch kindly gave us a quantity of this bacillus, besides 

 various dead and dried tubercle bacilli, which latter had been given to him 

 by Prof. Bang about eight years previously. Many of these had already been 

 extracted by Bulloch and MacLeod (40) when investigating the acid-fast 

 properties of the tubercle bacillus. The different bacillary powders were 



