1.911.] Cultivating the Mycobacterium enteritidis, etc. 



525 



sodium chloride solution can be used in preparing the medium, without any 

 detrimental effect. 



We found also that it was better to dry the growth of the tubercle bacillus 

 after killing it and before making it up into medium, a fact which may 

 be due to the formation of cracks or breaks in the continuity of the 

 covering, enabling the essential substance to diffuse more easily into the 

 medium. Experiments showed that \ to 1 per cent, of the dried tubercle 

 bacillus was the most suitable quantity to add. To obtain the best results, 

 the dried bacilli should be ground up with the glycerine which has been 

 mixed with an equal quantity of - 8 per cent, saline, and the remainder 

 of the saline added later. The emulsion so obtained should then be 

 steamed for 15 minutes, and, when cool, added to the egg. The probable 

 explanation for this is that the glycerine acts as a solvent for the essential 

 substance, and some experiments to be described later tend to confirm 

 this suggestion. We also tried some media similar to the above, in which 

 the normal alkalinity of the egg was wholly or partially neutralised by 

 hydrochloric acid; these were found to be unsuitable. This proves the 

 necessity of maintaining a distinctly alkaline reaction. 



In another series of experiments the egg was replaced by various other 

 substances, such as broth or agar. These, as a rule, did not give such 

 good results, although ordinary glycerine peptone bouillon, made distinctly 

 alkaline, and containing \ to 1 per cent, of dried tubercle bacilli, gave a 

 fairly satisfactory growth. This, with other experiments to be described 

 later, proved that Johne's bacillus can grow quite well in the absence of 

 albumen. 



We next proceeded to test our strains of Johne's bacilli on media in which 

 the dead tubercle bacillus was replaced by various other micro-organisms. 

 We soon found that some strains of human tubercle bacilli were more 

 suitable than others ; and, further, that if the human tubercle bacillus was 

 replaced by the bovine type, no growth of Johne's bacillus took place, and 

 that this was so even when sub-cultured from strains which had been growing 

 outside the animal body for a year. Several strains of tubercle bacilli 

 isolated from cats were also tested, but gave negative results. 



The question then arose as to whether these results were clue to the 

 absence of some substance in the bovine tubercle bacillus, or to the presence 

 of some toxic substance not found in the human type. This was tested by 

 preparing four batches of medium, one containing \ per cent, of dried 

 human tubercle bacilli, another \ per cent, of dried bovine bacilli, a third 

 | per cent, of both the human and bovine types, and a fourth \ per cent, of 

 both types. Several tubes of each were inoculated with pure cultures of 



