CULTIVATION OP LEPROSY BACILLUS. 407 



All "other tiibes, including those made from the spleen, were unchanged 

 and remained so. Transplants were made from the two tubes showing 

 a growth of the leprosy bacillus with the amoeba and the cholera vibrio, 

 to fresh agar tubes and kept at the same temperature. Smear prepara- 

 tions made at the end of the third day showed a great increase in the 

 number of the acid-fast bacilli and the same change ol; morphology as 

 was observed in the previous case. Negative results wei'e obtained in 

 transplants from both sets of control tubes, namely, from tliose containing 

 amoebae and cholera vibrios, but no leprosy material, and from those 

 containing plain agar inoculated with material from the nodule. The 

 tubes which contained amoebas and cholera vibrios and which were in- 

 oculated with the splenic material were also negative. Weekly trans- 

 plants were then made from the two tubes showing a growth of the 

 acid-fast bacillus with the mixed cirtture of amoeba and cholera. After 

 having been growing in this mixed symbiosis for six weeks, these cul- 

 tures were subjected to a temperature of 60° C. for thirty minutes, 

 and as in the previous case, the acid-fast organisms were obtained in pure 

 culture. 



Case H. — A mixed type of leprosy. The autopsy was performed three hours 

 three hours after death. The lungs showed tubercular lesions, but there was no 

 evidence of tuberculosis in the other organs. 



The spleen was removed by the method described in Case F and cultures were 

 made in a similar manner. Smear preparations made from this spleen showed 

 a small number of leprosy bacilli present. After three weeks' incubation smear 

 preparations from the cultures showed no cliange in the morphology of the 

 leprosy bacilli inoculated and no evidence of growth. 



Case I. — A well-advanced ease of tubercular leprosy. The autopsy was per- 

 formed twenty-four hours after death. The lungs showed the presence of tuber- 

 cular lesions; there was no evidence of other tuberculous lesions in the body. 

 The spleen was removed aseptically and cultures were made on media containing 

 amoeba- and cholera vibrios and al.so on media composed of plain sterile agar. 

 After six days' incubation a slight growth was observed in one of the tubes 

 containing the amoebaj and cholera vibrios. No development occurred, however, 

 in transplants from this tube. 



CULTURAL CHARACTERISTICS. 



The separation of the icid-fast bacilli from the amoebte and cholera 

 vibrios was accomplished, as already indicated, in a very simple manner; 

 viz, by killing the two latter organisms by subjecting the mixed cultures 

 for half an hour to a temperature of G0° C. At this temperature the 

 acid-fast bacteria are not killed in the first half hour. 



From these heated cultures transplants were made upon amoeba-cholera 

 cultures and upon plain agar slants without the amceboe. In both cases 

 a growth of the acid-fast organisms took place. 



Cultures were made from these 'original pure colonies upon ordinary 

 nutrient agar, plain agar, potato, alkaline litmus-milk, glucose-agar, 



.<i1 s.^.^ 2 



