CULTIVATION OF LEPROSY BACILLUS. 405 



which they started and take up those forming the rings. In from twenty- 

 four to forty-eight hours the amoebae will have passed to the outer ring 

 and from this location they may be taken for transplantation to 

 fresh ringed plates with a medium suitable for their growth, the same 

 procedure being repeated until by plating it is shown that the amoeba is 

 growing only with the bacterium desired. In continuing the work on the 

 ciiltivation of the leprosy bacillus, as has been mentioned, an amceba was 

 used which had been isolated from the city water supply and was growing 

 in symbiosis with the cholera vibrio. 



The mixed culture of the amceba and the cholera vibrio was inoculated 

 on slant tubes of plain sterile agar-agar without the beef extract, but 

 containing the same amount of sodium chloride and having the same 

 reaction as the amceba medium previously described — and after twenty- 

 four hours' growth of the mixed cultu.re on this medium, material con- 

 taining leprosy bacilli was added; at the same time control inoculations 

 of the leprous material were made upon the same medium without the 

 amoeba and the cholera vibrio. The tubes were incubated for one week 

 at 37°, at the end of which period examination showed an increase in 

 numbers and change in morphology of the acid-fast bacilli in three of 

 the amoeba cultures, whereas on the plain agar these bacilli had changed 

 neither in number nor in morphology. 



After having been transplanted once a week upon amosba-cholera cul- 

 tures for six weeks in one case and three months in another, thg acid- 

 fast organisms were obtained in pure culture by heating; in this manner 

 the cholera vibrios and amoebas were killed by a temperature of 60° C. for 

 half an hour, while the acid-fast bacilli survived and grew readily alone, 

 when transplanted. 



The acid-fast organism was obtained from the two following cases : 



Case F. — A well-marked case of tubercular leprosy. The autopsy was per- 

 formed three hours after death. The lungs showed the presence of tubercular 

 cavities and extensive adhesions to the chest wall. No lesions of tuberculosis 

 were found in the other organs. The spleen was removed under aseptic conditions 

 by the following method: A flap of the skin on the left side covering an area 

 extending from the median line to the vertebral column and from the axilla to 

 the crest of the ilium was removed. Alcohol was then poured over the muscular 

 layer and burned, after which the peritoneal cavity was opened with sterilized 

 instruments and the spleen removed and placed in a sterile vessel. Cultures were 

 then made in the usual way, after first burning the capsule of the spleen and 

 puncturing it with a sterile knife. 



Ten tubes, each containing a 24-hour growth of amoebae and the 

 cholera vibrio, were inoculated with the splenic pulp as were also ten 

 control tubes containing sterile agar. All the tubes were placed in the 

 incubator and kept at a temperature of 37° C. for seven days. At the 

 end of this period smears from the tubes containing the mixed culture 

 of amceba and cholera vibrio showed, when stained by the Ziehl-Neelson 



