Cultivation 743 



one tube opening into the autoclave and reaching nearly to the bottom of the 

 bottle, and the other leading from the top of the bottle into a condenser adjoining. 

 When the cover of the autoclave is adjusted and the steam admitted, then in the 

 case of each bottle, the steam passed by the one tube to the bottom of the bottle, 

 and rismg through the pieces of pumice stone, the steam, carrying with it the 

 volatile constituents of the meat-extract, reaches the condenser by the second 

 tube. The vapor in the condenser yields the salt-free nutrient medium in the 

 proportion of 2 liters to each ounce of meat-extract originally used. The medium 

 is collected from the condenser in sterilized Pasteur flasks which are kept plunged 

 during the process in a freezing mixture in order to condense some of the volatile 

 alkaloids from the beef that would otherwise escape. The nutrient fluid is now 

 inoculated with the bacillus of leprosy and the flasks kept at 37°C. for from four 

 to six weeks; at the end of this period when examined the flasks should present a 

 turbid appearance with a stringy white deposit." 



Clegg* announced the cultivation of lepra bacilli from human 

 leprous tissue in symbiosis with ameba and other bacteria. The 

 organisms thus cultured he kept alive in subcultures. The method 

 devised by Clegg was the starting-point of a more extended re- 

 search by Duval,t who, after confirming the work of Clegg, found 

 that the bacillus could be cultivated directly from human lesions 

 upon culture-media containing tryptophan, without the symbiotic 

 ameba or other bacteria. The initial culture was somewhat difficult 

 to secure, but once the bacilli grew, transplantation was easily 

 and successfully carried on for indefinite generations. He further 

 found that the lepra bacillus could be successfully started to grow 

 upon the ordinary laboratory media if bits of leprous tissue were 

 placed upon them, and at the same time some symbiotic organism, 

 such as the colon, typhoid, proteus, or other bacilli, added. Or 

 if the tissues were already contaminated the lepra bacilli proceeded 

 to multiply. Duval interprets this to mean that the lepra bacillus 

 is unable to effect the destruction of the albumin molecule alone, and 

 hence explains the advantage of adding tryptophan. The medium 

 most successfully employed by Duval was as follows : 



"Egg-albumin or human blood-serum is poured into sterile Petri dishes and 

 inspissated for three hcJurs at 7o°C. The excised leprous nodule is then cut into 

 thin slices, 2 to 4 mm. in breadth and 0.5 to i mm. in thickness, which are dis- 

 tributed over the surface of the coagulated albumin. By means of a pipet the 

 medium thus seeded with bits of tissue is bathed in a i per cent, sterile solution of 

 trypsin, care being taken not to submerge the pieces of leprous tissue. SuflScient 

 fluid is added to moisten thoroughly the surface of the medium. The Petri dishes 

 are now placed in a moist chamber at 37°C., and allowed to incubate for a week or 

 ten days. They are removed from the plates from time to time, as evaporation 

 necessitates, for the addition of more trypsin. It will be noted that after a week 

 or ten days the tissue bits are partially sunken below the surface of the medium 

 and are softened to a thick, creamy consistence, fragments of which are readily 

 removed with a platinum needle. On microscopic examination of this material it 

 is noted that the leprosy bacilli have increased to enormous numbers and scarcely 

 a trace of the tissue remains. Separate lepra bacillus colonies are also discernible 

 on and around the softened tissue masses . . . The colonies are at first gray- 

 ish white, but after several days they assume a distinct orange-yellow tint. . . 

 Subcultures may be obtained by transferring portions of the growth to a second 

 series of plates or to slanted culture-tubes that contain the special aibumin- 



* "Philippine Journal of Science," 1909, iv, 403. 



t "Journal of Experimental Medicine," 1910, xii, 649; 1911, xril, 365. 



