DISEASES CAUSED BY SPIROCH.ETES 857 



anaerobic jar. After ten days at 33.5 C. the spirochaetes had 

 multiplied considerably, in all but one case, together with bacteria. 

 He obtained pure cultures from these initial cultivations after much 

 difficulty, by a number of methods. At first he succeeded only by 

 allowing the spirochgetes to grow through Berkefeld filters, which 

 they did on the fifth day. A better method more recently adopted 

 by him consists in preparing high tubes of three parts of very 

 slightly alkaline or neutral agar to which a piece of sterile tissue 

 has been added. These tubes are then inoculated from the impure 

 cultures with a long pipette. Close to the tissue and along the 

 stab the spirochaetes and bacteria will grow and, after about ten 

 days to two weeks, the spirochastes will have wandered away from 

 the stab and will be visible as hazy colonies. They can then be 

 fished, after cutting the tubes, and directly transplanted to other 

 serum-a gar-tissue tubes prepared as before, and eventually will 

 grow in pure culture. By this method Noguchi has also cultivated 

 pure cultures from lesions in monkeys. 



The writer, with Hopkins, has successfully applied Noguchi 's 

 method and has found that, after once cultivated artificially,, the 

 treponema pallidum can be obtained in quantity best by cultivation 

 in flasks containing heated or unheated rabbit kidney with ascitic 

 broth and sealed with paraffin. Recently we have been using modifi- 

 cations of a method worked out in our laboratory by Miss Gilbert, 

 in which slanted egg, with or without glycerin, made as for tubercle 

 cultivation, is used instead of kidney tissue. This is put up in high 

 tubes and ascitic broth and paraffin oil added. By this method, 

 large quantities of culture pallida are obtained within two weeks 

 and can be concentrated in large quantities. 



Animal Pathogenicity. Until very recently, all experimental 

 inoculation of animals was unsuccessful. During the year 1903 

 Metchnikoff and Roux 27 finally succeeded in transmitting the disease 

 to monkeys. The monkey first used by these observers was a female 

 chimpanzee. At the point of inoculation, the clitoris, there appeared, 

 twenty-six days after inoculation, a typical indurated chancre, which 

 was soon followed by swelling of the inguinal glands. Fifty-six 

 days after the inoculation there appeared a typical secondary erup- 

 tion, together with swelling of the spleen and of the lymph nodes. 

 Similar successful experiments were made soon after this by Lassar. 28 



"Metchnikoff et Eoux, Ann. de 1'inst. Pasteur, 1903, 1904, and 1905. 

 28 Lassar, Berl. klin. Woch., xl, 1903. 



