227 



]\rnltii)lirn(ioii iiii|>;ii-OMtl.v hcuiiis in the iiciiililuirlinod (if s('\only-(\v(i 

 hours aftoi- iiiofuliitioii .-iikI tlic in;i\iiiiiiiii ,m<i\\tli is iciclicil in I'roMi six 

 to eight days. Miitcrial Cor cxaiuinini.' the ciiltmcs is dlitaincil liy intro- 

 ducing a capillary pipette'"' near tiie iiottdni of the liiiics. 



The organisms arc ]>erpctuat('(l ity transfcn'ini,' al»Mit one lialf cnliic 

 centimeter of the cultures from I he positive tubes to fr(>sii undcli- 

 brinated blood ascitic fluid mediiuu. preferably just before the cnltures 

 reach their maximum growth (live to seven days). These transjilants are 

 made by sucking up the material in a capillary pipette which is intro- 

 duced about one inch from the bottom of the culture tube, thus avoiding 

 a large number of so-called skeleton forms. 



By the means above described we have been able to (iillivatc Spiiodiaeta 

 Xovyi and to carry the same thru six generations witbdut the use of sterile 

 tissue. 



If the positive tubes are covered with paralhn oil and placed in the ice 

 box (0-10° centigrade) just before the maximinn growth results, successful 

 subcultures may be made ten to twelve days later. These iced paraffin 

 oil covered cultvu'es will also exhibit pathogenic properties two or three 

 weeks later. As a matter of fact, whenever motile forms are present, 

 although the greater part of the culture has degenerated into granules and 

 skeleton forms, they are pathogenic. It is advisable to warm the cidtures in 

 order to ascertain motion. 



One hundred percent infection resulted when rats were injected with 

 generations one to six inclusive. The jieriod of incubation is sduicwliat 

 longer in the case of culture infected rats than in rats receiving the blond 

 type of spirochaetes. The period of incubation in the former genersilly is 

 from seventy-two to ninety-six hours, while that of the latter is usually 

 eighteen to twenty -four hours. Once the infection is established the cul- 

 tural spirochaete form is identical in appearance with the straight blood 

 type. Besides the period of incubation being somewhat longer, the dis- 

 ease thus induced seems also to be less fatal for out of twelve rats receiving 

 cultiu'e material of various genei'ations, (one to six) no deaths resulted. 



In cultures approximately ninety-six hours old there are actively motile 

 spirochaetes of various description. Some have two or three irregular 

 curves, while others have six or eight. Degenerated skeleton and granular 

 forms and highly refractive granules are also present at this time but 

 more especially in tubes a week or more old. The spirochaetes may occur 

 singly, in pairs or chains, but there is a tendency for them to form agglut- 

 inated masses. These cultures do not stain as easily nor as distinctly as 

 the blood type by the Wright or Honianowsky methods. 



Nr.l/.l/.l/.'V. 



Spirochaeta Xovyi has lieen cultivated in vitro by employing a medium 

 devoid of tissue obtained from animal organs. 



Undefibrinated blood, ascitic fluid free from bile and capable of forming 

 a loose fibrin are neces.sary for obtaining and maintaining such cultures. 



