226 



resort to strict anaerobiosis, but it was however, necessary to> employ the 

 use of sterile animal tissue in all cases. In the cultivation of pallidum, 

 macrodentiuui, microdentiuni. mucosum, refringens, and phagedenis strict 

 anaerobic conditions wciv rciiuired. (juite the opposite seems to be true 

 for tlie relapsing -fever spirochaotes. 



Noguclu's method of obtaining cultures of the latter is briefly as follows : 

 A piece of sterile fresh rabbit kidney is placed in a sterile test-tube 

 to which is added a few drops of citrated infected hearts blood of a rat or 

 mouse. About fifteen cubic centimeters of sterile ascitic or hydrocle fluid is 

 then added. Some of the medium is covered with sterile paraffin oil. The 

 culture tubes thus prepared are incubated at 35-37° Centigrade. Maximum 

 growth occurred on the fourth to the ninth day. 



The use of sterile tissue being employed in all of this cultural work, 

 of course, entails in many cases the killing of rabbits for their kidney tissue 

 only. P^or this reason as well as being intensely intei'ested along these 

 lines in view of the fact that we attempted the cultivation of the relapsing- 

 fevcr organisms three years previous to Noguchi's first publication and 

 after obtaining cultures without difticulty by Noguchi's method, we deemed 

 it advisable to attempt culti^'ation without the use of sterile tissue from 

 animal organs. After various attempts we were finally able to obtain 

 initial cultures without such tissue. These cultures as well as those 

 which were obtained by the employment of the tissue medium, could be 

 transplanted, with success, to ascitic fluid to which a small amount of 

 sterile undefibrinated blood only, had been added. 



The medium employed by us differs considerably from that used by 

 Noguchi. Approximately eighteen cubic centimeters of sterile Ascitic 

 fluid are transferred to each of a number of sterile test-tubes (tubes 

 20 by 1.5 centimeters were used). The pli>ette for holding and measuring 

 sterile fluids^^ may be conveniently used in transferring this fluid to the 

 tubes. It is very important as Noguchi also notes that the ascitic fluid 

 does not contain bile, but forms a loose fibrin. Specimens of this fluid 

 which do not possess this property or which have been sterilized by pass- 

 ing thru a Berkefleld filter are entirely worthless for this work. 



Rats wei-e inoculated with a small amount (about one-eighth cubic centi- 

 meter) of spirochaetal blood obtained from the heart or tail of an infected 

 rat. The blood of these rats is conveniently examined by clipping off the 

 end of their tails. When it reveals the presence of from twenty-five to one 

 hundred spirochaetes per field, (one-twelfth oil immersion lense), before 

 large agglutinating masses of the organisms are seen, usually requiring 

 from eighteen to twenty-four hours after inoculation, they are bled from 

 the heart by means of a small bulb capillary pipette". 



The blood thus obtained is transferred before coagulation takes place 

 to the Iiottcmi of the tubes containing ascitic fluid which have been previous- 

 ly warmed to a temiierature of thirty-seven degrees centigrade. It is most 

 important that whole undefibrinated blood is used. The inoculated tubes 

 are then incubated at thirty-seven degrees centigrade. No perceptible 

 growth occurs at twenty-five degrees centigrade. 



