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resort to strict anaerobiosis, but it was however, necessary to empicey the 
use of sterile animal tissue in all cases. In the cultivation of pallidum, 
macrodentium, microdentium, mucosum, refringens, and phagedenis strict 
anaerobic conditions were required. (Quite the opposite seems to be true 
for the relapsing-fever spirochaetes. 
Noguchi’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. For this reason as well as being intensely interested along these 
lines in view of the fact that we attempted the cultivation of the relapsing- 
fever organisms three years previous to Noguchi's first publication and 
after obtaining cultures without difficulty by Noguchi’s method, we deemed 
it advisable to attempt cultivation 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 aseitic 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 pipette 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 ascitie 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 Berkefield filter are entirely worthless for this work. 
Rats were inoculated with a small amount (about one-eighth cubie 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 hefore coagulation takes place 
to the bottom of the tubes containing ascitic fluid which have been previous- 
ly warmed to a temperature 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. 
