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Multiplication apparently begins in the neighborhood of seventy-two 
hours after inoculation and the maximum growth is reached in from six 
to eight days. Material for examining the cultures is obtained by intro- 
ducing a capillary pipette’ near the bottom of the tubes. 
The organisms are perpetuated by transferring about one-half cubic 
centimeter of the cultures from the positive tubes to fresh undefi- 
brinated blood ascitic fluid medium, preferably just before the cultures 
reach their maximum growth (five to seven days). These transplants 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 cultivate Spirochaeta 
Novyi and to carry the same thru six generations without the use of sterile 
tissue. 
If the positive tubes are covered with paraffin oil and placed in the ice 
box (0-10° centigrade) just before the maximum growth results, successful 
subcultures may be made ten to twelve days later. These iced paraffin 
oil covered cultures 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 cultures in 
order to ascertain motion. 
One hundred percent infection resulted when rats were injected with 
generations one to six inclusive. The period of incubation is somewhat 
longer in the case of culture infected rats than in rats receiving the blood 
type of spirochaetes. The period of incubation in the former generally 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 
culture material of various generations, (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 haye 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 Romanowsky methods. 
SUMMARY. 
Spirochaeta Novyi has been 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 necessary for obtaining and maintaining such cultures. 
