816 



SCIENCE 



[N.S. Vol. XXV. No. 647 



specific germicidal bodies. On the other 

 hand, in 'onset blood' drawn during the 

 early stage of the disease the spirilla may 

 li"ve for several weeks. Thus, we have seen 

 living spirilla in such blood kept for 30 

 to 37 days and have been able to infect 

 rats with blood kept for 40 days. More- 

 over, we have been able to make use of this 

 fact in shipping the virus to distant points, 

 to Dr. Todd at Liverpool and to Professor 

 C. Fraenkel at Halle. 



In our first series of attempts at culti- 

 vating the spirilla on blood agar we were, 

 as a rule, unable to keep the organisms 

 alive for more than two or three days. 

 Since then, however, we have been some- 

 what more successful and have kept them 

 on blood agar for 22 to 28 days, and in 

 some experiments now in progress they are 

 still alive and numerous on the thirtieth 

 day. As yet, however, no evidence has 

 been obtained of actual multiplication m 

 vitro. The organisms which are found to 

 persist we prefer to regard as mere sur- 

 vivals until actual subcultures have been 

 obtained. 



The successful results obtained by Lava- 

 diti in the cultivation of 8. gallinarum, 8. 

 Duttoni, and 8. refringens in coUodium 

 sacs led us to apply this method to our 

 spirillum. With this object in view the 

 collodium sacs were filled with rat or rab- 

 bit blood, or corresponding sera, heated 

 and unheated, and after inoculation with 

 spirillar blood these sacs were placed in 

 the peritoneal cavity of rabbits. After 

 three to seven days the sacs were removed 

 and contents were examined with uniformly 

 negative results. Apparently the rabbit 

 is unsuited for sac cultures. 



We were finally led to make the trials 

 under conditions approachng the natural 

 state as much as possible. For this pur- 

 pose, the collodium sacs were filled with 

 uncoagulated rat blood and after inocula- 



tion were placed at once in the peritoneal 

 cavity of a white rat. Three days later, 

 on removal, the sacs were found to contain 

 active spirilla and in increased numbers. 

 From the sacs, transplants were made to 

 new ones and the result was equally satis- 

 factory. The spirilla were found to be in 

 an extremely active condition and were 

 undoubtedly multiplying. 



From this time on the transplantations 

 were made regularly, every three or four 

 days, from sac to sac. After a few pas- 

 sages the uncoagulated blood was replaced 

 by defibrinated rat blood or by rat serum. 

 Defibrinated rabbit blood has also been 

 employed to some extent, but whether it 

 will continue to be a favorable medium we 

 are unable to state. Two sacs were inoc- 

 ulated each time and placed in the perito-' 

 neal cavity of a rat. Each sac had a 

 capacity of from 2.5-3.0 c.c. and was sealed 

 so as to leave within as little air as possible. 

 It is a noteworthy fact that on removal 

 from the rat the sacs are invariably greatly 

 distended as a result of osmotic changes. 

 Furthermore, the air which was originally 

 present is in large part, and at times 

 wholly absorbed. 



Since October 13, the spirilla have been 

 carried through 20 consecutive passages in 

 68 days, and presumably they can be kept 

 multiplying under these conditions indefi- 

 nitely. The spirilla in the sac culture are 

 never as numerous as in the blood of rats. 

 They rarely exceed more than 5 to 10 per 

 field of the one twelfth inch objective, as 

 contrasted with several hundred per field 

 met with in the blood of rats during the 

 maximum period of infection. The inocu- 

 lation of the sac contents (blood or serum) 

 into rats, it is interesting to note, is fol- 

 lowed by a mild infection in which the 

 spirilla are not much more numerous than 

 in the sacs. Moreover, in such infection 



