472 THE SPIROCHETES 



pig. Sometimes the gas-forming organisms were eliminated by one guinea pig passage; some- 

 times two or three successive passages were necessary to reduce the number sufficiently for 

 successful cultivation of the spirochetes. 



Another organism which grows on the same media as the saprophytic treponemas is the 

 one which I called Spirochacta pJiagedcnis because of its presence in a phagedenic ulcer.' 

 This organism is not a treponema, and its taxonomic relationships are uncertain. 



None of the smegma spirochetes are odor-producing in culture; in this respect they differ 

 from T. mucosmn and T. microdeiitium. T. calligyrum grows much more readily than the 

 pallidum and can be cultivated in media not containing fresh tissue. It is easily mistaken 

 for T. pallidum, from which it differs in being non-pathogenic and by its presence in non- 

 specific lesions. In cultures in which T. pallidum is associated with calligyrum and refringens, 

 microdcntium, or mucosum, it is easy to eliminate the pallidum by subculturing on media not 

 containing fresh tissue and neglecting anaerobic precautions. 



Treponema vincenti was cultivated by Tunnicliff^ in 1906 under anaerobic conditions 

 on a medium consisting of glucose agar with or without serum. Miihlens and Hartmann^ 

 about the same time cultivated T. dentium on a similar medium. Repaci,'' in 1909, isolated 

 a number of spirochetes from the human mouth by means of Veillon's medium. 



AEROBIC GROUP 



Cultivation of the blood spirochetes (T. rccurr cutis, T. duttoni, T. kochi, T. novyi, T. 

 anscrinum) was first accomplished^ in ascitic fluid containing fresh tissue, incubated at 37° C. 

 under aerobic conditions. If the ascitic fluid is suitable the presence of the tissue will cause 

 the formation of a loose-meshed fibrin net throughout the fluid. The fluid must be free from 

 bile. A small amount of citrated blood from the heart of an infected rat or mouse is added 

 to the tubes either before or after the addition of the ascitic fluid. The organisms multiply 

 steadil}^ until after eight to nine days every field will show numerous motile specimens either 

 singly, in pairs, or in chains of three or more individuals. Sterilization or filtration appears 

 to impair the nutrient quality of the medium and invasion by bacteria kills the spirochetes. 

 In subculturing 0.5-1 cc. of culture is inoculated into the fresh medium. Subcultures are 

 best made every seven days. 



Kliglcr and Robertson^ found that a semisolid serum-agar medium may be used for the 

 blood spirochetes providing the hydrogen-ion concentration is adjusted to a pH of 7.4-7.8. 

 This medium is analogous to that which the writer used for the cultivation of the leptospira 

 group. The writer's term "aerotropic anaerobiosis"' was actually an erroneous interpreta- 

 tion of the conditions which e.xist in the ascitic fluid tissue medium. The presence of the 

 fresh tissue gave rise to the necessary semisolid condition by causing fibrin formation, and it 

 also brought the reaction to about pH 7.5. The paraffin-oil cover admitted oxygen enough 

 and at the same time prevented evaporation of the medium. 



The simplest and easiest of all spirochetes to cultivate are the members of the leptospira 

 group. Cultivation was first accomplished by Inada, Ido, and their collaborators in 1916,* 

 when Leptospira ictcrohacmorrliagiae, discovered by them in 1914-15 was cultivated on the 



' Noguchi, H.: J. Expcr. Med., 16, 261. 1912. 



^Tunnicliff, R.: /. Infect. Dis., 3, 148. 1906. 



3 Miihlens, P., and Hartmann, M.: Zlsehr. f. Ilyg. n. Infekllonskranldi., 55, Si. 1906. 



^ Repaci, G.: Compt. rend. Soc. dc biol., 69, 7S4. 191 1. 



5 Noguchi, H.: J. E.xper. Med., 16, 199, 620. 1912. 



^ Kligler, I. J., and Robertson, O. H. : ibid., 35, 303. 1922, 



7 Noguchi, IL: Harvey Lectures (9th ser.), p. 236. 1915-16. 



8 Inada, R., Ido, Y., Hoki, R., Kancko, R., and Ito, H.: loc. cit. 



