The buff coagulum is cut into small pieces, about 1 c.c. in volume. They must be forced with a sterile glass rod to the bottom of the semi-coagulated serum and saline mixture. The medium is inoculated with a small quantity of infected blood and kept at 37° C. In the case of _S. recurrentis_, growth of spirochætes is observed on the second day, reaching a maximum in five to seven days. The growth of the organisms proceeds rather more slowly, they live for a longer period and maintain their virulence better than in Noguchi’s medium. *Treponema pertenue*, Castellani, 1905. Syn.: _Spirochæta pertenuis_; _S. pallidula_, Castellani, 1905. Castellani discovered the organism in 1905, in scrapings of yaws pustules. He first described it under the name of _Spirochæta pertenuis_. [Illustration: FIG. 58.--_Treponema pertenue_. (After Castellani and Chalmers.)] _Treponema pertenue_ (fig. 58), though delicate and slender, shows great morphological variation both in length and thickness. It may be short, _e.g._, 7 µ, but can attain 18 µ to 20 µ in length and may be even larger. In cultures made by Noguchi, thick, medium and thin forms were found, each giving rise to a different type of frambœsial lesion when inoculated into the testicles of rabbits, thus suggesting the possibility of the occurrence of varieties of _T. pertenue_. The organism is difficult to stain, but occasionally deeper staining granules are found along its body. They may represent a diffuse nucleus. Granule formation similar to that of _T. pallidum_ has been observed by Ranken, using dark-ground illumination. Many experiments have been made with a view to establishing the identity of the organism of yaws and also of differentiating between the causative agents of yaws and syphilis. Both monkeys and the human subject have been experimentally inoculated with yaws material and have developed the disease. In an early experiment, negroes were inoculated with the secretion from lesions of yaws. All of them developed the disease, nodules appearing, chiefly at the seat of inoculation, in from twelve to twenty days, followed by the usual eruption. Similar results were obtained with thirty-two Chinese prisoners, who were inoculated with yaws, twenty-eight becoming infected. A naturally infected yaws patient when inoculated with syphilis, contracted that infection, thus showing that yaws does not confer immunity to syphilis. This has also been observed naturally, when yaws patients have contracted syphilis. Experiments with monkeys have been successfully performed. The incubation period varies from sixteen to ninety-two days. Lesions appear first at the seat of inoculation, and in some monkeys the eruption is localized to this spot, though the infection is general, _T. pertenue_ occurring in the spleen, lymphatics, etc. Monkeys inoculated with splenic blood of a yaws patient, and also sometimes with blood from the general circulation, have become infected.