A piece of fresh, sterile tissue, usually rabbit kidney, is placed in a sterile test-tube. A few drops of citrated blood from the heart of an infected animal, _e.g._, rat or mouse, is added, and about 15 c.c. of sterile ascitic or hydrocœle fluid is poured quickly into the tube. Some of the tubes are covered with a layer of sterile paraffin oil, others are left uncovered. The tubes are incubated at 37° C. The best results are obtained if the blood is taken from an animal forty-eight to seventy-two hours after it has been inoculated, that is, before the spirochætes reach their maximum multiplicative period in the blood. The presence of some oxygen seems indispensable for these blood spirochætes, and they fail to develop _in vacuo_ or in an atmosphere of hydrogen. For subcultures, 0·5 c.c. of a culture is added to the medium instead of citrated blood, and it is useful to add a little fresh, normal blood, either human or from an animal, such as a rat. Noguchi found that the events in cultures were:-- _S. duttoni_,[159] maximum multiplication on the eighth to ninth day; disintegration beginning on the tenth day, spirochætes disappeared after about the fifteenth day. No diminution of virulence was found at the ninth day. [159] _Journ. Exptl. Med._, xvi, p. 202. _S. rossii_ (= _S. kochi_).[160] Maximum development on the ninth day, after which the virulence diminishes. The incubation period is also prolonged. [160] _Ibid._, p. 205. _S. recurrentis_[161] (= _S. obermeieri_). Maximum growth on the seventh day. [161] _Ibid._, p. 205. _S. novyi._[162]--Maximum development on the seventh day. It is more difficult to grow than the preceding forms. [162] _Ibid._, p. 208. All the above spirochætes showed undoubted longitudinal division and transverse division was observed in part. _S. gallinarum_[163] can be cultivated as above, but transverse division was usual here. Maximum growth occurred in the culture about the fifth day. [163] _Ibid._, p. 620. TREPONEMATA. The genus _Treponema_ (Schaudinn, 1905), includes minute, thread-like organisms, with spirally coiled bodies, the spirals being preformed or fixed. No membrane or crista is present, according to Schaudinn, though a slight one is said by Blanchard to be present in the organism of yaws. The ends of the organisms are tapering and pointed. Multiplication is by longitudinal and transverse division. The most important members of the genus are _T. pallidum_, the agent of syphilis, and _T. pertenue_, which is responsible for frambœsia or yaws. *Treponema pallidum*, Schaudinn, 1905. Syn.: _Spirochæta pallida_.