A piece of fresh, sterile rabbit’s kidney is placed in the lower tube, which is filled with ascitic fluid, or ascitic fluid and bouillon mixture. The tube is inoculated with syphilitic material and corked by inserting the upper tube. In the bottom of the upper tube a piece of sterile rabbit’s kidney is placed and syphilitic material poured over it. A mixture of one part ascitic fluid and two parts of slightly alkaline agar is then poured over the tissue and allowed to solidify. When solid, a layer of sterile paraffin oil is poured on top of it, and the top plugged with cotton wool (fig. 57). The whole is then incubated at 37° C. for two or three weeks. The tissue removes traces of oxygen from the lower levels of the medium and also probably provides a special form of nourishment. At first _T. pallidum_ grows in the solid medium, and then when the cultural conditions in the lower fluid portion become favourable, the organisms migrate thither and multiply abundantly. At first the culture is impure, but after several transferences a pure culture is obtained readily. The syphilitic material for culture is prepared by cutting off pieces of tissue from the lesions, washing in sterile salt solution containing 1 per cent. sodium citrate, and then emulsifying the tissue in a mortar with sodium citrate. Good cultures show rapid multiplication, which is invariably by longitudinal division. In his various cultivation experiments Noguchi[166] found morphological and pathogenic variations in _T. pallidum_. Three forms of the organism were found, namely, thicker, average and thinner types. The lesions caused in the testicle of the rabbit differ according to the variety inoculated, but more work is necessary on the subject. [166] _Journ. Exptl. Med._, xv, p. 201. Noguchi[167] has cultivated a separate organism, _T. calligyrum_, from the surface of human genital or anal lesions, either syphilitic or non-syphilitic. It is apparently non-pathogenic, and is 6 µ to 14 µ long. [167] _Journ. Exptl. Med._, xvii, p. 89. Hata (1913)[168] has modified the Noguchi technique for the cultivation of spirochætes and treponemes, with a view to simplification and convenience. Hata substitutes normal horse serum for ascitic fluid and the “buffy coat” of the clot of horse blood in place of the small pieces of rabbit’s kidney. It is unnecessary to place sterile paraffin on the surface of the medium. [168] _Centralbl. f. Bakt._, Orig., lxxii, p. 107. The horse serum is mixed with twice its volume of physiological saline solution. The mixture is placed in tubes which are heated on a water-bath at 58° C., the temperature being raised gradually until it reaches 70° or 71° C. in three hours. The tubes are then heated at 71° C. for half an hour. After cooling, the contents will consist of an opaque semi-coagulated mass. This semi-coagulated serum and saline mixture may be substituted for Noguchi’s ascitic fluid.