Publisher Summary This chapter discusses the culture of Treponerna pallidum ( T. pallidum ). T. pallidum can be cultivated in a variety of tissue culture vessels; 24-, 12-, and 6-well culture plates, and 25-, 75-, and 150-cm 2 tissue culture flasks, have been used successfully to cultivate T. pallidum . Cultivation in suspension cultures of Sf1Ep cells in Magna–Flex flasks has also been successful. The amount of cells varies for each type of vessel. The cultures are set up 2 days before infection and confluency is between 20 and 25%. Methods have been developed to cultivate T. pallidum on Sf1Ep cells attached to microcarrier beads. The rationale for this development was to provide a better vehicle for serial passage experiments. Suspension cultures would be much easier to transfer from flask to flask because harvesting using TV would not be required. Thus, the cells and the treponemes would experience minimal disturbance during the suspension culture passage process as compared with more rigorous harvesting using TV. The chapter illustrates the typical growth curve for T. pallidum cultivated in vitro . When incubated at 34 ° in a microaerophilic atmosphere of 3-4% oxygen, the typical lag period for treponemal growth is about 2 days. Small increases (2- to 3-fold) can be detected as early as 3 days. In tissue cultures, generation times between 35 and 40 hr are routinely observed. The stationary phase of growth in treponemal cultures is relatively short, typically lasting only 1 to 2 days.