Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system

Abstract

Limited multiplication of Treponema pallidum subsp. pallidum (Nichols strain) can be obtained in the presence of Sf1Ep rabbit epithelial cell cultures, but continuous culture has not yet been achieved. In the system currently employed, growth is exponential for the first 10 to 15 days of culturing, after which multiplication and the percentage of motile organisms decrease. In an effort to identify culture conditions which may adversely affect treponemal viability and growth, eight culture parameters were monitored over a 12-day period of incubation. Several of these parameters, including pH, redox potential, dissolved oxygen concentration, and glucose levels were found to change dramatically during the course of incubation, indicating that they may be responsible for the cessation of treponemal multiplication. The feasibility of extending the period of growth by subculturing was also investigated. In preparation for planned serial subcultivation experiments, several subculture procedures were tested and found to be effective in allowing the transfer of T. pallidum from 3-day-old primary cultures to secondary cultures without loss of motility or growth potential. Increases of up to 55-fold were observed in secondary cultures, but increased growth due to subculturing was not a consistent finding. Use of subculture intervals of ≥ 6 days resulted in a progressive decrease in treponemal multiplication in secondary cultures, although retention of motility was extended in the subcultures compared with motility in the primary cultures. These results indicate that the lack of continued multiplication of T. pallidum in subcultures is not due to damage to the treponemes during subculture. Prolonged multiplication of T. pallidum may be obtained through the stabilization of culture conditions by either performing subcultures at regular intervals or by medium replacement techniques. It was also found that primary T. pallidum cultures could be established by using as the inoculum treponemes that had been stored at -70°C in a medium containing 15% glycerol.