In Vitro Cultivation of the Syphilis Spirochete Treponema pallidum

Abstract

For over a century, investigation of Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, was hindered by an inability to culture the organism in vitro. A recent breakthrough has enabled continuous in vitro growth of this organism in co-culture with mammalian tissue culture cells. This article contains the protocols needed to culture T. pallidum in the standard laboratory environment. In addition, protocols for growing and maintaining the required tissue culture cells, for generating isogenic strains by limiting dilution, and for quantitating T. pallidum by darkfield microscopy are included. © 2021 Wiley Periodicals LLC. Basic Protocol 1: In vitro cultivation of Treponema pallidum Basic Protocol 2: Generation of isogenic strains Support Protocol 1: Alternate harvest procedure Support Protocol 2: Culture of Sf1Ep cells Support Protocol 3: Assessment of T. pallidum number and viability.

Keywords: Treponema pallidum; cell culture; in vitro cultivation; laboratory growth; spirochetes; syphilis.

Figure 1

Diagram of the arrangement to evacuate and fill a Brewer jar with a low ‐oxygen gas mixture. Airtight tubing is connected to the vent of a Brewer jar. The tubing connects via a T‐joint to a vacuum supply (usually house vacuum) and to gas cylinders containing custom gas mixtures (5% CO₂, balance nitrogen and 1.5% O₂, 5% CO₂, balance nitrogen). The vacuum is drawn in the jar (about 12 to 18 in. Hg) and is measured using an in‐line vacuum gauge. The evacuated jar is then slowly refilled with the gas mixtures: the Brewer jar is refilled four times with 5% CO₂, balance nitrogen before a final vacuum and refill with 1.5% O₂, 5% CO₂, balance nitrogen. Culture or medium is then removed from the jar and quickly transferred to a low‐oxygen incubator. Alternatively, the tubing between the Brewer jar and the first T‐joint can be clamped tightly, the tubing disconnected from the T‐joint, and the entire Brewer jar transferred to a 34°C incubator.

Figure 2

Helber chamber grid. The Helber chamber grid consists of 16 large squares, and each of these large squares is further subdivided into 25 squares. One large square is outlined in red. The top row and the left column of each large square are bisected by another etched line to aid in orientation.

Figure 3

Overview of the T. pallidum in vitro cultivation procedure.