In Vitro Cultivation of the Syphilis Spirochete Treponema pallidum.

In Vitro Cultivation of the Syphilis Spirochete Treponema pallidum.

Current protocols (2021-02-19)

Diane G Edmondson, Steven J Norris

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.

MATERIALS

Product Number

Brand

Product Description

T4049

Sigma-Aldrich

Trypsin-EDTA solution, 0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA, 4Na per liter of Hanks′ Balanced Salt Solution with phenol red

R7017

Sigma-Aldrich

Resazurin sodium salt, powder, BioReagent

S8636

Sigma-Aldrich

Sodium pyruvate solution, 100 mM, sterile-filtered, BioReagent, suitable for cell culture

D9779

Sigma-Aldrich

DL-Dithiothreitol, Molecular Biology, ≥98% (HPLC), ≥99% (titration)

H6034

Sigma-Aldrich

L-Histidine, suitable for cell culture, meets EP, USP testing specifications, from non-animal source

G7513

Sigma-Aldrich

L-Glutamine solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture

D8537

Sigma-Aldrich

Dulbecco′s Phosphate Buffered Saline, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture

S8761

Sigma-Aldrich

Sodium bicarbonate solution, solution (7.5%), sterile-filtered, BioReagent, suitable for cell culture

G6152

Sigma-Aldrich

D-(+)-Glucose, suitable for mouse embryo cell culture, ≥99.5% (GC)

D2650

Sigma-Aldrich

Dimethyl sulfoxide, Hybri-Max^™, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%

M4655

Sigma-Aldrich

Minimum Essential Medium Eagle, With Earle′s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture