Laboratory Maintenance of Treponema denticola

Abstract

This unit describes methods, media, and equipment required for routine laboratory culture and handling of the oral anaerobic spirochete Treponema denticola. Topics discussed include nutrient requirements, recommended formulations for growth in liquid and solid media, and expected growth kinetics, as well as methods and equipment necessary to maintain anaerobic conditions. Protocols on isolation of T. denticola from clinical samples are included. With the notable exception of specific individual nutrient requirements and media formulations, these protocols are also appropriate for working with other species of cultivable commensal treponemes.

Basic protocols included in this chapter cover topics ranging from isolation of T. denticola from clinical samples (including isolation of pure cultures from single colonies) through culturing techniques applicable to biochemical analysis and genetic manipulation of T. denticola. The isolation protocols include a relatively standard method of dispersion and serial dilution on agar plates as well as a method using liquid medium in a 96-well plate format. Both of these methods employ rifampicin as a selective agent to enrich for treponemes in mixed clinical samples. Several commonly used liquid media and related solid media formulations are described, with comments on relative characteristics and performance of each. Methods, equipment, and conditions for inoculation, incubation, passage, and storage of cultures are discussed. Together, this information should provide the technical background required for routine growth and manipulation of T. denticola in a well-equipped microbiology laboratory.