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.CAUTION: Treponema denticola is a Biosafety Level 1 (BSL-1) organism. Such organisms are not known to consistently cause disease in healthy adult humans, and are of minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these organisms. See UNIT 1A.1 and other pertinent resources (see APPENDIX 1B) for more information.NOTE: Most strains of oral spirochetes require strictly anaerobic conditions during transfer and growth of cultures. Unless otherwise specified, all incubations and, where feasible, manipulations described in this unit are to be performed under strictly anaerobic conditions (i.e., nonhumidified 85% N 2 /10% H 2 /5% CO 2 atmosphere) in an anaerobic glove box chamber at 37 • C (see Strategic Planning). STRATEGIC PLANNING Media General requirementsSeveral complex broth media formulations are in common use for growth of T. denticola strains (see Reagents and Solutions). These share common features including sources of peptides, amino acids, and trace nutrients (e.g., tryptone, brain heart infusion, yeast extract), as well as reducing agent(s) (L-cysteine and/or thioglycolate), volatile fatty acids, and heat-inactivated serum. All these media provide very low redox potential (Eh = −185 to −220), which is a critical factor in growth of T. denticola (Socransky et al., 1964). These media were orig...

Treponema denticolaincreases MMP-2 expression and activation in the periodontium via reversible DNA and histone modifications

“…Treponema denticola ATCC 35405 was grown as described previously under anaerobic conditions at 37°C in New Oral Spirochete (NOS) broth medium (Haapasalo et al , 1991, Fenno, 2005). Purity of spirochete cultures was confirmed by darkfield microscopy prior to use in experiments.…”

“…Cells passaged three to six times were used for experimentation. The cell counting kit-8 4.3 | Culture of T. denticola T. denticola ATCC 35405 was grown as described previously under anaerobic conditions at 37°C in new oral spirochete broth medium (Fenno, 2005;Haapasalo, Singh, McBride, & Uitto, 1991). Purity of spirochete cultures was confirmed by darkfield microscopy prior to use in experiments.…”

Lyme disease and relapsing feverBorreliaelongate through zones of peptidoglycan synthesis that mark division sites of daughter cells

“…L. interrogans serovar Copenhageni strain Fiocruz L1-130 (43) was expanded at 30°C with gentle agitation in EMJH complete medium (44). T. denticola ATCC 34505 was propagated at 37°C in supplemented TYGVS medium (45) In Vitro Identification of PBPs. B. burgdorferi B31 MI (100 mL) was cultured to midlog exponential growth, harvested, and washed with, and resuspended in, PBS.…”

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

“…T. denticola strains, including ATCC 33520 and ATCC 35405 as well as isogenic 35405 mutant 35405-⌬TDE0911 (3), were grown as previously described (9) under anoxic conditions in NOS broth or semisolid medium containing 0.8% Noble agar, supplemented with erythromycin (Em; 40 g ml Ϫ1 ) and chloramphenicol (Cm; 10 g ml Ϫ1 ) as appropriate. All growth media were incubated under anoxic conditions for at least 18 h prior to use (10).…”