Laboratory Maintenance of Borrelia burgdorferi

Abstract

This unit describes the methods for growth and storage of Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. It provides the recipe of the standard culture medium, Barbour-Stoenner-Kelly-II (BSK-II) and details its use in broth and solid culture. Methods for short and long-term storage of Borrelia cells are also provided. CAUTION: B. burgdorferi is considered a Biosafety Level 2 (BSL-2) category organism. Work should be performed in a BSL-2 biosafety hood. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. The use of a biosafety hood also further protects cultures from potential contaminants, which will readily outgrow the spirochetes in the rich culture medium (see Commentary). STRATEGIC PLANNING Borrelia burgdorferi characteristicsBorrelia burgdorferi are slender, highly motile spirochetes 0.2-to 0.5-µm in diameter and up to 30 µm in length. They can be visualized by phase-contrast or dark-field microscopy (UNIT 2A.1). B. burgdorferi stain weakly Gram negative; other stains such as Giemsa or Wright are more appropriate. B. burgdorferi are motile in both low-and high-viscosity media due to periplasmic bundles of seven to eleven flagella attached at each end of the cell cylinder. Intriguingly, these flagella also determine the spirochete's flat-wave cell shape (Motaleb et al., 2000). Opaque white colonies form within or on the surface of solid medium (see Basic Protocol 3). In general, colonies are regularly shaped, up to 3 mm in diameter, with a dense center and a diffuse border. However, colony size and morphology can vary from strain to strain or even within clones of the same isolate (Elias et al., 2002). Borrelia sensitivity to detergentsThe use of disposable labware is recommended since Borrelia cells are sensitive to detergents, residues of which might be present on reusable plastic and glass labware. All reusable labware, such as beakers for medium preparation, should be rinsed with double-distilled water before use. Stock solutionsThe formulation for the standard Borrelia growth medium BSK-II, provided in Reagents and Solutions, follows the recipe described by Barbour in 1984. The medium is made in two steps: an incomplete or basal medium, which can be stored indefinitely at 4 • C or lower, and a complete medium, which is stored up to 2 to 3 weeks at 4 • C. In addition to the basal BSK-II medium, gelatin or agarose stock solutions for liquid or solid BSK-II, respectively, are prepared beforehand and stored indefinitely at room temperature. Rabbit serum is aliquoted in ready-to-use volumes and stored indefinitely at −20 • C or lower. CMRL-1066CMRL-1066 without L-glutamine is available as liquid or powder from a limited number of companies, e.g., Invitrogen (1× solution), or U.S. Biological (powder). The 10× solutions previously sold by different suppliers (e.g., Invitrogen and Sigma) have been discontinued. In powder form, CMRL-1066 should be stored protected from light and humidity (e.g., in a dessicator) at room temperature. Solutions should be stored protected f...

Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface

“…B31-A3 was confirmed to contain all linear and circular plasmids characteristic of strain B31, with the exception of lp5, using a set of multiplex-PCRcompatible oligonucleotide primers (111) (data not shown). B. burgdorferi B31-e2 and B31-A3 were maintained in BSK-II complete medium at 34°C containing 300 g/ml kanamycin, as necessary (141,142). Recovery of B. burgdorferi transformants was performed using semisolid BSK-II medium as described previously (142), with plates incubated at 34°C in a humidified 5% CO 2 atmosphere until the development of colonies.…”

“…Clones that expressed epitope-tagged protein were then expanded in BSK-II medium and used in subsequent assays. Verified B. burgdorferi clones were stored in BSK-II medium containing 10% (vol/vol) dimethyl sulfoxide (DMSO) at Ϫ80°C (142).…”

Evaluation of RevA, a Fibronectin-Binding Protein of Borrelia burgdorferi, as a Potential Vaccine Candidate for Lyme Disease

“…B. burgdorferi strain B31 MI-16 is an infectious clone of the sequenced type strain (27,28) which contains all parental plasmids (29). Bacteria were grown at 34°C to cell densities of approximately 1 ϫ 10 7 bacteria/ml in modified Barbour-Stoenner-Kelly (BSK-II) medium supplemented with 6% rabbit serum (30). Total DNA (genomic and plasmids) was isolated using a DNeasy blood and tissue kit (Qiagen, Valencia, CA).…”

Borrelia burgdorferi RevA Significantly Affects Pathogenicity and Host Response in the Mouse Model of Lyme Disease

“…B. burgdorferi strain B31-A3 is an infectious clone of the sequenced type strain (40,41) that contains all parental plasmids except cp9 (42). Bacteria were grown at 34°C to densities of approximately 1 ϫ 10 7 cells/ml in modified Barbour-Stoenner-Kelly (BSK-II) medium supplemented with 6% rabbit serum (43). Total DNA (genomic and plasmid DNA) was isolated using a DNeasy blood and tissue kit (Qiagen, Valencia, CA).…”