Laboratory Maintenance of Borrelia burgdorferi

Wolfram R. Zückert1

1 University of Kansas Medical Center, Kansas City, Kansas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12C.1
DOI:  10.1002/9780471729259.mc12c01s4
Online Posting Date:  February, 2007
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This unit describes the propagation of Borrelia burgdorferi as well as other “cultivable” Borrelia species in Barbour‐Stoenner‐Kelly‐II (BSK‐II) medium. It supplies a detailed recipe for BSK‐II, as well as protocols for standard liquid culture, the generation of frozen Borrelia stocks, and the plating of B. burgdorferi cells in solid BSK‐II medium.

Keywords: Borrelia; spirochete; Barbour‐Stoenner‐Kelly‐II medium; broth culture; plating; frozen stocks

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Cultivation and Storage of Borrelia burgdorferi in BSK‐II
  • Basic Protocol 2: Preparation of Frozen Borrelia Stocks
  • Basic Protocol 3: Plating of B. burgdorferi
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Cultivation and Storage of Borrelia burgdorferi in BSK‐II

  • Complete BSK‐II medium (see recipe)
  • Late‐exponential‐phase B. burgdorferi culture or frozen stock (see protocol 2)
  • 8‐ml polystyrene round‐bottom screw cap tubes (e.g., Falcon 352027)
  • 34°C incubator
  • Petroff‐Hausser counting chamber (optional; appendix 4A)

Basic Protocol 2: Preparation of Frozen Borrelia Stocks

  • Late‐exponential‐phase B. burgdorferi BSK‐II culture ( protocol 1)
  • Sterile 100% DMSO or 80% (v/v) glycerol solution
  • 2‐ml polypropylene cryogenic vial (Corning cat no. 430659), sterile

Basic Protocol 3: Plating of B. burgdorferi

  • 2% (top) and 3% (bottom) agarose stock (see recipe)
  • 2× BSK‐II (see recipe), 37° and 45°C
  • B. burgdorferi cell suspension (in BSK‐II or PBS; protocol 1)
  • 45° and 65°C water baths
  • 50‐ml disposable polypropylene tube
  • 10‐cm diameter polystyrene petri dishes
  • 34°C, 5% CO 2 incubator or anaerobic jar with hydrogen‐ and CO 2‐generating packs (e.g., BD GasPacks Plus)
  • 15‐ml disposable polypropylene tubes
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Literature Cited

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Key References
   Barbour, 1984. See above
  Describes the cultivation of Borrelia in BSK‐II medium, including its original recipe.
   Hinnebusch & Barbour, 1992. See above
  Describes the cloning of B. burgdorferi by subsurface plating in BSK‐II agar.
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