Laboratory Cultivation and Maintenance of Borrelia miyamotoi

Brandee L. Stone1, Catherine A. Brissette1

1 Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12F.1
DOI:  10.1002/cpmc.12
Online Posting Date:  August, 2016
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Abstract

Borrelia miyamotoi is a relapsing fever tick‐borne pathogen found in Ixodes spp. (hard) ticks. In vitro culturing has proven difficult despite initial reports of cultures maintained in Barbour‐Stoenner‐Kelly‐II (BSK‐II) medium. The ability to culture in vitro opens many avenues for investigating the genetics and physiology of bacterial species. This unit describes methods for the maintenance and cultivation of B. miyamotoi in liquid medium. © 2016 by John Wiley & Sons, Inc.

Keywords: Borrelia miyamotoi; in vitro cultivation; Ixodes; pathogen; spirochete

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cultivation of Borrelia miyamotoi IN MKP‐F
  • Basic Protocol 2: Preparation of frozen B. miyamotoi Stocks
  • Support Protocol 1: Preparation of Complete Modified Kelly‐Pettenkofer Medium (MKP‐F)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Cultivation of Borrelia miyamotoi IN MKP‐F

  Materials
  • MKP‐F, prewarmed to 34°C (see protocol 3Support Protocol)
  • Late‐exponential phase B. miyamotoi culture or frozen stock (see protocol 2)
  • 16‐ml polystyrene round bottom tube with screw cap (e.g., Falcon, cat. no. 352025)
  • 34°C incubator with or without 5% CO 2
  • Dark‐field microscope
  • Petroff‐Hausser counting chamber (optional; see appendix 4A)

Basic Protocol 2: Preparation of frozen B. miyamotoi Stocks

  Materials
  • Mid‐ to late‐exponential phase B. miyamotoi culture in MKP‐F (see protocol 1)
  • 50% (v/v) glycerol, filter‐sterilized or autoclaved
  • 1.8‐ml polyethylene cryogenic tubes (e.g., Thermo Scientific, cat. no.375418)
  • Vortex (optional)

Support Protocol 1: Preparation of Complete Modified Kelly‐Pettenkofer Medium (MKP‐F)

  Materials
  • CMRL‐1066 without L‐glutamine powder (US Biological Life Sciences, cat. no. C5900‐01)
  • Neopeptone
  • Sodium bicarbonate
  • HEPES
  • Glucose
  • Sodium pyruvate
  • Sodium citrate
  • N‐acetyl‐glucosamine
  • Heat‐inactivated rabbit serum
  • 5 N NaOH
  • Heat‐inactivated fetal bovine serum (FBS)
  • 65.57 g/liter bovine serum albumin (BSA), pH 7.6, sterile (see recipe)
  • 7% (w/v) gelatin, pH 7.7, sterile (see recipe)
  • 1‐liter beaker
  • 0.22‐μm filter units
  • Vacuum filtration system
  • Tissue culture hood
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Literature Cited

Literature Cited
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