Isolation and Maintenance of Brachyspira Species

Thad B. Stanton1

1 Food Safety and Enteric Pathogen Research Unit, National Animal Disease Center, USDA‐ARS, Ames, Iowa
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12D.1
DOI:  10.1002/9780471729259.mc12d01s22
Online Posting Date:  August, 2011
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Abstract

Brachyspira species are anaerobic spirochetes inhabiting intestinal tracts of animals and humans. Several species cause transmissible intestinal diseases of swine and birds. This unit describes methods for the isolation of Brachyspira from fecal samples, cultivation on liquid and solid media, and long term and short term preservation of Brachyspira species. Curr. Protoc. Microbiol. 22:12D.1.1‐12D.1.14. © 2011 by John Wiley & Sons, Inc.

Keywords: Brachyspira hyodysenteriae; spirochete; anaerobic; pathogen; intestine; swine; poultry; human

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Culturing Brachyspira Species in BHIS Broth
  • Alternate Protocol 1: Large‐Scale Culture of Brachyspira
  • Support Protocol 1: Preparing BHIS Culture Broth
  • Basic Protocol 2: Isolation of Brachyspira Species from Feces and Other Samples Using TSB Agar Plates
  • Support Protocol 2: Estimation of Brachyspire Viable Cell Densities
  • Support Protocol 3: Preparation of Selective Trypticase Soy Blood (TSB) Agar Plates for Growth of Brachyspira Species
  • Basic Protocol 3: Short‐Term Brachyspira Stock Culture
  • Basic Protocol 4: Long‐Term Brachyspira Stock Culture
  • Basic Protocol 5: Shipping Stock Cultures
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culturing Brachyspira Species in BHIS Broth

  Materials
  • BHI basal broth in 18 × 142−mm tubes (see protocol 3)
  • Calf or fetal calf serum (heat‐treated at 56°C for 30 min)
  • Brachyspire broth culture or isolated colony from TSB ± antibiotic plate
  • Sterile magnetic, Teflon‐coated stir bar “fleas” (∼3 × 10 mm)
  • Spectrophotometer accommodating 18‐mm tubes (e.g., Bausch & Lomb Spectronic 20 or Spectronic 70; for measuring culture absorbance, optical density, at 620 nm)
  • Phase‐contrast microscope (for counting brachyspire cells, detecting contaminant bacteria, and observing brachyspire cell morphology; Fig. )
  • Petroff‐Hausser counting chamber (hemocytometer)

Alternate Protocol 1: Large‐Scale Culture of Brachyspira

  Materials
  • Dehydrated brain‐heart infusion broth (BHI, Difco Laboratories)
  • 0.1% (w/v) resazurin in distilled water (a redox indicator dye; Sigma‐Aldrich)
  • 0.01 N NaOH
  • 3.3% (w/v) L‐cysteine‐HCl in distilled water, pH 7.0 (a chemical reducing agent)
  • Sterile, heat‐treated (56°C, 30 min) fetal calf or calf serum
  • 250‐ml Florence flask with stopper
  • 18 × 142−mm glass tubes (Bellco Glass)
  • Neoprene (green) or butyl (black) no. 1 rubber stoppers
  • Plastic, stretchable 1‐in yellow tape (3M Scotch Brand #47)
  • Anaerobic chamber (see Strategic Planning) inflated with 85:10:5 (v/v/v) N 2/H 2/CO 2
  • Autoclave with aluminum tube press and autoclavable rubber or foam pads
NOTE: To remove traces of oil and solvent, new Neoprene or butyl rubber stoppers should be autoclaved in a laboratory detergent solution (e.g., 1% Alconox) and rinsed thoroughly before use.

Support Protocol 1: Preparing BHIS Culture Broth

  Materials
  • Swab sample containing Brachyspira cells
  • TSB plates (see protocol 6)
  • Sterile inoculating loops
  • Phase‐contrast microscope

Basic Protocol 2: Isolation of Brachyspira Species from Feces and Other Samples Using TSB Agar Plates

  Materials
  • Intestinal samples or Brachyspira cultures
  • TSB (containing glucose) agar plates
  • Coy anaerobic chamber
  • Sterile plastic spreaders (Lazy‐L spreader, Sigma‐Aldrich)
  • Inoculating turntable (VWR)

Support Protocol 2: Estimation of Brachyspire Viable Cell Densities

  Materials
  • Trypticase‐soy agar containing glucose (BBL, Becton Dickinson)
  • Defibrinated bovine blood, prewarmed to 45°C
  • Antibiotics of choice, filter sterilized
  • 45°C water bath
  • 100‐mm Petri plates

Support Protocol 3: Preparation of Selective Trypticase Soy Blood (TSB) Agar Plates for Growth of Brachyspira Species

  Materials
  • Brachyspira broth cultures
  • BHIS broth
  • Dimethyl sulfoxide (DMSO)
  • Nunc cryovials
  • Coy anaerobic chamber
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Figures

  •   FigureFigure 12.D0.1 (A) B. alvinipulli C1T cells. (B) B. hyodysenteriae B78T cells. Phase‐contrast photomicrographs of wet‐mount preparations. Bars, 10 µm (Stanton et al., ).
  •   FigureFigure 12.D0.2 Components of a gas delivery system for Brachyspira broth cultures. Pure nitrogen gas from a gas cylinder flows through butyl rubber tubing into the bottom of a heated glass column containing copper filings (A). Residual oxygen in the nitrogen gas is removed by reacting with the heated copper metal. The nitrogen gas is carried from the top of the column through rubber tubing to individual sterile glass syringes (B). The 3‐ml glass gassing syringes are packed loosely with cotton and fitted with bent 18‐G Luer‐Lok needles that can be flamed to ensure sterility. The syringes with needles can be removed from the gas lines and autoclaved. Prior to autoclaving, anaerobic broth medium is dispensed into individual glass tubes flushed with oxygen‐free nitrogen gas. Stoppered tubes are placed in an aluminum press (C) and autoclaved (15 to 20 min, fast exhaust). Based on methods described by Hungate (). Further details of a gassing station for preparing broth media and culturing anaerobes can be found in APPENDIX.
  •   FigureFigure 12.D0.3 Small‐ and large‐volume broth cultures of Brachyspira species. (A) Multiple 7‐ml B. hyodysenteriae broth cultures can be continually mixed on a modified magnetic stirrer. The top of the stirring platform has a Plexiglass rack supporting up to 14 culture tubes. An unmodified magnetic stirrer can be used by supporting the tubes in a laboratory beaker on top of the stirring platform. (B) Freshly inoculated 500‐ml Brachyspira sp. culture in a 2‐L flat‐bottom Florence flask. To release metabolic gasses (H2, CO2), the rubber stopper sealing the flask is fitted with a Bunsen valve (a rubber dropper bulb with a lengthwise 1‐cm slit made with a razor blade, attached to a disposable sterile 0.45‐µm Millipore filter unit, inserted into a sterile 1.5‐in disposable 20‐G needle passing through the rubber stopper). Stirring was stopped for the purpose of photography.

Literature Cited

Literature Cited
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