Burkholderia thailandensis: Growth and Laboratory Maintenance

Erin C. Garcia1, Peggy A. Cotter1

1 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 4C.1
DOI:  10.1002/cpmc.15
Online Posting Date:  August, 2016
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Burkholderia thailandensis is a nonpathogenic Gram‐negative bacterium found in tropical soils. Closely related to several human pathogens, its ease of genetic manipulation, rapid growth in the laboratory, and low virulence make B. thailandensis a commonly used model organism. This unit describes the fundamental protocols for in vitro growth and maintenance of B. thailandensis in the laboratory. © 2016 by John Wiley & Sons, Inc.

Keywords: Burkholderia thailandensis; Burkholderia pseudomallei; growth; model organism

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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of B. thailandensis from a Frozen Stock
  • Basic Protocol 2: Growth of B. thailandensis in Liquid Medium
  • Basic Protocol 3: Preparation of B. thailandensis Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: Growth of B. thailandensis from a Frozen Stock

  • B. thailandensis frozen stock (see protocol 3)
  • Low salt LB agar plate (see recipe), with antibiotics if necessary (see Table 4.1.1)
  • Wooden or plastic applicator stick or inoculating loop, sterile
  • 37°C incubator
Table 4.0.1   MaterialsAntibiotic Concentrations for Use with B. thailandensis

Antibiotic Solvent Stock concentration a Final concentration b
Chloramphenicol Ethanol 20 mg/ml 20 μg/ml
Glyphosate c N/A 50% (w/v) 0.04% (w/v)
Kanamycin Water 125 mg/ml 250 μg/ml
Tetracycline d Ethanol 10 mg/ml 50 μg/ml
Trimethoprim d DMSO 50 mg/ml 50 μg/ml

 aAll stock solutions should be filter‐sterilized (0.22‐μm filter) and stored at −20°C.
 bFinal working concentration in liquid medium or agar plates. Media should be cooled to 55°C before addition of antibiotics.
 cGlyphosate is a nonantibiotic herbicide optimized for use with B. pseudomallei, B. mallei, and B. thailandensis. It is only compatible for use with minimal medium and can be purchased as a 50% (w/v) solution from hardware stores (sold as Roundup; Norris et al., ).
 dLight‐sensitive. Antibiotic stock solution and prepared media should be protected from light.

Basic Protocol 2: Growth of B. thailandensis in Liquid Medium

  • Low salt LB medium (see recipe) or M63 minimal medium (see recipe)
  • Selective antibiotics (see Table 4.1.1)
  • B. thailandensis grown on agar plates (see protocol 1)
  • Capped test tubes, sterile (glass or plastic)
  • Sterile wooden or plastic applicator stick or heat‐sterilized inoculating loop
  • 37°C incubator with shaker or drum roller

Basic Protocol 3: Preparation of B. thailandensis Frozen Stocks

  • 20% (v/v) glycerol LB medium (see recipe)
  • B. thailandensis grown on agar plates (see protocol 1)
  • Screw‐cap cryovials
  • Sterile wooden or plastic applicator stick
  • Vortex
  • −80°C freezer
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Literature Cited

  Anderson, M.S., Garcia, E.C., and Cotter, P.A. 2012. The Burkholderia bcpAIOB genes define unique classes of Two‐Partner secretion and contact dependent growth inhibition systems. PLoS Genet. 8:e1002877. doi: 10.1371/journal.pgen.1002877.
  Anderson, M.S., Garcia, E.C., and Cotter, P.A. 2014. Kind discrimination and competitive exclusion mediated by contact‐dependent growth inhibition systems shape biofilm community structure. PLoS Path. 10:e1004076. doi: 10.1371/journal.ppat.1004076.
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  Brett, P.J., Deshazer, D., and Woods, D.E. 1998. Burkholderia thailandensis sp. nov., a Burkholderia pseudomallei‐like species. Int. J. Sys. Bacteriol. 48:317‐320. doi: 10.1099/00207713‐48‐1‐317.
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Internet Resources
  The Burkholderia Genome Database website contains a comprehensive collection of sequences and annotations for completed genomes within the Burkholderia genus (including B. thailandensis), as well as comparative tools.
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