Laboratory Maintenance of Brucella abortus

Yao‐Hui Sun1, Andreas B. den Hartigh1, Renée M. Tsolis1, Thomas A. Ficht2

1 University of California at Davis, Davis, California, 2 Texas A&M University, College Station, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3B.1
DOI:  10.1002/9780471729259.mc03b01s00
Online Posting Date:  October, 2005
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Abstract

This unit provides protocols for growth of Brucella abortus on solid or in liquid media and for long‐term storage of laboratory stocks. Two issues affecting the culture and storage of isolates of this slow‐growing bacterium are emphasized: contamination of cultures and outgrowth of attenuated variants lacking a complete lipopolysaccharide. Laboratories planning to work with B. abortus should be aware that Biosafety Level 3 facilities are required. Furthermore, this organism is classified in the U.S. as a Select Agent, which restricts its use to laboratories registered with the U.S. government's Select Agent programs.

Keywords: Brucella; brucellosis; biosafety; culture; laboratory stocks; strain collection; growth phase; bacteriological media; select agent

     
 
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Table of Contents

  • Basic Protocol 1: Growth of B. abortus in Liquid Culture
  • Basic Protocol 2: Maintenance of Laboratory Stocks of Brucella by Freezing
  • Alternate Protocol 1: Preservation of Brucella Stocks by Lyophilization
  • Basic Protocol 3: Detection of Rough Brucella Colonies by Crystal Violet Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Growth of B. abortus in Liquid Culture

  Materials
  • B. abortus strain
  • Agar plates:
    • Tryptic soy agar (TSA) plates (see recipe)
    • TSA blood agar plates (see recipe)
    • Brucella agar plates (e.g., BBL, Oxoid, EMD), prepare according to manufacturer's instructions
    • Potato infusion agar plates (PIA; see recipe)
  • Tryptic soy broth (TSB; see recipe)
  • 70% (v/v) ethanol in a spray bottle
  • 10% (v/v) bleach or 1% (w/v) Virkon S (Dupont) in a spray bottle
  • Peptone saline maintenance medium (see recipe)
  • 15‐ and 50‐ml disposable polypropylene centrifuge tubes (e.g., Falcon or Sarstedt), sterile
  • 1‐µl inoculating loops
  • 10‐mm, screw‐topped, glass tubes
  • Closed container (e.g., large plastic box with lid)
  • Spectrophotometer (e.g., Spectronic 20)
  • 1‐liter polypropylene straight‐sided jar with screw top (e.g., Nalgene, 2118‐0032)
  • Styrofoam tube racks from 50‐ml conical tube package
  • Shaking incubator
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Maintenance of Laboratory Stocks of Brucella by Freezing

  Materials
  • B. abortus strain to be stored
  • TSA blood agar plates (see recipe)
  • TSB/50% (v/v) glycerol (see recipe)
  • 15‐ml disposable polypropylene centrifuge tubes, sterile
  • 1.5‐ml freezer vials

Alternate Protocol 1: Preservation of Brucella Stocks by Lyophilization

  • Peptone saline maintenance medium (see recipe)
  • Bleach
  • Stabilizing medium (see recipe)
  • 50‐ml disposable, polypropylene centrifuge tubes, sterile
  • Centrifuge with an aerosol‐proof rotor
  • Sterile 10‐dram glass vials
  • Sterile rubber stoppers (autoclaved separately from vials)
  • Tray
  • Lyophilizer equipped with an internal stoppering device
  • Metal crimp closures

Basic Protocol 3: Detection of Rough Brucella Colonies by Crystal Violet Staining

  Materials
  • B. abortus growing on solid medium
  • Crystal violet stock solution (see recipe)
  • Agar plate containing well‐spaced individual colonies of Brucella
  • 10% (v/v) bleach solution in a beaker
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Figures

Videos

Literature Cited

   Alton, G.G., Jones, L.M., and Pietz, D.E. 1975. Laboratory Techniques in Brucellosis, 2nd ed. World Health Organization, Geneva.
   Fiori, P.L., Mastrandrea, S., Rappelli, P., and Cappuccinelli, P. 2000. Brucella abortus infection acquired in microbiology laboratories. J. Clin. Microbiol. 38:2005‐2006.
   Moreno, E., Clockaert, A., and Moriyon, I. 2002. Brucella evolution and taxonomy. Vet. Microbiol. 90:209‐227.
Key References
   Alton et al., 1975. See above.
  This manual contains useful information on culturing different Brucella species. It is out of print, but may be obtained through library collections.
   http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm
  The CDC's Biosafety in Microbiological and Biomedical Laboratories (BMBL), should be consulted to obtain information on precautions and regulations for work at biosafety level 3.
Internet Resources
   http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm
  This site contains the CDC's Biosafety in Microbiological and Biomedical Laboratories (BMBL).
   http://serine.urbm.fundp.ac.be/∼seqbruce/GENOMES
  The University of Namur (Belgium) Bioinformatics group's site contains information on the B. melitensis genome.
   http://bbp.vbi.vt.edu/index.php
  The Brucella Bioinformatics Portal at Virginia Tech contains updates, links to the sequenced Brucella gemomes and to sites of interest to researchers on brucellosis.
   http://www.moag.gov.il/brunet
  The BruNet contains epidemiological information about brucellosis and its control
   http://www.cdc.gov/od/sap
  The CDC site contains information about its Select‐Agent Program
   http://www.aphis.usda.gov/vs/cvb/selectagent.htm
  This site contains information about the USDA's Select Agent Registration Program
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