Laboratory Maintenance and Cultivation of Bacteroides Species

Melissa K. Bacic1, C. Jeffrey Smith1

1 Dept. of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 13C.1
DOI:  10.1002/9780471729259.mc13c01s9
Online Posting Date:  May, 2008
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Abstract

Bacteroides species are Gram‐negative, obligate anaerobic bacteria. They are the predominant indigenous bacterial species in the human intestinal tract, where they play an important role in the normal physiology of the host, but they can also be significant opportunistic pathogens. The fact that these are obligate anaerobes is the prevailing feature that affects the methodology used for their cultivation. Several techniques are described for anaerobic culturing; the anaerobic glove box and anaerobic jars are the two techniques most adaptable to a range of research needs. Straightforward methods are presented for propagation on solid media and in broth cultures, and for the long‐term storage and maintenance of stock cultures. The Bacteroides species are saccharolytic, which is the second feature of their physiology that impacts cultivation methodology. Several flexible media formulations, including a defined minimal media, are provided that allow the researcher to choose the carbon source best suited for his or her work. Curr. Protoc. Microbiol. 9:13C.1.1‐13C.1.21. © 2008 by John Wiley & Sons, Inc.

Keywords: anaerobic culturing; Bacteroides; anaerobic jars; anaerobic chambers

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparing Liquid Cultures of Bacteroides in an Anaerobic Glove Box
  • Basic Protocol 2: Preparing Liquid Cultures of Bacteroides in an Anaerobic Jar
  • Establishing and Maintaining an Anaerobic Environment
  • Support Protocol 1: Sealed Brewer's Jars
  • Support Protocol 2: Vented Anaerobe Jars
  • Support Protocol 3: Anaerobe Chamber
  • Support Protocol 4: Anaerobic Gassing Cannula
  • Basic Protocol 3: Long‐Term Storage of Bacteroides at Room Temperature
  • Support Protocol 5: Chopped Meat Medium
  • Basic Protocol 4: Frozen Glycerol Stock Cultures of Bacteroides
  • Basic Protocol 5: Frozen Skim Milk Stock Cultures of Bacteroides
  • Basic Protocol 6: Determination of the Growth Curve
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparing Liquid Cultures of Bacteroides in an Anaerobic Glove Box

  Materials
  • Supplemented brain heart infusion medium (BHIS; see recipe) or Tryptone yeast extract glucose medium (TYG; see recipe), 5 ml in 13 × 100–mm Hungate screw‐cap tubes
  • Chopped meat stock culture of Bacteroides strain in chopped meat medium (see protocol 7)
  • Glove box ( protocol 5)
  • Neoprene gloves
  • Disposable antiseptic wipes (Allegiance Antiseptic Towelettes)
  • 6″ hemostat
  • 1‐ml disposable sterile pipet

Basic Protocol 2: Preparing Liquid Cultures of Bacteroides in an Anaerobic Jar

  Materials
  • BHIS medium (see recipe) or TYG medium (see recipe), 5 ml in 13 × 100–mm screw cap tubes in rack
  • Bacteroides strain streaked on BHIS or TYG plate (see )
  • Sterile culture tubes
  • Inoculating loop
  • Bunsen burner
  • Anaerobic jar (Support Protocols protocol 31 and protocol 42)
  • 37°C incubator

Support Protocol 1: Sealed Brewer's Jars

  Materials
  • Palladium catalyst (Coy Laboratories; in wire mesh basket attached to jar lid)
  • Cultures (rack of culture tubes or streaked petri plates)
  • 10 ml tap or dH 2O
  • Polycarbonate GasPak jar or equivalent with lid
  • GasPak anaerobic system envelope
  • 10‐ml pipet or syringe
  • GasPak anaerobic indicator
  • 37°C incubator
NOTE: GasPak is a trademark for the Becton, Dickinson and Company (BD). The Oxoid Company also supplies Brewer‐type jars and anaerobic gas‐generating envelope systems that work equally well for the growth of Bacteroides species and the Mitsubishi Gas Chemical‐America Company supplies gas‐generating envelopes.

Support Protocol 2: Vented Anaerobe Jars

  Materials
  • Palladium catalyst (in wire mesh basket attached to jar lid)
  • Cultures (rack of culture tubes or petri plates)
  • Polycarbonate GasPak jar or equivalent with lid
  • GasPak anaerobic indicator
  • Standard vacuum pump with gauge, tubing, and three‐way stopcock (Fisher Scientific)
  • Gas Mixture cylinder (85% N 2, 10% CO 2, and 5% H 2) and regulator
  • 37°C incubator
NOTE: An automatic evacuation/replacement system, Anoxomat (see Internet Resources), that precisely evacuates and fills jars by a microprocessor controlled pumping unit is now available. The standard Anoxomat configuration has one gas connection and one jar connection but options are available for connecting up to 5 jars at one time.

Support Protocol 3: Anaerobe Chamber

  Materials
  • Palladium catalyst, 4 packs (wire screen enclosure containing ∼165 g catalyst)
  • Silica gel desiccant type IV, 2 trays each containing ∼ 2.5 kg desiccant (Sigma Chemical)
  • Bacterial cultures
  • Coy flexible glove box (2 person) with 37°C forced air incubator (Coy Laboratories)
  • Oxygen/hydrogen meter (Coy Laboratories)
  • Nitrogen gas cylinder and regulator (Coy Laboratories)
  • Gas mixture cylinder (80% N 2, 10% CO 2, and 10% H 2) and regulator (Coy Laboratories)

Support Protocol 4: Anaerobic Gassing Cannula

  Materials
  • Tube of chopped meat medium ( protocol 8)
  • Bacteroides strain streaked on BHIS or TYG plate (see )
  • Gas mixture cylinder (90% N 2, 10% CO 2, 10% H 2) and regulator (Coy Laboratories)
  • Gassing cannula with 6″, 16‐G blunted needle bent at 90° (Popper & Sons)
  • Bunsen burner
  • Test tube rack
  • Inoculating loop
  • 6″ hemostat
  • 37°C incubator

Basic Protocol 3: Long‐Term Storage of Bacteroides at Room Temperature

  Materials
  • Chopped meat medium stock cultures ( protocol 8)
  • Bacteroides strain streaked on BHIS or TYG plate (see )
  • 37°C incubator

Support Protocol 5: Chopped Meat Medium

  Materials
  • Extra lean ground beef
  • 1 M NaOH
  • Tryptone
  • Yeast extract
  • KH 2PO 4
  • K 2HPO 4
  • Hemin solution (see recipe)
  • Resazurin solution (see recipe)
  • Cysteine (free base, Sigma)
  • 10% NaHCO 3 (see recipe)
  • 2‐liter beaker
  • Stir bar
  • Bunsen burner
  • Aluminum foil
  • Cheese cloth
  • Graduated cylinder
  • 2‐liter flask
  • Gassing cannula ( protocol 6)
  • Gas cylinder (either a gas mix or 100% N 2) and regulator (Coy Laboratories)
  • Laboratory tape
  • Anaerobe chamber
  • Hungate tubes (Bellco Biotechnology), 16 × 125–mm with septum stoppers and screw caps

Basic Protocol 4: Frozen Glycerol Stock Cultures of Bacteroides

  Materials
  • BHIS medium and plate (see recipe)
  • Glycerol
  • Overnight culture ( protocol 1)
  • 125‐ml bottle
  • Glass rod or rubber spatula
  • 2‐ml Cryogenic vials (Nalgene)
  • Dry ice/ethanol bath

Basic Protocol 5: Frozen Skim Milk Stock Cultures of Bacteroides

  Materials
  • Bacto skim milk (Difco)
  • BHIS plate containing the appropriate antibiotics (see recipe)
  • Overnight culture ( protocol 1)
  • Glass rod or rubber spatula
  • 2 ml Cryogenic vial (Nalgene)
  • Dry ice/ethanol bath

Basic Protocol 6: Determination of the Growth Curve

  Materials
  • 100 ml bottle of defined minimal medium (see recipe)
  • Chopped meat stock culture of Bacteroides in chopped meat medium (see protocol 7)
  • Anaerobic glove box
  • 13 × 100–mm sterile screw cap tubes in rack
  • Antiseptic wipes
  • Hemostat
  • 37°C incubator
  • 125‐ml side arm flask (sterile, aluminum foil cap)
  • 1‐ and 10‐ml pipets, disposable
  • Sterile #3 rubber stopper (in covered beaker)
  • Spectrophotometer
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Figures

Videos

Literature Cited

   Aranki, A., Syed, S.A., Kenney, E.B., and Freter, R. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17: 568‐578.
   Baughn, A.D. and Malamy, M.H. 2004. The strict anaerobe Bacteroides fragilis grows in and benefits from nanomolar concentrations of oxygen. Nature 427: 441‐444.
   Brewer, J.H. and Allgeier, D.L. 1966. Safe self‐contained carbon dioxide‐hydrogen anaerobic system. Appl. Microbiol. 14: 985‐988.
   Breznak, J.A. and Costilow, R.N. 1994. Physiochemical factors in growth. In Methods for General and Molecular Bacteriology (P. Gerhards, R.G.E. Murray, W.A. Wood, and N.R. Krieg, eds.) pp. 137‐154. American Society for Microbiology, Washington, D.C.
   Bryant, M.P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Am. J. Clin. Nutr. 25: 1324‐1328.
   Hecht, D.W. 2006. Anaerobes: Antibiotic resistance, clinical significance, and the role of susceptibility testing. Anaerobe 12: 115‐121.
   Holdeman, L.V., Cato, E.P., and Moore, W.E.C. 1977. Anaerobic Laboratory Manual, 4th ed., Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va.
   Hooper, L.V., Wong, M.H., Thelin, A., Hansson, L., Falk, P.G., and Gordon, J.I. 2001. Molecular analysis of commensal host‐microbial relationships in the intestine. Science 291: 881‐884.
   Mazmanian, S.K., Liu, C.H., Tzianabos, A.O., and Kasper, D.L. 2005. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122: 107‐118.
   Smith, C.J., Rocha, E.R., and Paster, B.J. 2006. The medically important Bacteroides spp. in health and disease. In The Prokaryotes (M. Dworkin, S. Falkow, E. Rosenberg, K.H Schleifer, and E. Stackebrandt eds.) pp. 381‐427. Springer‐Verlag, N.Y.
   Smith, C.J., Tribble, G.D., and Bayley, D. 1998. Genetic elements of the Bacteroides: A moving story. Plasmid 40: 12‐29.
   Turnbaugh, P.J., Ley1, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. 2006. An obesity‐associated gut microbiome with increased capacity for energy harvest. Nature 444: 1027‐1031.
   Varel, V.H. and Bryant, M.P. 1974. Nutritional features of Bacteroides fragilis sp. fragilis. Appl. Microbiol. 28: 251‐257.
Internet Resources
   http://www.anoxomat.com/english/standardpage.php[?ArtikelID=46
  The above three Web sites contain information about anaerobic systems.
   http://www.anaerobesystems.com
  The above three Web sites provide information on commercially available gas packs.
   http://www.bellcoglass.com/product_detail.php?product_id=276
  The above four Web sites provide information on anaerobic chambers.
   http://www.bd.com/ds/productCenter/BblGaspakProductsAndAccessories.asp
   http://www.mgc‐a.com/Pages/anaeropac.html
   http://www.oxoid.com/us/index.asp?mpage=ipsearch&c=US
   http://www.coylab.com/anaerobic_chamber.html
   http://www.shellab.com/bactron.html
   http://www.biotrace.com/content.php?hID=2&nhID=75
   http://www.800ezmicro.com/productImages.asp?mb=01&ez=9
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