Growing and Analyzing Biofilms in Fermenters

Bronwyn E. Ramey1, Matthew R. Parsek1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1B.3
DOI:  10.1002/9780471729259.mc01b03s00
Online Posting Date:  October, 2005
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Abstract

One of the most daunting challenges of biofilm research is comparing experimental results produced by multiple laboratories, each of which uses different techniques to generate, analyze, and interpret biofilm data. The heterogeneity inherent to biofilm communities contributes to the difficulty in obtaining reproducible results between experiments within a single laboratory, but the problem is compounded further by a lack of standardization in techniques. A number of biofilm culture methods are presented in this unit to provide a set of standards for biofilm study. Each model system differs in growth conditions, applied variables, and experimental output, all of which must be carefully considered when designing an experiment and, most critically, during data interpretation. In this unit, two methods of biofilm culture that are known to reliably provide reproducible, statistically clean results in determining the viability and antimicrobial susceptibility of biofilm communities are described. The spinning disc model provides multiple biofilm samples from the same biofilm reactor, significantly reducing data variability. The tube biofilm method, in addition to providing this benefit, can be used for expression analysis, and thus can yield informative data on both macro‐ and micro‐scales. These methods also utilize continuous culture, or chemostat, conditions to maintain a quasi‐steady state.

Keywords: biofilm; chemostat; spinning disc; silicone tubing; tube biofilm; fermenter

     
 
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Table of Contents

  • Basic Protocol 1: Biofilm Culture on Spinning Discs
  • Alternate Protocol 1: Continuous Flow Culture Inoculation of Spinning Discs
  • Basic Protocol 2: Biofilm Culture in Tubes
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Biofilm Culture on Spinning Discs

  Materials
  • Silicone glue
  • Starter culture (mid‐log phase bacterial culture)
  • Rich medium (e.g., LB, TBS, or other; appendix 2C)
  • 95% ethanol
  • Antibiotic solution
  • PBS or KPBS ( appendix 2A)
  • Agar medium plates (for viable counting; see appendix 2C)
  • 1‐liter glass (e.g., Pyrex) beaker with overflow effluent port (see Fig. )
  • Rubber stopper (no. 15; 100‐mm top diameter, 81‐mm bottom diameter) with four predrilled holes
  • Glass tubing, 5‐mm o.d. or sized to fit holes in stopper, of appropriate length
  • Flexible silicone tubing, autoclavable: 12‐mm o.d./6‐mm i.d. and 6‐mm o.d./3‐mm i.d., and of size appropriate to fit through peristaltic pump (Marprene Manifold tubing from Watson‐Marlow, cat. no. 978.0102.000, if Watson‐Marlow peristaltic pump is used)
  • 0.22‐µm filter cartridges
  • Small test tube or vial with bottom removed
  • Medium reservoir and waste bottle: 2‐liter, 4‐liter, or larger‐capacity autoclavable plastic bottles (e.g., Nalgene jugs)
  • Elbow joint (Cole‐Parmer), 0.25‐in. (∼0.625 cm)
  • Peristaltic pump (e.g., Watson Marlow; http://www.watson‐marlow.com)
  • Incubator with shaker
  • Aluminum foil
  • Sandblasted ground‐glass biofilm chips
  • Spinning disc (Fig. )
  • Magnetic stir bar
  • Tube clamps
  • Stir plate
  • 96‐well microtiter plate
  • Hemostats and forceps, sterile
  • 1.5‐ml microcentrifuge tubes
  • Bath sonicator or tissue homogenizer

Alternate Protocol 1: Continuous Flow Culture Inoculation of Spinning Discs

  Materials
  • 30% (v/v) hydrogen peroxide (H 2O 2)
  • 1:8 diluted LB or other rich medium ( appendix 2C)
  • Mid‐log phase bacterial culture
  • 95% ethanol
  • PBS or KPBS ( appendix 2A)
  • Petri plates containing appropriate solid medium
  • 30% H 2O 2
  • Medium reservoir (see protocol 1)
  • Silicone tubing (2‐ to 3‐mm i.d.)
  • Mini tube fittings, barbed (Cole‐Parmer)
  • Multichannel peristaltic pump (e.g. Watson‐Marlow)
  • Marprene Manifold tubing from Watson‐Marlow, cat. no. 978.0102.000, if Watson‐Marlow peristaltic pump is used (or other size silicone tubing compatible with peristaltic pump used)
  • Bubble traps (unit 1.2; optional)
  • Aluminum foil
  • Tube clamps
  • Incubator with shaker
  • Spectrophotometer
  • 1‐ml syringe with a 12‐G needle attached
  • Petri dishes
  • Razor or scalpel blades
  • 1.5‐ml microcentrifuge tubes
  • 50‐ml conical tubes (optional)
  • Bath sonicator (optional)
  • 96‐well microtiter plate
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Figures

Videos

Literature Cited

Literature Cited
   Adair, C.G., Gorman, S.P., Feron, G.M., Byers, L.M., Jones, D.S., Goldsmith, C.E., Moore, J.E., Kerr, J.R., Curran, M.D., Hogg, G., Webb, C.H., McCarthy, G.J., and Milligan, K.R. 1999. Implications of endotracheal tube biofilm for ventilator‐associated pneumonia. Intensive Care Med. 25:1072‐1076.
   Bagge, N., Hentzer, M., Andersen, J.B., Ciofu, O., Givskov, M., and Hoiby, N. 2004a. Dynamics and spatial distribution of beta‐lactamase expression in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 48:1168‐1174.
   Bagge, N., Schuster, M., Hentzer, M., Ciofu, O., Givskov, M., Greenberg, E.P., and Hoiby, N. 2004b. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta‐lactamase and alginate production. Antimicrob. Agents Chemother. 48:1175‐1187.
   Beyenal, H. and Lewandowski, Z. 2002. Internal and external mass transfer in biofilms grown at various flow velocities. Biotechnol. Prog. 18:55‐61.
   Boles, B.R., Thoendel, M., and Singh, P.K. 2004. Self‐generated diversity produces “insurance effects” in biofilm communities. Proc. Natl. Acad. Sci. U.S.A. 101:16630‐16635.
   Characklis, W.G. 1990. Laboratory biofilm reactors. In Biofilms (W.G. Characklis and K.C. Marshall, eds.) pp. 55‐89. John Wiley and Sons, New York.
   Hentzer, M., Teitzel, G.M., Balzer, G.J., Heydorn, A., Molin, S., Givskov, M., and Parsek, M.R. 2001. Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J. Bacteriol. 183:5395‐5401.
   Lawrence, J.R., Swerhone, G.D.W., and Neu, T.R. 2000. A simple rotating annular reactor for replicated biofilm studies. J. Microbiol. Methods 42:215‐224.
   Lee, C.F., Lee, C.J., Chen, C.T., and Huang, C.T. 2004. Delta‐aminolaevulinic acid mediated photodynamic antimicrobial chemotherapy on Pseudomonas aeruginosa planktonic and biofilm cultures. J. Photochem. Photobiol. B Biol. 75:21‐25.
   Leibovitz, A., Dan, M., Zinger, J., Carmeli, Y., Habot, B., and Segal, R. 2003. Pseudomonas aeruginosa and the oropharyngeal ecosystem of tube‐fed patients. Emerg. Infect. Dis. 9:956‐959.
   Leon, O.A., Horn, H., and Hempel, D.C. 2004. Behaviour of biofilm systems under varying hydrodynamic conditions. Water Sci. Technol. 49:345‐351.
   Lin, H.Y., Chen, C.T., and Huang, C.T. 2004. Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells. Appl. Environ. Microbiol. 70:6453‐6458.
   Manz, B., Volke, F., Goll, D., and Horn, H. 2003. Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI). Biotechnol. Bioeng. 84:424‐432.
   Okabe, S., Itoh, T., Satoh, H., and Watanabe, Y. 1999a. Analyses of spatial distributions of sulfate‐reducing bacteria and their activity in aerobic wastewater biofilms. Appl. Environ. Microbiol. 65:5107‐5116.
   Okabe, S., Satoh, H., and Watanabe, Y. 1999b. In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes. Appl. Environ. Microbiol. 65:3182‐3191.
   Perkins, J., Mouzakes, J., Pereira, R., and Manning, S. 2004. Bacterial biofilm presence in pediatric tracheotomy tubes. Arch. Otolaryngol. Head Neck Surg. 130:339‐343.
   Pitts, B., Willse, A., McFeters, G.A., Hamilton, M.A., Zelver, N., and Stewart, P.S. 2001. A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms. J. Appl. Microbiol. 91:110‐117.
   Saint, S. and Chenoweth, C.E. 2003. Biofilms and catheter‐associated urinary tract infections. Infect. Dis. Clin. North. Am. 17:411‐432.
   Singh, P.K., Parsek, M.R., Greenberg, E.P., and Welsh, M.J. 2002. A component of innate immunity prevents bacterial biofilm development. Nature 417:552‐555.
   Smeets, E., Kooman, J., Van Der Sande, F., Stobberingh, E., Frederik, P., Claessens, P., Grave, W., Schot, A., and Leunissen, K. 2003. Prevention of biofilm formation in dialysis water treatment systems. Kidney Int. 63:1574‐1576.
   Sternberg, C., Christensen, B.B., Johansen, T., Toftgaard, N.A., Andersen, J.B., Givskov, M., and Molin, S. 1999. Distribution of bacterial growth activity in flow‐chamber biofilms. Appl. Environ. Microbiol. 65:4108‐4117.
   Teitzel, G.M. and Parsek, M.R. 2003. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol. 69:2313‐2320.
   Walker, J.T., Bradshaw, D.J., Bennett, A.M., Fulford, M.R., Martin, M.V., and Marsh, P.D. 2000. Microbial biofilm formation and contamination of dental‐unit water systems in general dental practice. Appl. Environ. Microbiol. 66:3363‐3367.
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