Growing Oral Biofilms in a Constant Depth Film Fermentor (CDFF)

Jonathan Pratten1

1 University College London, Eastman Dental Institute, London, England
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1B.5
DOI:  10.1002/9780471729259.mc01b05s6
Online Posting Date:  August, 2007
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Abstract

In order to grow organisms in such a manner as to mimic their physiological growth state in vivo, it is often desirable to grow them as biofilms in the laboratory. There are numerous systems available to accomplish this; however, some are more suited to the growth of oral biofilms (dental plaque) than others. The operating parameters of one such model, the constant depth film fermentor (CDFF), are given in this unit. This model is particularly suited to studying the varied biofilms which exist in the oral cavity because environmental factors such as the substratum, nutrient source, and gas flow can be altered. Curr. Protoc. Microbiol. 6:1B.5.1‐1B.5.18. © 2007 by John Wiley & Sons, Inc.

Keywords: constant depth film fermentor; oral biofilms; dental plaque

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Inoculating the Constant Depth Film Fermentor
  • Support Protocol 1: Setup for the Constant Depth Film Fermentor (CDFF)
  • Support Protocol 2: Constant Depth Film Fermentor Pan Assembly
  • Support Protocol 3: Dismantle the Constant Depth Film Fermentor and Prepare for Reuse
  • Support Protocol 4: Preparing the Substratum
  • Support Protocol 5: Inoculating the Constant Depth Film Fermentor
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Inoculating the Constant Depth Film Fermentor

  Materials
  • Bacterial growth medium (appropriate for the organisms being used)
  • 10 ml single‐species inoculum or 2 ml pooled saliva (see protocol 6)
  • 70% (v/v) ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Air filters (e.g., Hepa‐vent; Whatman)
  • Inoculum vessel: 500‐ml Erlenmeyer flask with silicon rubber stopper having two metal tubes going through
  • 3‐ and 5‐mm bore silicone tubing (depending upon bore of stainless steel tubing), 15‐cm and other lengths (depending upon setup)
  • Connectors and clips for silicone tubing (e.g., Sigma‐Aldrich Z12, 654‐3 and Z12, 651‐9)
  • Couplers for connecting silicone tubing of different size bores (e.g., Value Plastics N220/210‐6)
  • Nylon straps (for securing tubing)
  • Foil
  • Magnetic stir bar
  • Incubator, appropriate for inoculum used (see protocol 6)
  • Magnetic stirrer
  • CDFF (sterilized; see protocol 2), with motor and power supply unit
  • Peristaltic pump, precalibrated to desired flow rate
  • 10‐ or 20‐liter Pyrex glass bottles (for effluent collection and medium reservoir; size depending on flow rate used) with silicone rubber stoppers having two metal tubes going through
  • Glass grow‐back traps (Hampshire Glassware)
  • Retort stand and clamp
  • Portable butane burner
  • Sample tool (wrapped in foil and autoclaved; provided with CDFF; see protocol 2)
  • Forceps, sterile (e.g., flame sterilized in 70% v/v ethanol just before use or autoclaved)

Support Protocol 1: Setup for the Constant Depth Film Fermentor (CDFF)

  Materials
  • Spray lubricant (e.g., WD‐40), optional
  • Silicon high‐vacuum grease (VWR)
  • Constant depth film fermentor (CDFF; see Fig. ; contact Professor A. Peters for purchasing information; ) including:
    • Motor
    • Power supply
    • 15 PTFE pans, each with 5 plugs (see protocol 3 for preparation)
    • Recess tools
  • 8‐G × 12 in. stainless steel needle tubing for CDFF inlet ports and vessels (e.g., SLS Scientific SRY700 8H)
  • 3‐ and 5‐mm bore silicone tubing (depending upon bore of stainless steel tubing), 15‐cm and other lengths (depending upon setup)
  • Connectors and clips for metal tubing (e.g., Sigma‐Aldrich Z12, 654‐3 and Z12, 651‐9)
  • Air filters (Hepa‐vent; Whatman)
  • Nylon straps for securing tubing
  • Couplers for connecting pump tubing to 3‐mm tubing (e.g., Value Plastics N220/210‐6)
  • Foil
  • Autoclave tape
  • Incubator with ports at the top and side (for media in and effluent out) and gas tanks, as required (see protocol 6) or 37°C room

Support Protocol 2: Constant Depth Film Fermentor Pan Assembly

  Materials
  • Silicone grease
  • Plugs: PTFE (supplied with CDFF; see protocol 2)
  • PTFE pans (supplied with CDFF; see protocol 2): drilling a 2‐mm small hole in the bottom of the threaded hole recommended
  • Recess tool (supplied with CDFF; see protocol 2)
  • Substratum (e.g., see protocol 5)
  • CDFF turntable (supplied with CDFF; see protocol 2)
  • Flat tool (supplied with CDFF; see protocol 2)

Support Protocol 3: Dismantle the Constant Depth Film Fermentor and Prepare for Reuse

  Materials
  • CDFF with biofilm‐containing pans removed ( protocol 1)
  • (0.01% v/v) Decon acid rinse (Decon Laboratories)
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Figures

Videos

Literature Cited

   Allan, I., Newman, H., and Wilson, M. 2002. Particulate Bioglass reduces the viability of bacterial biofilms formed on its surface in an in vitro model. Clin. Oral Implants Res. 13:53‐58.
   Busscher, H.J., and van der Mei, H.C. 1995. Use of flow chamber devices and image analysis methods to study microbial adhesion. Methods Enzymol. 253:455‐477.
   Coombe, R.A., Tatevossian, A., and Wimpenny, J.W.T. 1982. Bacterial thin films as in vitro models for dental plaque. In Surface and Colloid Phenomena in the Oral Cavity: Methodological Aspects, (R.M. Frank and S.A. Leach, eds.) pp. 239‐249. IRL, London.
   Dalwai, F., Spratt, D.A., and Pratten, J. 2006. Modeling shifts in microbial populations associated with health or disease. App. Environ. Microbiol. 72:3678‐3684.
   Deng, D.M., van Loveren, C., and ten Cate, J.M. 2005. Caries‐preventive agents induce remineralization of dentin in a biofilm model. Caries Res. 39:216‐223.
   Guggenheim, B., Guggenheim, M., Gmür, R., Giertsen, E., and Thurnheer, T. 2004. Application of the Zürich Biofilm Model to problems of cariology. Caries Res. 38:212‐222.
   Kinniment, S.L., Wimpenny, J.W., Adams, D., and Marsh, P.D. 1996. Development of a steady‐state oral microbial biofilm community using the constant‐depth film fermenter. Microbiology 142:631‐638.
   Lamfon, H., Al‐Karaawi, Z., McCullough, M., Porter, S.R., and Pratten, J. 2005. Composition of in vitro denture plaque biofilms and susceptibility to antifungals. FEMS Microbiol. Lett. 242:345‐351.
   Leung, D., Spratt, D.A., Pratten, J., Gulabivala, K., Mordan, N.J., and Young, A.M. 2005. Chlorhexidine‐releasing methacrylate dental composite materials. Biomaterials 26:7145‐7153.
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   Pratten, J., and Wilson, M. 1999. Antimicrobial susceptibility and composition of microcosm dental plaques supplemented with sucrose. Antimicrob. Agents Chemother. 43:1595‐1599.
   Pratten, J., Wills, K., Barnett, P., and Wilson, M. 1998. In vitro studies of the effect of antiseptic‐containing mouthwashes on the formation and viability of Streptococcus sanguis biofilms. J. Appl. Microbiol. 84:1149‐1155.
   Pratten, J., Pasu, M., Jackson, G., Flanagan, A., and Wilson, M. 2003. Modeling oral malodor in a longitudinal study. Arch. Oral Biol. 48:737‐743.
   Ready, D., Pratten, J., Watts, E., Mordan, N., and Wilson, M. 2007. The effect of amalgam exposure on mercury and antibiotic‐resistant bacteria. Int. J. Antimicrob. Agents 30:34‐39.
   Roberts, A.P., Pratten, J., Wilson, M., and Mullany, P. 1999. Transfer of a conjugative transposon, Tn5397 in a model oral biofilm. FEMS Microbiol. Lett. 177:63‐66.
   Roberts, A.P., Cheah, G., Ready, D., Pratten, J., Wilson, M., and Mullany, P. 2001. Transfer of TN916‐like elements in microcosm dental plaques. Antimicrob. Agents Ch. 45:2943‐2946.
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Internet Resources
  http://www.imt.net/∼mitbst/Products.html
  BioSurface Technologies produces several of the biofilm reactors described, including flow cells, annular reactors, and drip flow reactors.
  http://www.tylerresearch.com/instr/biofilm.shtml
  Tyler research produces several biofilm‐generating devices, including high‐pressure and laminar devices as well as chemostats.
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