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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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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Basic Protocol 1: Biofilm Culture on Spinning Discs

  • 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‐
  • 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

  • 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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Literature Cited

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