Growing and Analyzing Static Biofilms

Judith H. Merritt1, Daniel E. Kadouri2, George A. O'Toole3

1 Glycobia Inc., Ithaca, New York, 2 University of Medicine and Dentistry of New Jersey, Newark, New Jersey, 3 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1B.1
DOI:  10.1002/9780471729259.mc01b01s22
Online Posting Date:  August, 2011
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Abstract

Many bacteria can exist as surface‐attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small‐ or relatively large‐scale studies. Unlike assays involving continuous‐flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms. Curr. Protoc. Microbiol. 22:1B.1.1‐1B.1.18. © 2011 by John Wiley & Sons, Inc.

Keywords: biofilm; bacterial adhesion; microscopy; static biofilm assay; attachment; microcolony; flow cell

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Microtiter Plate Biofilm Assay
  • Alternate Protocol 1: Direct Enumeration of Bacteria in Biofilms
  • Basic Protocol 2: Air‐Liquid Interface Assay
  • Alternate Protocol 2: Air‐Liquid Interface Coverslip Assay
  • Basic Protocol 3: Colony Biofilm Assay
  • Basic Protocol 4: Kadouri Drip‐Fed Biofilm Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microtiter Plate Biofilm Assay

  Materials
  • Bacterial strains of interest
  • Appropriate media for bacteria under study ( appendix 2C)
  • 70% ethanol
  • 0.1% (w/v) crystal violet in water
  • Solvent (e.g., 30% v/v acetic acid in water; see Table 1.1.1 for other options) for solubilizing dye and biofilm biomass
  • 96‐well microtiter plates, not tissue culture treated (Becton Dickinson, cat. no. 353911) with lids (Becton Dickinson, cat. no. 353913)
  • 96‐prong inoculating manifold, sterile (DanKar Scientific)
  • Small trays (e.g., large pipet tip boxes) sufficient in size to hold 96‐well microtiter plates
  • Optically clear flat‐bottom 96‐well plates, nonsterile
  • Plate reader or spectrophotometer

Alternate Protocol 1: Direct Enumeration of Bacteria in Biofilms

  • PBS ( appendix 2A), sterile
  • Agar plates of appropriate medium
  • Multichannel pipettor
  • 8‐ml plastic tubes with caps
  • Sonicator (e.g., Sonics and Materials VC‐505)
  • Additional reagents and equipment for counting viable cells (Phelan, )

Basic Protocol 2: Air‐Liquid Interface Assay

  Materials
  • Bacterial strains of interest
  • Appropriate media for bacteria under study ( appendix 2C)
  • Flat‐bottom 24‐well plate, sterile, with lid
  • Large paper clips or microcentrifuge tube rack and lab tape
  • Inverted microscope

Alternate Protocol 2: Air‐Liquid Interface Coverslip Assay

  • 0.1% (w/v) crystal violet in water
  • Flat‐bottom multiwell (e.g., 12‐well) plates, sterile, with lids
  • Glass or plastic coverslips (e.g., Fisher brand unbreakable 22‐mm2 coverslips; Fisher Scientific, cat. no. 12‐547)
  • Conventional, upright microscope

Basic Protocol 3: Colony Biofilm Assay

  Materials
  • Bacterial strain of interest
  • Appropriate liquid medium for bacterial strain under study ( appendix 2C)
  • Appropriate agar medium with and without antibiotic (or other) supplementation
  • PBS ( appendix 2A), sterile
  • Forceps, sterilized (e.g., autoclaved or flame‐sterilized in 70% v/v ethanol)
  • Poretics 25‐mm‐diameter black polycarbonate membranes with pore size of 0.22 µm (GE Osmonics, cat. no. K02BP02500)
  • 100‐mm‐diameter petri dishes, sterile
  • UV light source (e.g., UVP 8‐W multiple‐ray laboratory lamp)
  • 15‐ml tubes, sterile, with tightly fitting lids
  • Vortex mixer

Basic Protocol 4: Kadouri Drip‐Fed Biofilm Assay

  Materials
  • Silicone sealant
  • Sodium hypochlorite
  • 70% (v/v) ethanol
  • Bacterial strain of interest
  • Appropriate liquid medium for bacterial strain under study ( appendix 2C)
  • 20‐G needles, sterile (Becton Dickinson, cat. no. 305175)
  • Bunsen burner
  • Flat‐bottom 6‐well plates, sterile, with lids
  • 1/16 × 1/16–in. (∼0.16 × 0.16–cm) straight and 90°‐elbow, barbed, polypropylene fittings (such as those included in Cole‐Parmer, cat. no. 6365‐90)
  • 0.8‐ to 1.6‐mm‐i.d. silicone tubing (e.g., Watson‐Marlow, cat. no. 913.A008.016 or 913.A016.016)
  • 2‐liter flasks
  • Two‐hole rubber stopper (no. 8) equipped with a long glass tube (i.e., one that can reach almost to the bottom of a 2‐liter flask) and a short glass tube (for gas exchange)
  • Peristaltic pump (e.g., Watson‐Marlow PumpPro) equipped with Marprene manifold tubing (inner diameter, 0.8 mm; e.g., Watson‐Marlowe, cat. no. 978.0165.000)
  • Inverted microscope (unit 2.1)
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Figures

Videos

Literature Cited

   Anderl, J.N., Franklin, M.J., and Stewart, P.S. 2000. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 44:1818‐1824.
   Caiazza, N.C. and O'Toole, G.O. 2004. SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14. J. Bacteriol. 186:4476‐4485.
   Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M., and Beachey, E.H. 1985. Adherence of coagulase‐negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol. 22:996‐1006.
   Danhorn, R., Hentzer, M., Givskov, M., Parsek, M.R., and Fuqua, C. 2004. Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR‐PhoB regulatory system. J. Bacteriol. 186:4492‐4501.
   Hunt, S.M., Werner, E.M., Huang, B., Hamilton, M.A., and Stewart, P.S. 2004. Hypothesis for the role of nutrient starvation in biofilm detachment. Appl. Environ. Microbiol. 70:7418‐7425.
   Junker, L.M. and Clardy, J. 2007. High‐throughput screens for small‐molecule inhibitors of Pseudomonas aeruginosa biofilm development. Antimicrob. Agents Chemother. 51:3582‐3590.
   O'Toole, G.A., Pratt, L.A., Watnick, P.I., Newman, D.K., Weaver, V.B., and Kolter, R. 1999. Genetic approaches to study of biofilms. Methods Enzymol. 310:91‐109.
   Mack, D., Nedelmann, M., Krokotsch, A., Schwarzkopf, A., Heesemann, J., and Laufs, R. 1994. Characterization of transposon mutants of biofilm‐producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: Genetic identification of a hexosamine‐containing polysaccharide intracellular adhesin. Infect. Immun. 62:3244‐3253.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Sawyer, L.K. and Hermanowicz, S.W. 1998. Detachment of biofilm bacteria due to variations in nutrient supply. Water Sci. Technol. 37:211‐214.
   Stepanovic, S., Vukovic, D., Jezek, P., Pavlovic, M., and Svabic‐Vlahovic, M. 2001. Influence of dynamic conditions on biofilm formation by Staphylococci. Eur. J. Clin. Microbiol. Infect. Dis. 20:502‐504.
   Walters, M.C. 3rd, Roe, F., Bugnicourt, A., Franklin, M.J., and Stewart, P.S. 2003. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob. Agents Chemother. 47:317‐323.
   Zegans, M.E., Wagner, J.C., Cady, K.C., Murphy, D.M., Hammond, J.H., and O'Toole, G.A. 2009. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J. Bacteriol. 191:210–219.
Internet Resources
   http://www.jove.com/Details.stp?ID=2437
  Microtiter dish biofilm formation assay video by author (George O'Toole). Subscription required.
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