Growing and Analyzing Biofilms in Flow Chambers

Tim Tolker‐Nielsen1, Claus Sternberg2

1 University of Copenhagen, Copenhagen, Denmark, 2 Technical University of Denmark, Lyngby, Denmark
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1B.2
DOI:  10.1002/9780471729259.mc01b02s21
Online Posting Date:  May, 2011
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This unit describes the setup of flow chamber systems for the study of microbial biofilms, and methods for the analysis of structural biofilm formation. Use of flow chambers allows direct microscopic investigation of biofilm formation. The biofilms in flow chambers develop under hydrodynamic conditions, and the environment can be carefully controlled and easily changed. The protocols in this unit include construction of the flow chamber and the bubble trap, assembly and sterilization of the flow chamber system, inoculation of the flow chambers, running of the system, image capture and analysis, and disassembly and cleaning of the system. In addition, embedding and fluorescent in situ hybridization of flow chamber–grown biofilms are addressed. Curr. Protoc. Microbiol. 21:1B.2.1‐1B.2.17. © 2011 by John Wiley & Sons, Inc.

Keywords: biofilm; flow chamber; confocal laser scanning microscopy; bubble trap; fluorescent in situ hybridization; Comstat

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Table of Contents

  • Introduction
  • Basic Protocol 1: Use of Flow Chambers for Growth and Analysis of Biofilms
  • Support Protocol 1: Construction of a Flow Chamber System
  • Support Protocol 2: Construction of the Bubble Trap
  • Support Protocol 3: Embedding and Fluorescent In Situ Hybridization of a Flow Chamber–Grown Biofilm
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Use of Flow Chambers for Growth and Analysis of Biofilms

  • Inoculum, e.g., fresh overnight culture of desired microorganisms
  • 70% and 96% (v/v) ethanol
  • Medium (e.g., see appendix 2C)
  • Silicone glue (3M Super Silicone Sealant Clear)
  • Flow chamber system (DTU Systems Biology, Technical University of Denmark, or see protocol 2)
  • Syringes with needles (e.g., Terumo LU‐100, 27‐G (0.4 × 12 mm), 0.5 ml)
  • Clamps
  • Microscope (e.g., Zeiss LSM710; Chapter 2)
  • Scalpels
  • Computer software:
    • Imaris (Bitplane;
    • COMSTAT version 2 (DTU‐Systems Biology, Technical University of Denmark;
    • Java runtime environment (needed for Comstat v.2;

Support Protocol 1: Construction of a Flow Chamber System

  • Silicone glue (3M Super Silicone Sealant Clear)
  • Medium appropriate for organisms and type of biofilm being grown (see appendix 2C)
  • 70% and 96% (v/v) ethanol
  • 0.5% (w/v) sodium hypochlorite
  • H 2O, sterile
  • 1% hydrogen peroxide
  • Polycarbonate sheet plastic, 6‐ and 35‐mm thick
  • CNC tooling machine, or a drilling machine mounted on an upright stand and equipped with a milling drill‐tool (3 mm)
  • Substratum: 50 × 24–mm glass coverslips or other appropriate material
  • 2‐ml syringe (Terumo)
  • Tubing:
    • Silicone, 3‐mm outer diameter, 1‐mm inner diameter
    • Silicone, 4‐mm outer diameter, 2‐mm inner diameter
    • Silicone, 7‐mm outer diameter, 5‐mm inner diameter
    • Marprene, 3‐mm outer diameter, 1‐mm inner diameter (Watson‐Marlow)
  • Peristaltic pump (Watson‐Marlow, cat. no. 205S)
  • Medium bottles (Schott or BIO 101)
  • Rubber stoppers (to fit medium bottles) with holes for glass tubes
  • Glass tubes, 6‐mm outer diameter, 3‐mm inner diameter
  • Air filter, autoclavable (optional)
  • Clear polypropylene plastic connectors and T‐connectors (Cole Parmer, cat. no. E‐06365), 1/8 in. (3.175 mm) and 1/16 in. (1.588 mm)
  • Bubble traps (DTU Systems Biology, Technical University of Denmark, or see protocol 3)
  • Microscope (unit 2.1)
  • Rolling cart for flow systems and pumps (optional)
  • Waste container

Support Protocol 2: Construction of the Bubble Trap

  • 10‐mm‐thick polycarbonate sheet plastic (simple trap) or 35 × 80 × 45–mm polycarbonate block (advanced trap)
  • Drilling machine mounted on an upright stand and equipped with a 3‐mm milling drill‐tool and an 8‐mm pointed drill (simple trap) or CNC tooling machine (advanced trap)
  • 2‐ml syringes with inner diameter of 8.5 mm (simple trap) or 5‐ml syringes with inner diameter of 12.5 mm (advanced trap)
  • 9 × 2–mm rubber gaskets (advanced trap; M‐seals, 221355; http://www.m‐
  • Silicone glue (3M Super Silicone Sealant Clear)
  • Silicone tubing, 3‐mm outer diameter (simple trap)
  • Stoppers (e.g., EFD; PN 7017976;‐us/divisions/efd) or use the leftover needle protective cover from the inoculation needles used for inoculating the flow chambers (see above)

Support Protocol 3: Embedding and Fluorescent In Situ Hybridization of a Flow Chamber–Grown Biofilm

  • Biofilms adhered to the substratum of flow chamber system (see protocol 1)
  • Paraformaldehyde fixative (see recipe)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 20% (w/v) acrylamide monomer (200:1 acrylamide:bisacrylamide)
  • N,N,N,N‐tetramethylethylenediamine (TEMED)
  • 1% (w/v) ammonium persulfate (APS) activator, freshly prepared
  • Fluorescently labeled oligonucleotide probes (e.g., FITC, Cy3, Cy5; e.g., Eurogentec)
  • FISH prehybridization buffer (see recipe)
  • FISH hybridization buffer (prehybridization buffer with an appropriate concentration of formamide; see Stahl and Amann, )
  • FISH washing buffer (see recipe)
  • Anti‐fade agent (e.g., SlowFade, Molecular Probes)
  • Clamps
  • 50‐ml beaker
  • 2‐ml microcentrifuge tubes
  • Humidified storage container (e.g., petri dish with wetted paper or cotton)
  • Microscope slides with 8‐ to 10‐mm diameter wells for FISH (Novakemi AB)
  • Incubation chamber (50‐ml polypropylene centrifuge tube with FISH hybridization buffer–saturated paper toweling)
  • Microscope slides and coverslips
  • Rubber gaskets, 0.7‐mm thick
  • Additional reagents and equipment for calibrating oligonucleotide probes (e.g., Stahl and Amann, )
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Literature Cited

   Caldwell, D.E. and Lawrence, J.R. 1986. Growth kinetics of Pseudomonas fluorescens microcolonies within the hydrodynamic boundary layers of surface microenvironments. Microb. Ecol. 12:299‐312.
   Hentzer, M., Riedel, K., Rasmussen, T.B., Heydorn, A., Andersen, J.B., Parsek, M.R., Rice, S.A., Eberl, L., Molin, S., Hoiby, N., Kjelleberg, S., and Givskov, M. 2002. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148:87‐102.
   Heydorn, A., Ersboll, B.K., Hentzer, M., Parsek, M.R., Givskov, M., and Molin, S. 2000a. Experimental reproducibility in flow‐chamber biofilms. Microbiology 146:2409‐2415.
   Heydorn, A., Nielsen, A.T., Hentzer, M., Sternberg, C., Givskov, M., Ersbøll, B.K., and Molin, S. 2000b. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146:2395‐2407.
   Klausen, M., Aaes‐Jorgensen, A., Molin, S., and Tolker‐Nielsen, T. 2003. Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol. Microbiol. 50:61‐68.
   Korber, D.R., Lawrence, J.R., Hendry, M.J., and Caldwell, D.E. 1992. Programs for determining statistically representative areas of microbial biofilms. Binary 4:204‐210.
   Kuehn, M., Hausner, M., Bungartz, H.J., Wagner, M., Wilderer, P.A., and Wuertz, S. 1998. Automated confocal laser scanning microscopy and semiautomated image processing for analysis of biofilms. Appl. Environ. Microbiol. 64:4115‐4127.
   Lawrence, J.R., Korber, D.R., Hoyle, B.D., Costerton, J.W., and Caldwell, D.E. 1991. Optical sectioning of microbial biofilms. J. Bacteriol. 173:6558‐6567.
   McCoy, W.F., Bryers, J.D., Robbins, J., and Costerton, J.W. 1981. Observations of fouling biofilm formation. Can. J. Microbiol. 27:910‐917.
   Neu, T., Swerhone, G.D., and Lawrence, J.R. 2001. Assessment of lectin‐binding analysis for in situ detection of glycoconjugates in biofilm systems. Microbiology 147:299‐313.
   Nickel, J.C., Ruseska, I., Wrigth, J.B., and Costerton, J.W. 1985. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob. Agents Chemother. 27:619‐624.
   Nielsen, A.T., Tolker‐Nielsen, T., Barken, K.B., and Molin, S. 2000. Role of commensal relationships on the spatial structure of a surface‐attached microbial consortium. Environ. Microbiol. 2:59‐68.
   Pamp, S.J., Sternberg, C., and Tolker‐Nielsen, T. 2009. Insight into the microbial multicellular lifestyle via flow‐cell technology and confocal microscopy. Cytometry A 75:90‐103.
   Sauer, K., Camper, A.K., Ehrlich, G.D., Costerton, J.W., and Davies, D.G. 2002. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184:1140‐1154.
   Stahl, D.A. and Amann, R.I. 1991. Development and application of nucleic acid probes. In Nucleic Acid Techniques in Bacterial Systematics (E. Stackebrandt and M. Goodfellow, eds.) pp. 205‐248. John Wiley & Sons, New York.
   Stoodley, P., Lewandowski, Z., Boyle, J.D., and Lappin‐Scott, H.M. 1999. The formation of migratory ripples in a mixed species bacterial biofilm growing in turbulent flow. Environ. Microbiol. 1:447‐455.
   Wolfaardt, G.M., Lawrence, J.R., Robarts, R.D., Caldwell, S.J., and Caldwell, D.E. 1994. Multicellular organization in a degradative biofilm community. Appl. Environ. Microbiol. 60:434‐446.
   Xavier, J.B., White, D.C., and Almeida, J.S. 2003. Automated biofilm morphology quantification from confocal laser scanning microscopy imaging. Water Sci. Technol. 47:31‐37.
   Yang, X., Beyenal, H., Harkin, G., and Lewandowski, Z. 2000. Quantifying biofilm structure using image‐analysis. J. Microbiol. Methods 39:109‐119.
Key References
   Wolfaardt et al., 1994. See above.
  The first paper reporting the use of the flow chamber, which is described here and widely used.
   Lawrence et al., 1991. See above.
  The first paper reporting the use of CLSM in biofilm research.
   Heydorn et al., 2000b. See above.
  A paper describing development and application of the COMSTAT image‐analysis program.
Internet Resources
  Web site for Comstat with download instructions and support.
  Contains useful information for the biofilm researcher.
  Home of the commercial software package Imaris, which is suitable for presenting 3D images of biofilms grown in flow chambers.
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