Chapter 20 : Methods of Studying Biofilms

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This chapter reviews many commonly used organisms, experimental approaches, and techniques used for the growth and study of biofilms. Here, the authors focus on commonly used approaches with a greater emphasis on currently used techniques. They have summarized several diverse techniques for growing and evaluating biofilms below. These include examples of biofilm techniques for exploring naturally occurring biofilms, medically important biofilms, industrial applications, laboratory biofilm techniques, imaging and spectroscopy techniques, and broad-based genetic techniques. Overall, the authors concentrate on several techniques that are presently used in current biofilm research. Included in this discussion are biofilm growth strategies, a brief mention of genetic strategies, imaging techniques, and data analysis. The authors hope that this discussion will serve as an informative reference for the biofilm research community. In the field of dental microbiology, the constant depth fermenter is a device in which oral microorganisms are cultured on hydroxyapatite disks that are coated with saliva. Such standardized methods will permit a meaningful and rational comparison of data among individual laboratories. Finally, microbiologists now realize the many diverse environments and situations in which biofilms occur. As a result, there will be a continuing need for innovation in the experimental designs used to study biofilms.

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Image of FIGURE 1

Schematic representation of growth in presence of a limiting nutrient. To ensure a linear response during chemostat culture, the limiting nutrient concentration should be restricted to the region within the box as shown.

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Image of FIGURE 2

Field studies of biofilms can be quite beautiful, yet challenging, as one may need to manipulate and collect samples using aseptic technique in a remote location such as Midwestern State University's Dalquest Research Site, near Big Bend National Park, Texas.

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Image of FIGURE 3

Photograph (A) and schematic representation (B) of Calgary biofilm device ( ). Planktonic cultures are placed in wells into which are inserted prongs (left arrow in A, schematic representation in Ba). Biofilms grown on inserted prongs can then be placed into antibioticcontaining wells (Bb) for susceptibility testing. Figure 3A provided courtesy of H. Ceri, University of Calgary.

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Image of FIGURE 4

(A) The original Robbins' device (RD) ( ) consists of a brass pipe with removable sections of the wall from which biofilm samples may be acquired. (B) The modified RD (MRD) ( ) allowed biofilm testing to be conducted on a variety of materials (typically 7- mm-diameter plugs). (C) A further modification ( ) involving the use of a metal plug and platinum wire enables one to study the impact of electric fields on biofilm control.

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Image of FIGURE 5

Schematic representation of microtiter biofilm assay ( ). Cultures of wild-type (wt) and biofilm defective () mutant strains are grown in wells of microtiter plate. After growth, they are stained with crystal violet, the excess stain and planktonic cultures are removed, and then the stain is released from the wall-adherent biofilm by a solvent such as ethanol. The crystal violet stain intensity can be measured (optical density at 600 nm) and correlates with the amount of biofilm present.

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Image of FIGURE 6

Schematic representation (A) and photograph (B) of chemostat used for biofilm growth ( ). Nutrient-limited medium is pumped (P1) from a reservoir (R) into a chemostat (C). Once the culture is stabilized, it is pumped (P2) through a biofilm device such as an MRD, flow cell, or Tygon tubing as shown in panel B. Excess culture is removed to waste (W).

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Image of FIGURE 7

Example of a microscope flow cell commonly used for growing biofilms. As described in the text, we have noticed biofilms grown in a recirculating mode (A) to be much more evenly distributed within a flow cell than are those grown in a onceflowthrough design (B) (C. L. Bates and R. J. C. McLean, unpublished data). Flow cell was provided courtesy of K. Mathee, Florida International University, Miami, Fla.

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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1. Adams, J. L.,, and R. J. C. McLean. 1999. The impact of rpoS deletion on Escherichia coli biofilms. Appl. Environ. Microbiol. 65: 4285 4287.
2. Allison, D. G.,, B. Ruiz,, C. SanJose,, A. Jaspe,, and P. Gilbert. 1998. Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbiol. Lett. 167: 179 184.
3. Amann, R. I.,, B. J. Binder,, R. J. Olson,, S. W. Chisholm,, R. Devereux,, and D. A. Stahl. 1990a. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56: 1919 1925.
4. Amann, R. I.,, L. Krumholz,, and D. A. Stahl. 1990b. Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172: 762 770.
5. Amann, R. I.,, W. Ludwig,, and K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143 169.
6. Amann, R. I.,, J. Stromley,, R. Devereux,, R. Key,, and D. A. Stahl. 1992. Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl. Environ. Microbiol. 58: 614 623.
7. Anderl, J. N.,, J. Zahler,, F. Roe,, and P. S. Stewart. 2003. Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 47: 1251 1256.
8. Andersen, J. B.,, A. Heydorn,, M. Hentzer,, L. Eberl,, O. Geisenberger,, B. B. Christensen,, S. Molin,, and M. Givskov. 2001. gfp-based N-acyl homoserine lactone sensor systems for detection of bacterial communication. Appl. Environ. Microbiol. 67: 575 585.
9. Andersen, J. B.,, C. Sternberg,, L. K. Poulsen,, S. P. Bjørn,, M. Givskov,, and S. Molin. 1998. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64: 2240 2246.
10. Andersson, B.,, B. M. Gray, , H. C. Dillon, Jr.,, A. Bahrmand, , and C. Svanborg Edén. 1988. Role of adherence of Streptococcus pneumoniae in acute otitis media. Pediatr. Infect. Dis. J. 7: 476 480.
11. Arrage, A. A.,, N. Vasishtha,, D. Sundberg,, G. Bausch,, H. L. Vincent,, and D. C. White. 1995. On-line monitoring of antifouling and fouling- release surfaces using bioluminescence and fluorescence measurements during laminar flow. J. Ind. Microbiol. 15: 277 282.
12. Auerbach, I. D.,, C. Sorensen,, H. G. Hansma,, and P. A. Holden. 2000. Physical morphology and surface properties of unsaturated Pseudomonas putida biofilms. J. Bacteriol. 182: 3809 3815.
13. Balzer, G. J.,, and R. J. C. McLean. 2002. The stringent response genes relA and spoT are important for Escherichia coli biofilms under slow-growth conditions. Can. J. Microbiol. 48: 675 680.
14. Bardouniotis, E.,, W. Huddleston,, H. Ceri,, and M. E. Olson. 2001. Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system. FEMS Microbiol. Lett. 203: 263 267.
15. Battin, T. J.,, A. Wille,, B. Sattler,, and R. Psenner. 2001. Phylogenetic and funtional heterogeneity of sediment biofilms along environmental gradients in a glacial stream. Appl. Environ. Microbiol. 67: 799 807.
16. Beachey, E. H.,, C. S. Giampapa,, and S. N. Abraham. 1988. Bacterial adherence. Adhesin receptor-mediated attachment of pathogenic bacteria to mucosal surfaces. Am. Rev. Respir. Dis. 138: S45 S48.
17. Beveridge, T. J., 1989. The structure of bacteria, p. 1 65. In J. S. Poindexter, and E. R. Leadbetter (ed.), Bacteria in Nature, vol. 3. Plenum Publishing Corporation, New York, N.Y.
18. Blattner, F. R., , G. Plunkett III,, C. A. Bloch,, N. T. Perna,, V. Burland,, M. Riley,, J. Collado-Vides,, J. D. Glasner,, C. K. Rode,, G. F. Mayhew,, J. Gregor,, N. W. Davis,, H. A. Kirkpatrick,, M. A. Goeden,, D. J. Rose,, B. Mau,, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277: 1453 1474.
19. Blenkinsopp, S. A.,, A. E. Khoury,, and J. W. Costerton. 1992. Electrical enhancement of biocide efficacy against Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 58: 3770 3773.
20. Bond, P. L.,, S. P. Smriga,, and J. F. Banfield. 2000. Phylogeny of microorganisms populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site. Appl. Environ. Microbiol. 66: 3842 3849.
21. Borchardt, S. A.,, E. J. Allain,, J. J. Michels,, G. W. Stearns,, R. F. Kelly,, and W. F. McCoy. 2001. Reaction of acylated homoserine lactone bacterial signaling molecules with oxidized halogen antimicrobials. Appl. Environ. Microbiol. 67: 3174 3179.
22. Bradshaw, D. J.,, P. D. Marsh,, K. M. Schilling,, and D. Cummins. 1996. A modified chemostat system to study the ecology of oral biofilms. J. Appl. Bacteriol. 80: 124 130.
23. Brink, D. E.,, I. Vance,, and D. C. White. 1994. Detection of Desulfobacter in oil field environments by non-radioactive DNA probes. Appl. Microbiol. Biotechnol. 42: 469 475.
24. Brock, T. D.,, M. T. Madigan,, J. M. Martinko,, and J. Parker. 1994. Biology of Microorganisms, 7th ed. Prentice Hall, Englewood Cliffs, N.J.
25. Brown, D. A.,, D. C. Kamineni,, J. A. Sawicki,, and T. J. Beveridge. 1994. Minerals associated with biofilms occurring on exposed rock in an granitic underground research laboratory. Appl. Environ. Microbiol. 60: 3182 3191.
26. Brummer, I. H. M.,, W. Fehr,, and I. Wagner- Dobler. 2003. Biofilm community structure in polluted rivers: abundance of dominant phylogenetic groups over a complete annual cycle. Appl. Environ. Microbiol. 66: 3078 3082.
27. Busalmen, J. P.,, and S. R. De Sanchez. 2003. Changes in the electrochemical interface as a result of the growth of Pseudomonas fluorescens biofilms on gold. Biotechnol. Bioeng. 82: 619 624.
28. Caldwell, D. E.,, D. R. Korber,, and J. R. Lawrence. 1993. Analysis of biofilm formation using 2D versus 3D digital imaging. J. Appl. Bacteriol. 74(Suppl): 52S 66S.
29. Camper, A. K.,, W. L. Jones,, and J. T. Hayes. 1996. Effect of growth conditions and substratum composition on the persistence of coliforms in mixed-population biofilms. Appl. Environ. Microbiol. 62: 4014 4018.
30. Casamayor, E. O.,, H. Schäfer,, L. Bañeras,, C. Pedrós-Alió,, and G. Muyzer. 2000. Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 66: 499 508.
31. Ceri, H.,, M. E. Olson,, C. Stremick,, R. R. Read,, D. W. Morck,, and A. Buret. 1999. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J. Clin. Microbiol. 37: 1771 1776.
32. Chandra, J.,, D. M. Kuhn,, P. K. Mukherjee,, L. L. Hoyer,, T. McCormick,, and M. A. Ghannoum. 2001. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J. Bacteriol. 183: 5385 5394.
33. Cheng, K.-J.,, J. P. Fay,, R. N. Coleman,, L. P. Milligan,, and J. W. Costerton. 1981. Formation of bacterial microcolonies of feed particles in the rumen. Appl. Environ. Microbiol. 41: 298 305.
34. Chiang, S. L.,, J. J. Mekalanos,, and D. W. Holden. 1999. In vivo genetic analysis of bacterial virulence. Annu. Rev. Microbiol. 53: 129 154.
35. Choo-Smith, L. P.,, K. Maquelin,, T. van Vreeswijk,, H. P. Bruining,, G. J. Puppels,, N. A. Ngo Thi,, C. Kirchner,, D. Naumann,, D. Ami,, A. M. Villa,, F. Orsini,, S. M. Doglia,, H. Lamfarraj,, G. D. Sockalingum,, M. Manfait,, P. Allouch,, and H. P. Endtz. 2003. Investigating microbial (micro)colony heterogeneity by vibrational spectroscopy. Appl. Environ. Microbiol. 67: 1461 1469.
36. Christensen, B. B.,, J. A. J. Haagensen,, A. Heydorn,, and S. Molin. 2002. Metabolic commensalism and competition in a two-species microbial consortium. Appl. Environ. Microbiol. 68: 2495 2502.
37. Christensen, B. B.,, C. Sternberg,, J. B. Andersen, , R. J. Palmer, Jr.,, A. Toftgaard Nielsen, , M. Givskov, , and S. Molin. 1999. Molecular tools for study of biofilm physiology. Methods Enzymol. 310: 20 42.
38. Corbin, B. D.,, R. J. C. McLean,, and G. M. Aron. 2001. Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm. Can. J. Microbiol. 47: 680 684.
39. Costerton, J. W.,, K.-J. Cheng,, G. G. Geesey,, T. I. Ladd,, J. C. Nickel,, M. Dasgupta,, and T. J. Marrie. 1987. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41: 435 464.
40. Costerton, J. W.,, B. Ellis,, K. Lam,, F. Johnson,, and A. E. Khoury. 1994. Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria. Antimicrob. Agents Chemother. 38: 2803 2809.
41. Costerton, J. W.,, Z. Lewandowski,, D. E. Caldwell,, D. R. Korber,, and H. M. Lappin- Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49: 711 745.
42. Costerton, J. W.,, P. S. Stewart,, and E. P. Greenberg. 1999. Bacterial biofilms: a common cause of persistent infections. Science 284: 1318 1322.
43. Cote, R. J.,, and R. L. Gherna,. 1994. Nutrition and media, p. 155 178. In P. Gerhardt, , R. G. E. Murray, , W. A. Wood, , and N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, D.C.
44. Cramton, S. E.,, C. Gerke,, N. F. Schnell,, W. W. Nichols,, and F. Götz. 1999. The intercellular adhesion ( ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect. Immun. 67: 5427 5433.
45. Cramton, S. E.,, M. Ulrich,, F. Götz,, and G. Döring. 2001. Anaerobic conditions induce expression of polysaccharide intracellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis. Infect. Immun. 69: 4079 4085.
46. Czerkawski, J. W.,, and G. Breckenridge. 1977. Design and development of a long-term rumen simulation technique (Rusitec). Br. J. Nutr. 38: 371 384.
47. Danese, P. N.,, L. A. Pratt,, and R. Kolter. 2000. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J. Bacteriol. 182: 3593 3596.
48. Darby, C.,, J. W. Hsu,, N. Ghori,, and S. Falkow. 2002. Caenorhabditis elegans: plague bacteria biofilm blocks food intake. Nature 417: 243 244.
49. Davey, M. E.,, N. C. Caiazza,, and G. A. O’Toole. 2003. Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J. Bacteriol. 185: 1027 1036.
50. Davies, D. G.,, M. R. Parsek,, J. P. Pearson,, B. H. Iglewski,, J. W. Costerton,, and E. P. Greenberg. 1998. The involvement of cell-tocell signals in the development of a bacterial biofilm. Science 280: 295 298.
51. de Beer, D.,, and A. Schramm. 1999. Micro-environments and mass transfer phenomena in biofilms studied with microsensors. Water Sci. Technol. 39: 173 178.
52. de Beer, D.,, P. Stoodley,, and Z. Lewandowski. 1997. Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy. Biotechnol. Bioeng. 53: 151 158.
53. De Kievit, T. R.,, M. D. Parkins,, R. J. Gillis,, R. Srikumar,, H. Ceri,, K. Poole,, B. H. Iglewski,, and D. G. Storey. 2001. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 45: 1761 1770.
54. Donlan, R. M. 2001. Biofilms and device-associated infections. Emerg. Infect. Dis. 7: 277 281.
55. Donlan, R. M.,, and J. W. Costerton. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin.Microbiol.Rev. 15: 167 193.
56. Doolittle, M. M.,, J. J. Cooney,, and D. E. Caldwell. 1995. Lytic infection of Escherichia coli biofilms by bacteriophage T4. Can. J. Microbiol. 41: 12 18.
57. Doyle, R. J. 1999. Biofilms. Methods Enzymol. 310: 1 720.
58. Doyle, R. J. 2001a. Microbial growth in biofilms. Part A: developmental and molecular biological aspects. Methods Enzymol. 336: 1 469.
59. Doyle, R. J. 2001b. Microbial growth in biofilms, Part B: special environments and physicochemical aspects. Methods Enzymol. 337: 1 469.
60. Egan, S.,, S. James,, C. Holmstrom,, and S. Kjelleberg. 2002. Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata. Environ. Microbiol. 4: 433 442.
61. Egan, S.,, T. Thomas,, C. Holmstrom,, and S. Kjelleberg. 2000. Phylogenetic relationship and antifouling activity of bacterial epiphytes from the marine alga Ulva lactuca. Environ. Microbiol. 2: 343 347.
62. Espinosa-Urgel, M.,, A. Salido,, and J. L. Ramos. 2000. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J. Bacteriol. 182: 2363 2369.
63. Ferris, F. G.,, W. S. Fyfe,, T. Witten,, S. Schultze,, and T. J. Beveridge. 1989a. Effect of mineral substrate hardness on the population density of epilithic microorganisms in two Ontario rivers. Can. J. Microbiol. 35: 744 747.
64. Ferris, F. G.,, S. Schultze,, T. C. Witten,, W. S. Fyfe,, and T. J. Beveridge. 1989b. Metal interactions with microbial biofilms in acidic and neutral pH environments. Appl. Environ. Microbiol. 55: 1249 1257.
65. Finelli, A.,, C. V. Gallant,, K. Jarvi,, and L. L. Burrows. 2003. Use of in-biofilm expression technology to identify genes involved in Pseudomonas aeruginosa biofilm development. J. Bacteriol. 185: 2700 2710.
66. Flemming, H. C.,, T. Griebe,, and G. Schaule. 1996. Antifouling strategies in technical systems: a short review. Water Sci. Technol. 34: 517 524.
67. Flora, J. R. V.,, M. T. Suidan,, P. Biswas,, and G. D. Sayles. 1995. Modeling algal biofilms: Role of carbon, light, cell surface charge, and ionic species. Water Environ. Res. 67: 87 94.
68. Francois, P.,, P. Tu Quoc,, C. Bisognano,, W. L. Kelley,, D. P. Lew,, J. Schrenzel,, S. E. Cramton,, F. Gotz,, and P. Vaudaux. 2003. Lack of biofilm contribution to bacterial colonisation in an experimental model of foreign body infection by Staphylococcus aureus and Staphylococcus epidermidis. FEMS Immunol. Med. Microbiol. 35: 135 140.
69. Frangeul, L.,, K. E. Nelson,, C. Buchrieser,, A. Danchin,, P. Glaser,, and F. Kunst. 1999. Cloning and assembly strategies in microbial genome projects. Microbiology 145: 2625 2634.
70. Freeman, C.,, and M. A. Lock. 1995. Isotope dilution analysis and rates of 32P incorporation into phospholipid as a measure of microbial growth rates in biofilms. Water Res. 29: 789 792.
71. Friedmann, E. I.,, and R. Weed. 1987. Microbial trace fossil formation, biogenous, and abiotic weathering in the Antarctic cold desert. Science 236: 703 705.
72. Frirdich, E.,, B. Lindner,, O. Holst,, and C. Whitfield. 2003. Overexpression of the waaZ gene leads to modification of the structure of the inner core region of Escherichia coli lipopolysaccharide, truncation of the outer core, and reduction of the amount of O polysaccharide on the cell surface. J. Bacteriol. 185: 1659 1671.
73. Gander, S.,, and P. Gilbert. 1997. The development of a small-scale biofilm model suitable for studying the effects of antibiotics on biofilms of gram-negative bacteria. J. Antimicrob. Chemother. 40: 329 334.
74. Geesey, G. G.,, R. Mutch,, J. W. Costerton,, and R. B. Green. 1978. Sessile bacteria: an important component of the microbial population in small mountain streams. Limnol. Oceanogr. 23: 1214 1223.
75. Gerhardt, P.,, and S. W. Drew,. 1994. Liquid culture, p. 224 247. In P. Gerhardt, , R. G. E. Murray, , W. A. Wood, , and N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. ASM Press,
76. Gherna, R. L., 1994. Culture preservation, p. 278 292. In P. Gerhardt, , R. G. E. Murray, , W. A. Wood, , and N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. ASM Press, Washington, D.C.
77. Ghigo, J. M. 2001. Natural conjugative plasmids induce bacterial biofilm development. Nature 412: 442 445.
78. Gibbs, J. T.,, and P. L. Bishop. 1995. A method for describing biofilm surface roughness using geostatistical techniques. Water Sci. Technol. 32: 91 98.
79. Gilbert, P.,, D. G. Allison,, D. J. Evans,, P. S. Handley,, and M. R. W. Brown. 1989. Growth rate control of adherent bacterial populations. Appl. Environ. Microbiol. 55: 1308 1311.
80. Gristina, A. G. 1987. Biomaterial-centered infection: microbial adhesion versus tissue integration. Science 237: 1588 1595.
81. Guo, D.,, Y. Wu,, and H. B. Kaplan. 2000. Identification and characterization of genes required for early Myxococcus xanthus developmental gene expression. J. Bacteriol. 182: 4564 4571.
82. Hamadeh, H.,, and C. A. Afshari. 2000. Gene chips and functional genomics. Am. Sci. 88: 508 515.
83. Hamilton, W. A., 1987. Biofilms: microbial interactions and metabolic activities, p. 361 385. In W. A. Hamilton (ed.), Ecology of Microbial Communities. University Press, Cambridge, United Kingdom.
84. Hatfield, G. W.,, S. P. Hung,, and P. Baldi. 2003. Differential analysis of DNA microarray gene expression data. Mol. Microbiol. 47: 871 877.
85. Hausner, M.,, and S. Wuertz. 1999. High rates of conjugation in bacterial biofilms as determined by quantitative in situ analysis. Appl. Environ. Microbiol. 65: 3710 3713.
86. Hein, I.,, A. Lehner,, P. Rieck,, K. Klein,, E. Brandl,, and M. Wagner. 2001. Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese. Appl. Environ. Microbiol. 67: 3122 3126.
87. Helmer, C.,, S. Kunst,, S. Juretschko,, M. C. Schmid,, K. H. Schleifer,, and M. Wagner. 1999. Nitrogen loss in a nitrifying biofilm system. Water Sci. Technol. 39: 13 21.
88. Hentzer, M.,, K. Riedel,, T. B. Rasmussen,, A. Heydorn,, J. B. Andersen,, M. R. Parsek,, S. A. Rice,, L. Eberl,, S. Molin,, N. Hoiby,, S. Kjelleberg,, and M. Givskov. 2002. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148: 87 102.
89. Herles, S.,, S. Olsen,, J. Afflitto,, and A. Gaffar. 1994. Chemostat flow cell system: An in vitro model for the evaluation of antiplaque agents. J. Dent. Res. 73: 1748 1755.
90. Heydorn, A.,, B. K. Ersbøll,, M. Hentzer,, M. R. Parsek,, M. Givskov,, and S. Molin. 2000a. Experimental reproducibility in flow-chamber biofilms. Microbiology 146: 2409 2415.
91. Heydorn, A.,, B. K. Ersbøll,, J. Kato,, M. Hentzer,, M. R. Parsek,, T. Tolker-Nielsen,, M. Givskov,, and S. Molin. 2002. Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression. Appl. Environ. Microbiol. 68: 2008 2017.
92. Heydorn, A.,, A. T. Nielsen,, M. Hentzer,, C. Sternberg,, M. Givskov,, B. K. Ersbøll,, and S. Molin. 2000b. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146: 2395 2407.
93. Hibma, A. M.,, S. A. Jassim,, and M. W. Griffiths. 1997. Infection and removal of L-forms of Listeria monocytogenes with bred bacteriophage. Int. J. Food Microbiol. 34: 197 207.
94. Hogan, D. A.,, and R. Kolter. 2002. Pseudomonas- Candida interactions: an ecological role for virulence factors. Science 296: 2229 2232.
95. Holden, P. A.,, M. G. LaMontagne,, W. G. Miller,, and S. E. Lindow. 2002. Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand. Appl. Environ. Microbiol. 68: 2509 2518.
96. Hoskins, B. C.,, L. Fevang,, P. D. Majors,, M. M. Sharma,, and G. Georgiou. 1999. Selective imaging of biofilms in porous media by NMR relaxation. J. Magn. Reson. 139: 67 73.
97. Huang, C. T.,, K. D. Xu,, G. A. McFeters,, and P. S. Stewart. 1998. Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation. Appl. Environ. Microbiol. 64: 1526 1531.
98. Huang, C. T.,, F. P. Yu,, G. A. McFeters,, and P. S. Stewart. 1995. Nonuniform spatial patterns of respiratory activity within biofilms during disinfection. Appl. Environ. Microbiol. 61: 2252 2256.
99. Hughes, K. A.,, I. W. Sutherland,, and M. V. Jones. 1998. Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. Microbiology 144: 3039 3047.
100. Jolley, J. G.,, G. G. Geesey,, and M. R. Hankins. 1989. Auger electron and X-ray photoelectron spectroscopy study of the biocorrosion of copper by alginic acid polysaccharide. Appl. Surf. Sci. 37: 469 480.
101. Jouenne, T.,, O. Tresse,, and G. A. Junter. 1994. Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics. FEMS Microbiol. Lett. 119: 237 242.
102. Kaplan, J. B.,, and D. H. Fine. 2002. Biofilm dispersal of Neisseria subflava and other phylogenetically diverse oral bacteria. Appl. Environ. Microbiol. 68: 4943 4950.
103. Kinniment, S. L.,, J. W. T. Wimpenny,, D. Adams,, and P. D. Marsh. 1996. Development of a steady-state oral microbial biofilm community using the constant-depth film fermenter. Microbiology 142: 631 638.
104. Kolenbrander, P. E.,, R. N. Andersen,, K. Kazmerzak,, R. Wu, , and R. J. Palmer, Jr. 1999. Spatial organization of oral bacteria in biofilms. Methods Enzymol. 310: 322 332.
105. Konhauser, K. O.,, S. Schultze-Lam,, F. G. Ferris,, W. S. Fyfe,, F. J. Longstaffe,, and T. J. Beveridge. 1994. Mineral precipitation by epilithic biofilms in the Speed River, Ontario, Canada. Appl. Environ. Microbiol. 60: 549 553.
106. Korber, D. R.,, J. R. Lawrence,, B. Sutton,, and D. E. Caldwell. 1989. Effect of laminar flow velocity on the kinetics of surface recolonization by Mot + and Mot Pseudomonas fluorescens. Microb. Ecol. 18: 1 19.
107. Korber, D. R.,, G. M. Wolfaardt,, V. Brözel,, R. MacDonald,, and T. Niepel. 1999. Reporter systems for microscopic analysis of microbial biofilms. Methods Enzymol. 310: 3 20.
108. Kurtz, H. D., Jr.,, and J. Smit. 1992. Analysis of a Caulobacter crescentus gene cluster involved in attachment of the holdfast to the cell. J. Bacteriol. 174: 687 694.
109. Lapaglia, C.,, and P. L. Hartzell. 1997. Stressinduced production of biofilm in the hyperthermophile Archaeoglobus fulgidus. Appl. Environ. Microbiol. 63: 3158 3163.
110. Lappin-Scott, H. M., 2000. Claude E. ZoBell: his life and contributions to biofilm microbiology , p. 45 50. In C. R. Bell, , M. Brylinsky, , and J. L. Johnson-Green (ed.), Proceedings of the 8th International Symposium on Microbial Ecology, Halifax. Atlantic Canada Society for Microbial Ecology.
111. Lappin-Scott, H. M.,, C. J. Bass,, K. M. McAlpine,, and P. F. Sanders. 1995. Survival mechanisms of hydrogen sulphide-producing bacteria isolated from extreme environments and their role in corrosion. Int. Biodeterior. Biodegrad. 34: 305 319.
112. Lappin-Scott, H. M.,, and J. W. Costerton. 1989. Bacterial biofilms and surface fouling. Biofouling 1: 323 342.
113. Lawrence, J. R.,, D. R. Korber,, B. D. Hoyle,, J. W. Costerton,, and D. E. Caldwell. 1991. Optical sectioning of microbial biofilms. J. Bacteriol. 173: 6558 6567.
114. Lawrence, J. R.,, D. R. Korber,, G. M. Wolfaardt,, and D. E. Caldwell. 1995. Behavioral strategies of surface-colonizing bacteria. Adv. Microb. Ecol. 14: 1 75.
115. Lawrence, J. R.,, D. R. Korber,, G. M. Wolfaardt,, and D. E. Caldwell,. 1997. Analytical imaging and microscopy techniques , p. 29 51. In C. J. Hurst, , G. R. Knudsen, , M. J. McInerney, , L. D. Stetzenbach, , and M. V. Walter (ed.), Manual of Environmental Microbiology. ASM Press, Washington, D. C.
116. Lawrence, J. R.,, B. Scharf,, G. Packroff,, and T. R. Neu. 2003. Microscale evaluation of the effects of grazing by invertebrates with contrasting feeding modes on river biofilm architecture and composition. Microb. Ecol. 44: 199 207.
117. Lenski, R. E.,, J. A. Mongold,, P. D. Sniegowski,, M. Travisano,, F. Vasi,, P. J. Gerrish,, and T. M. Schmidt. 1998. Evolution of competitive fitness in experimental populations of E. coli: what makes one genotype a better competitor than another? Antonie van Leeuwenhoek 73: 35 47.
118. Lewandowski, Z.,, S. A. Altobelli,, and E. Fukushima. 1993. NMR and microelectrode studies of hydrodynamics and kinetics in biofilms. Biotechnol. Prog. 9: 40 45.
119. Li, Y. H.,, P. C. Y. Lau,, J. H. Lee,, R. P. Ellen,, and D. G. Cvitkovitch. 2001. Natural genetic transformation of Streptococcus mutans growing in biofilms. J. Bacteriol. 183: 897 908.
120. Li, Y. H.,, P. C. Y. Lau,, N. Tang,, G. Svensäter,, R. P. Ellen,, and D. G. Cvitkovitch. 2002. Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans. J. Bacteriol. 18: 6333 6342.
121. Little, B.,, and P. Wagner. 1996. An overview of microbiologically influenced corrosion of metals and alloys. Can. J. Microbiol. 42: 367 374.
122. Little, B.,, P. Wagner,, and F. Mansfeld. 1991. Microbiologically influenced corrosion of metals and alloys. Int. Mater. Rev. 36: 253 272.
123. Loo, C. Y.,, D. A. Corliss,, and N. Ganeshkumar. 2000. Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes. J. Bacteriol. 182: 1374 1382.
124. Luppens, S. B. I.,, M. W. Reij,, R. W. L. vander Heijden,, F. M. Rombouts,, and T. Abee. 2003. Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants. Appl. Environ. Microbiol. 68: 4194 4200.
125. Mackie, E. B.,, K. N. Brown,, J. Lam,, and J. W. Costerton. 1979. Morphological stabilization of capsules of group B streptococci, types Ia, Ib, II, and III with specific antibody. J. Bacteriol. 138: 609 617.
126. MacLeod, F. A.,, H. M. Lappin-Scott,, and J. W. Costerton. 1988. Plugging of a model rock system by using starved bacteria. Appl. Environ. Microbiol. 54: 1365 1372.
127. Manefield, M.,, R. de Nys,, N. Kumar,, R. Read,, M. Givskov,, P. Steinberg,, and S. Kjelleberg. 1999. Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein. Microbiology 145: 283 291.
128. Marrie, T. J.,, and J. W. Costerton. 1984. Morphology of bacterial attachment to cardiac pacemaker leads and power packs. J. Clin. Microbiol. 19: 911 914.
129. Marrie, T. J.,, and J. W. Costerton. 1985. Mode of growth of bacterial pathogens in chronic polymicrobial human osteomyelitis. J. Clin. Microbiol. 22: 924 933.
130. Marshall, K. C. 1985. Bacterial adhesion in oligotrophic habitats. Microbiol. Sci. 2: 321 326.
131. Mattison, R. G.,, H. Taki,, and S. Harayama. 2002. The bacterivorous soil flagellate Heteromita globosa reduces bacterial clogging under denitrifying conditions in sand-filled aquifer columns. Appl. Environ. Microbiol. 68: 4539 4545.
132. Mattsby-Baltzer, I.,, M. Sandin,, B. Ahlström,, S. Allenmark,, M. Edebo,, E. Falsen,, K. Pedersen,, N. Rodin,, R. A. Thompson,, and L. Edebo. 1989. Microbial growth and accumulation in industrial metal-working fluids. Appl. Environ. Microbiol. 55: 2681 2689.
133. McCoy, W. F.,, J. D. Bryers,, J. Robbins,, and J. W. Costerton. 1981. Observations of fouling biofilm formation. Can. J. Microbiol. 27: 910 917.
134. McEldowney, S.,, and M. Fletcher. 1986. Variability of the influence of physicochemical factors affecting bacterial adhesion to polystyrene substrata. Appl. Environ. Microbiol. 52: 460 465.
135. McLean, R. J. C.,, and T. J. Beveridge,. 1990. Metal binding capacity of bacterial surfaces and their ability to form mineralized aggregates, p. 185 222. In H. L. Ehrlich, and C. L. Brierley (ed.), Microbial Mineral Recovery. McGraw-Hill Book Co., New York, N.Y.
136. McLean, R. J. C.,, J. M. Cassanto,, M. B. Barnes,, and J. Koo. 2001. Bacterial biofilm formation under microgravity conditions. FEMS Microbiol. Lett. 195: 115 119.
137. McLean, R. J. C.,, A. A. Hussain,, M. Sayer,, P. J. Vincent,, D. J. Hughes,, and T. J. N. Smith. 1993. Antibacterial activity of multilayer silver copper surface films on catheter material. Can. J. Microbiol. 39: 895 899.
138. McLean, R. J. C.,, J. R. Lawrence,, D. R. Korber,, and D. E. Caldwell. 1991. Proteus mirabilis biofilm protection against struvite crystal dissolution and its implications in struvite urolithiasis. J. Urol. 146: 1138 1142.
139. McLean, R. J. C.,, J. C. Nickel,, K.-J. Cheng,, and J. W. Costerton. 1988. The ecology and pathogenicity of urease-producing bacteria in the urinary tract. Crit. Rev. Microbiol. 16: 37 79.
140. McLean, R. J. C.,, M. Whiteley,, B. C. Hoskins,, P. D. Majors,, and M. M. Sharma. 1999. Laboratory techniques for studying biofilm growth, physiology, and gene expression in flowing systems and porous media. Methods Enzymol. 310: 248 264.
141. Mercier, J.,, and S. E. Lindow. 2000. Role of leaf surface sugars in colonization of plants by bacterial epiphytes. Appl. Environ. Microbiol. 66: 369 374.
142. Merritt, J.,, F. Qi,, S. D. Goodman,, M. H. Anderson,, and W. Shi. 2003. Mutation of luxS affects biofilm formation in Streptococcus mutans. Infect. Immun. 71: 1972 1979.
143. Mireles, J. R., II,, A. Toguchi, , and R. M. Harshey. 2001. Salmonella enterica serovar typhimurium swarming mutants with altered biofilmforming abilities: surfactin inhibits biofilm formation. J. Bacteriol. 183: 5848 5854.
144. Moeseneder, M. M.,, J. M. Arrieta,, G. Muyzer,, C. Winter,, and G. J. Herndl. 1999. Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 65: 3518 3525.
145. Møller, S.,, A. R. Pedersen,, L. K. Poulsen,, E. Arvin,, and S. Molin. 1996. Activity and threedimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy. Appl. Environ. Microbiol. 62: 4632 4640.
146. Møller, S.,, C. Sternberg,, J. B. Andersen,, B. B. Christensen,, J. L. Ramos,, M. Givskov,, and S. Molin. 1998. In situ gene expression in mixedculture biofilms: evidence of metabolic interactions between community members. Appl. Environ. Microbiol. 64: 721 732.
147. Morris, C. E.,, M. B. Barnes,, and R. J. C. McLean,. 2002a. Biofilms on leaf surfaces: implications for the biology, ecology and management of populations of epiphytic bacteria, p. 139 155. In S. E. Lindow, , E. I. Hecht-Poinar, , and V. J. Elliott (ed.), Phyllosphere Microbiology. American Phytopathology Society, St. Paul, Minn.
148. Morris, R. M.,, M. S. Rappe,, S. A. Connan,, K. L. Vergin,, W. A. Siebold,, C. A. Carlson,, and S. J. Giovannoni. 2002b. SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420: 806 810.
149. Neu, T. R.,, and J. R. Lawrence. 1997. Development and structure of microbial biofilms in river water studied by confocal laser scanning microscopy. FEMS Microbiol. Ecol. 24: 11 25.
150. Nichols, P. D.,, J. M. Henson,, J. B. Guckert,, D. E. Nivens,, and D. C. White. 1985. Fourier transform-IR spectroscopic methods for microbial ecology analysis of bacteria, bacteria-polymer mixtures and biofilms. J. Microbiol. Methods 4: 79 94.
151. Nichols, W. W., 1989. Susceptibility of biofilms to toxic compounds, p. 321 331. In W. G. Characklis, and P. A. Wilderer (ed.), Structure and Function of Biofilms. John Wiley & Sons, Inc., New York, N.Y.
152. Nickel, J. C.,, S. K. Grant,, K. Lam,, M. E. Olson,, and J. W. Costerton. 1991a. Bacteriologically stressed animal model of new closed catheter drainage system with microbicidal outlet tube. Urology 38: 280 289.
153. Nickel, J. C.,, M. E. Olson,, and J. W. Costerton. 1991b. Rat model of experimental prostatitis. Infection 19: S126 S130.
154. Nickel, J. C.,, M. E. Olson,, R. J. C. McLean,, S. K. Grant,, and J. W. Costerton. 1987. An ecologic study of infected urinary stone genesis in an animal model. Br. J. Urol. 59: 21 30.
155. Nickel, J. C.,, I. Ruseska,, J. B. Wright,, and J. W. Costerton. 1985. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob. Agents Chemother. 27: 619 624.
156. Niedhardt, F. C.,, P. L. Bloch,, and D. F. Smith. 1974. Culture medium for Enterobacteria. J. Bacteriol. 119: 736 747.
157. Nivens, D. E.,, D. E. Ohman,, J. Williams,, and M. J. Franklin. 2001. Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J. Bacteriol. 183: 1047 1057.
158. Nivens, D. E., , R. J. Palmer, Jr.,, and D. C. White. 1995. Continuous nondestructive monitoring of microbial biofilms: A review of analytical techniques. J. Ind. Microbiol. 15: 263 276.
159. Nyholm, S. V.,, B. Deplanke,, H. R. Gaskins,, M. A. Apicella,, and M. J. McFall Ngai. 2002. Roles of Vibrio fischeri and nonsymbiotic bacteria in the dynamics of mucus secretion during symbiont colonization of the Euprymna scolopes light organ. Appl. Environ. Microbiol. 68: 5113 5122.
160. Nyström, T.,, R. M. Olsson,, and S. Kjelleberg. 1992. Survival, stress resistance, and alterations in protein expression in the marine Vibrio sp. strain S14 during starvation for different individual nutrients. Appl. Environ. Microbiol. 58: 55 65.
161. Odham, G.,, A. Tunlid,, A. Valeur,, P. Sundin,, and D. C. White. 1986. Model system for studies of microbial dynamics at exuding surfaces such as the rhizosphere. Appl. Environ. Microbiol. 52: 191 196.
162. Oosthuizen, M. C.,, B. Steyn,, D. Lindsay,, V. Brözel,, and A. von Holy. 2001. Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm. FEMS Microbiol. Lett. 194: 47 51.
163. Oosthuizen, M. C.,, B. Steyn,, J. Theron,, P. Cosette,, D. Lindsay,, A. von Holy,, and V. Brözel. 2002. Proteomic analysis reveals differential protein expression by Bacillus cereus during biofilm formation. Appl. Environ. Microbiol. 68: 2770 2780.
164. Orvain, F.,, R. Galois,, C. Barnard,, A. Sylvestre,, G. Blanchard,, and P. G. Sauriau. 2003. Carbohydrate production in relation to microphytobenthic biofilm development: an integrated approach in a tidal mesocosm. Microb. Ecol. 45: 237 251.
165. Ostrowski, M.,, and A. Sklodowska. 1993. Bacterial and chemical leaching pattern on copper ores of sandstone and limestone type. World J. Microbiol. Biotechnol. 9: 328 331.
166. O’Toole, G. A.,, K. A. Gibbs,, P. W. Hager, , P. V. Phibbs, Jr.,, and R. Kolter. 2000a. The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa. J. Bacteriol. 182: 425 431.
167. O’Toole, G. A.,, H. B. Kaplan,, and R. Kolter. 2000b. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54: 49 79.
168. O’Toole, G. A.,, and R. Kolter. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS 365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol. Microbiol. 28: 449 461.
169. O’Toole, G. A.,, L. A. Pratt,, P. I. Watnick,, D. K. Newman,, V. B. Weaver,, and R. Kolter. 1999. Genetic approaches to study of biofilms. Methods Enzymol. 310: 91 109.
170. Owusu Ababio, G.,, J. A. Rogers,, D. W. Morck,, and M. E. Olson. 1995. Efficacy of sustained release ciprofloxacin microspheres against deviceassociated Pseudomonas aeruginosa biofilm infection in a rabbit peritoneal model. J. Med. Microbiol. 43: 368 376.
171. Paerl, H. W.,, and J. L. Pinckney. 1996. A minireview of microbial consortia: Their roles in aquatic production and biogeochemical cycling. Microb. Ecol. 31: 225 247.
172. Palmer, R. J., Jr.,, K. Kazmerzak, , M. C. Hansen, , and P. E. Kolenbrander. 2001. Mutualism versus independence: strategies of mixed-species oral biofilms in vitro using saliva as the sole nutrient source. Infect. Immun. 69: 5794 5804.
173. Parales, R. E.,, J. L. Ditty,, and C. S. Harwood. 2000. Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene. Appl. Environ. Microbiol. 66: 4098 4104.
174. Park, Y. S.,, and K. Toda. 1992. Multi-stage biofilm reactor for acetic acid production at high concentration. Biotechnol. Lett. 14: 609 612.
175. Parker, D. S.,, T. Jacobs,, E. Bower,, D. W. Stowe,, and G. Farmer. 1997. Maximizing trickling filter nitrification rates through biofilm control: research review and full scale application. Water Sci. Technol. 36: 255 262.
176. Parsek, M. R.,, and E. P. Greenberg. 2000. Acylhomoserine lactone quorum sensing in gramnegative bacteria: a signaling mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. USA 97: 8789 8793.
177. Perna, N. T., , G. Plunkett III,, V. Burland,, B. Mau,, J. D. Glasner,, D. J. Rose,, G. F. Mayhew,, P. S. Evans,, J. Gregor,, H. A. Kirkpatrick,, G. Posfai,, J. Hackett,, S. Klink,, A. Boutin,, Y. Shao,, L. Miller,, E. J. Grotbeck,, N. W. Davis,, A. Lim,, E. T. Dimalanta,, and K. D. Potamousis. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409: 529 533.
178. Perrot, F.,, M. Hébraud,, R. Charlionet,, G. A. Junter,, and T. Jouenne. 2000. Protein patterns of gel-entrapped Escherichia coli cells differ from those of free-floating organisms. Electrophoresis 21: 645 653.
179. Phipps, D.,, G. Rodriguez,, and H. Ridgway. 1999. Deconvolution fluorescence microscopy for observation and analysis of membrane biofilm architecture. Methods Enzymol. 310: 178 194.
180. Potter, K.,, R. L. Kleinberg,, F. J. Brockman,, and E. W. McFarland. 1996. Assay for bacteria in porous media by diffusion-weighted NMR. J. Magn. Reson. B 113: 9 15.
181. Prigent-Combaret, C.,, E. Brombacher,, O. Vidal,, A. Ambert,, P. Lejeune,, P. Landini,, and C. Dorel. 2001. Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene. J. Bacteriol. 183: 7213 7223.
182. Prigent-Combaret, C.,, G. Prensier,, T. T. Le Thi,, O. Vidal,, P. Lejeune,, and C. Dorel. 2000. Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid. Environ. Microbiol. 2: 450 464.
183. Prigent-Combaret, C.,, O. Vidal,, C. Dorel,, and P. Lejeune. 1999. Abiotic surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli. J. Bacteriol. 181: 5993 6002.
184. Prosser, B. L.,, D. Taylor,, B. A. Dix,, and R. Cleeland. 1987. Method of evaluating effects of antibiotics on bacterial biofilm. Antimicrob. Agents Chemother. 31: 1502 1506.
185. Prouty, A. M.,, W. H. Schwesinger,, and J. S. Gunn. 2002. Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp. Infect. Immun. 70: 2640 2649.
186. Purevdorj, B.,, J. W. Costerton,, and P. Stoodley. 2002. Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 68: 4457 4464.
187. Quignon, F.,, M. Sardin,, L. Kiene,, and L. Schwartzbrod. 1997. Poliovirus-1 inactivation and interaction with biofilm: a pilot-scale study. Appl. Environ. Microbiol. 63: 978 982.
188. Qureshi, F. M.,, M. Badar,, and N. Ahmed. 2001. Biosorption of copper by a bacterial biofilm on a flexible polyvinyl chloride conduit. Appl. Environ. Microbiol. 67: 4349 4352.
189. Rachid, S.,, K. Ohlsen,, U. Wallner,, J. Hacker,, M. Hecker,, and W. Ziebuhr. 2000. Alternative transcription factor σ B is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate. J. Bacteriol. 182: 6824 6826.
190. Ramos, C.,, L. Mølbak,, and S. Molin. 2000. Bacterial activity in the rhizosphere analyzed at the single-cell level by monitoring ribosome contents and synthesis rates. Appl. Environ. Microbiol. 66: 801 809.
191. Raskin, L.,, B. E. Rittmann,, and D. A. Stahl. 1996. Competition and coexistence of sulfate-reducing and methanogenic populations in anaerobic biofilms. Appl. Environ. Microbiol. 62: 3847 3857.
192. Rippere-Lampe, K. E.,, M. Lang,, H. Ceri,, M. Olson,, H. A. Lockman,, and A. D. O’Brien. 2001. Cytotoxic necrotizing factor type 1-positive Escherichia coli causes increased inflammation and tissue damage to the prostate in a rat prostatitis model. Infect. Immun. 69: 6515 6519.
193. Robinson, R. W.,, D. E. Akin,, R. A. Nordstedt,, M. V. Thomas,, and H. C. Aldrich. 1984. Light and electron microscopic examinations of methane-producing biofilms from anaerobic fixedbed reactors. Appl. Environ. Microbiol. 48: 127 136.
194. Rogers, J.,, A. B. Dowsett,, P. J. Dennis,, J. V. Lee,, and C. W. Keevil. 1994. Influence of plumbing materials on biofilm formation and growth of Legionella pneumophila in potable water systems. Appl. Environ. Microbiol. 60: 1842 1851.
195. Rumbaugh, K. P.,, J. A. Griswold,, B. H. Iglewski,, and A. N. Hamood. 1999. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections. Infect. Immun. 67: 5854 5862.
196. Sambrook, J.,, E. F. Fritsch,, and T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual, 2nd ed., p. 2.60 2.79. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
197. Sauer, K.,, and A. K. Camper. 2001. Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth. J. Bacteriol. 183: 6579 6589.
198. Schembri, M. A.,, K. Kjærgaard,, and P. Klemm. 2003. Global gene expression in Escherichia coli biofilms. Mol. Microbiol. 48: 253 267.
199. Shaw, J. C.,, B. Bramhill,, N. C. Wardlaw,, and J.W. Costerton. 1985. Bacterial fouling in a model core system. Appl. Environ. Microbiol. 49: 693 701.
200. Sibille, I.,, T. Sime-Ngando,, L. Mathieu,, and J. C. Block. 1998. Protozoan bacterivory and Escherichia coli survival in drinking water distribution systems. Appl. Environ. Microbiol. 64: 197 202.
201. Singh, P. K.,, M. R. Parsek,, E. P. Greenberg,, and M. J. Welsh. 2002. A component of innate immunity prevents bacterial biofilm development. Nature 417: 552 555.
202. Singh, P. K.,, A. L. Schaefer,, M. R. Parsek,, T. O. Moninger,, M. J. Welsh,, and E. P. Greenberg. 2000. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407: 762 764.
203. Smibert, R. M.,, and N. R. Krieg,. 1994. Phenotypic characterization, p. 607 654. In P. Gerhardt, , R. G. E. Murray, , W. A. Wood, , and N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, D.C.
204. Stahl, D. A., 1997. Molecular approaches for the measurement of density, diversity, and phylogeny, p. 102 114. In C. J. Hurst, , G. R. Knudsen, , M. J. McInerney, , L. D. Stetzenbach, , and M. V. Walter (ed.), Manual of Environmental Microbiology. ASM Press, Washington, D.C.
205. Stanley, N. R.,, R. A. Britton,, A. D. Grossman,, and B. A. Lazazzera. 2003. Identification of catabolite repression as a physiological regulator of biofilm formation by Bacillus subtilis by use of DNA microarrays. J. Bacteriol. 185: 1951 1957.
206. Sternberg, C.,, B. B. Christensen,, T. Johansen,, A. T. Nielsen,, J. B. Andersen,, M. Givskov,, and S. Molin. 1999. Distribution of bacterial growth activity in flow-chamber biofilms. Appl. Environ. Microbiol. 65: 4108 4117.
207. Stewart, P. S.,, F. Roe,, J. Rayner,, J. G. Elkins,, Z. Lewandowski,, U. A. Ochsner,, and D. J. Hassett. 2000. Effect of catalase on hydrogen peroxide penetration into Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 66: 836 838.
208. Steyn, B.,, M. C. Oosthuizen,, R. MacDonald,, J. Theron,, and V. S. Brözel. 2001. The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis. Proteomics 1: 871 879.
209. Stickler, D.,, and G. Hughes. 1999. Ability of Proteus mirabilis to swarm over urethral catheters. Eur. J. Clin. Microbiol. Infect. Dis. 18: 206 208.
210. Stickler, D. J.,, C. L. Clayton,, and J. C. Chawla. 1987. Assessment of antiseptic bladder washout procedures using a physical model of the catheterised bladder. Br. J. Urol. 60: 413 418.
211. Stickler, D. J.,, C. L. Clayton,, M. J. Harber,, and J. C. Chawla. 1988. Pseudomonas aeruginosa and long-term indwelling bladder catheters. Arch. Phys. Med. Rehabil. 69: 25 28.
212. Stickler, D. J.,, and R. J. C. McLean. 1995. Biomaterials associated infections: the scale of the problem. Cells Mater. 5: 167 182.
213. Stickler, D. J.,, N. S. Morris,, R. J. C. McLean,, and C. Fuqua. 1998. Biofilms on indwelling urinary catheters produce quorum-sensing signal molecules in situ and in vitro. Appl. Environ. Microbiol. 64: 3486 3490.
214. Stoodley, P.,, D. deBeer,, and Z. Lewandowski. 1994. Liquid flow in biofilm systems. Appl. Environ. Microbiol. 60: 2711 2716.
215. Stoodley, P.,, Z. Lewandowski,, J. D. Boyle,, and H. M. Lappin-Scott. 1999a. Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: An in situ investigation of biofilm rheology. Biotechnol. Bioeng. 65: 83 92.
216. Stoodley, P.,, Z. Lewandowski,, J. D. Boyle,, and H. M. Lappin-Scott. 1999b. The formation of migratory ripples in a mixed species bacterial biofilm growing in turbulent flow. Environ. Microbiol. 1: 447 455.
217. Stoodley, P.,, S. Wilson,, L. Hall-Stoodley,, J. D. Boyle,, H. M. Lappin-Scott,, and J. W. Costerton. 2001. Growth and detachment of cell clusters from mature mixed-species biofilms. Appl. Environ. Microbiol. 67: 5608 5613.
218. Stover, C. K.,, X. Q. Pham,, A. L. Erwin,, S. D. Mizoguchi,, P. Warrener,, M. J. Hickey,, F. S. Brinkman,, W. O. Hufnagel,, D. J. Kowalik,, M. Lagrou,, R. L. Garber,, L. Goltry,, E. Tolentino,, S. Westbrock-Wadman,, Y. Yuan,, L. L. Brody,, S. N. Coulter,, K. R. Folger,, A. Kas,, K. Larbig,, R. Lim,, K. Smith,, D. Spencer,, G. K. Wong,, Z. Wu,, I. T. Paulsen,, J. Reizer,, M. H. Saier,, R. E. W. Hancock,, S. Lory,, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406: 947 948.
219. Suci, P. A.,, M. W. Mittelman,, F. P. Yu,, and G. G. Geesey. 1994. Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 38: 2125 2133.
220. Suci, P. A.,, and B. J. Tyler. 2002. Action of chlorhexidine digluconate against yeast and filamentous forms in an early-stage Candida albicans biofilm. Antimicrob. Agents Chemother. 46: 3522 3531.
221. Taylor, J. H.,, and J. T. Holah. 1996. A comparative evaluation with respect to the bacterial cleanability of a range of wall and floor surface materials used in the food industry. J. Appl. Bacteriol. 81: 262 266.
222. Teitzel, G. M.,, and M. R. Parsek. 2003. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol. 69: 2313 2320.
223. Teplitski, M.,, J. B. Robinson,, and W. D. Bauer. 2000. Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria. Mol. Plant Microb. Interact. 13: 637 648.
224. Thar, R.,, M. Kühl,, and G. Holst. 2001. Fiberoptic fluorometer for microscale mapping of photosynthetic pigments in microbial communities. Appl. Environ. Microbiol. 67: 2823 2828.
225. Todd, S. J.,, A. J. Moir,, M. J. Johnson,, and A. Moir. 2003. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. J. Bacteriol. 185: 3373 3378.
226. Toledo-Arana, A.,, J. Valle,, C. Solano,, M. J. Arrizubieta,, C. Cucarella,, M. Lamata,, B. Amorena,, J. Leiva,, J. R. Penades,, and I. Lasa. 2001. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Appl. Environ. Microbiol. 67: 4538 4545.
227. Tomlin, K. L.,, O. P. Coll,, and H. Ceri. 2001. Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia. Can. J. Microbiol. 47: 949 954.
228. Tyler, B. 1997. XPS and SIMS studies of surfaces important in biofilm formation. Three case studies. Ann. N. Y. Acad. Sci. 831: 114 126.
229. Vallet, I.,, J. W. Olson,, S. Lory,, A. Lazdunski,, and A. Filloux. 2001. The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters ( cup) and their involvement in biofilm formation. Proc. Natl. Acad. Sci. USA 98: 6911 6916.
230. van Loosdrecht, M. C. M.,, and S. J. Heijnen. 1993. Biofilm bioreactors for waste-water treatment. Trends Biotechnol. 11: 117 121.
231. von Canstein, H.,, Y. Li,, E. Haase,, A. Felske,, W.-D. Deckwer,, and I. Wagner-Dobler. 2002. Spatially oscillating activity and microbial succession of mercury-reducing biofilms in a technical- scale bioremediation system. Appl. Environ. Microbiol. 68: 1938 1946.
232. Vroom, J. M.,, K. J. De Grauw,, H. C. Gerritsen,, D. J. Bradshaw,, P. D. Marsh,, G. K. Watson,, J. J. Birmingham,, and C. Allison. 1999. Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy. Appl. Environ. Microbiol. 65: 3502 3511.
233. Vuopio-Varkila, J.,, and G. K. Schoolnik. 1991. Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins. J. Exp. Med. 174: 1167 1177.
234. Walker, J. T.,, D. J. Bradshaw,, A. M. Bennett,, M. R. Fulford,, M. V. Martin,, and P. D. Marsh. 2000. Microbial biofilm formation and contamination of dental-unit water systems in general dental practice. Appl. Environ. Microbiol. 66: 3363 3367.
235. Walters, M. C., III,, F. Roe, , A. Bugnicourt, , M. J. Franklin, , and P. S. Stewart. 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.
236. Watnick, P.,, and R. Kolter. 2000. Biofilm, city of microbes. J. Bacteriol. 182: 2675 2679.
237. Watnick, P. I.,, C. M. Lauriano,, K. E. Klose,, C. Croal,, and R. Kolter. 2001. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol. Microbiol. 39: 223 235.
238. Welch, R. A.,, V. Burland, , G. Plunkett III,, P. Redford,, P. Roesch,, D. A. Rasko,, E. L. Buckles,, S. R. Liou,, A. Boutin,, J. Hackett,, D. Stroud,, G. F. Mayhew,, D. J. Rose,, S. Zhou,, D. C. Schwartz,, N. T. Perna,, H. L. T. Mobley,, M. S. Donnenberg,, and F. R. Blattner. 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 99: 17020 17024.
239. Wen, Z. T.,, and R. A. Burne. 2002. Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans. Appl. Environ. Microbiol. 68: 1196 1203.
240. Wentland, E. J.,, P. S. Stewart,, C. T. Huang,, and G. A. McFeters. 1996. Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm. Biotechnol. Prog. 12: 316 321.
241. Whiteley, M.,, M. G. Bangera,, R. E. Bumgarner,, M. R. Parsek,, G. M. Teitzel,, S. Lory,, and E. P. Greenberg. 2001a. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413: 860 864.
242. Whiteley, M.,, E. Brown,, and R. J. C. McLean. 1997. An inexpensive chemostat apparatus for the study of microbial biofilms. J. Microbiol. Methods 30: 125 132.
243. Whiteley, M., , J. R. Ott, , E. A. Weaver, , and R. J. C. McLean . 2001b. Effects of community composition and growth rate on aquifer biofilm bacteria and their susceptibility to betadine disinfection. Environ. Microbiol. 3: 43 52.
244. Woolfson, A. D.,, S. P. Gorman,, D. F. McCafferty,, and D. S. Jones. 1987. On the statistical evaluation of adherence assays. J. Appl. Bacteriol. 63: 147 151.
245. Yildiz, F. H.,, and G. K. Schoolnik. 1999. Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc. Natl. Acad. Sci. USA 96: 4028 4033.
246. Zhang, Y.,, and R. M. Miller. 1995. Effect of rhamnolipid (biosurfactant) structure on solubilization and biodegradation of n-alkanes. Appl. Environ. Microbiol. 61: 2247 2251.


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Examples of organisms used in biofilm research

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Examples of biofilm growth techniques used in field studies

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Medically important biofilms

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Industrial applications

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Common laboratory biofilm techniques

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Biofilm imaging, image analysis, and infrared spectroscopy

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20
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Broadly based biofilm genetics, genomics, and proteomics

Citation: McLean R, Bates C, Barnes M, McGowin C, Aron G. 2004. Methods of Studying Biofilms, p 379-413. In Ghannoum M, O'Toole G (ed), Microbial Biofilms. ASM Press, Washington, DC. doi: 10.1128/9781555817718.ch20