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New Technologies for Studying Biofilms

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  • Authors: Michael J. Franklin1, Connie Chang3, Tatsuya Akiyama5, Brian Bothner7
  • Editors: Mahmoud Ghannoum9, Matthew Parsek10, Marvin Whiteley11, Pranab Mukherjee12
    Affiliations: 1: Center for Biofilm Engineering, Montana State University-Bozeman, Bozeman, MT 59717; 2: Department of Microbiology and Immunology, Montana State University-Bozeman, Bozeman, MT 59717; 3: Center for Biofilm Engineering, Montana State University-Bozeman, Bozeman, MT 59717; 4: Department of Chemical and Biological Engineering, Montana State University-Bozeman, Bozeman, MT 59717; 5: Center for Biofilm Engineering, Montana State University-Bozeman, Bozeman, MT 59717; 6: Department of Microbiology and Immunology, Montana State University-Bozeman, Bozeman, MT 59717; 7: Center for Biofilm Engineering, Montana State University-Bozeman, Bozeman, MT 59717; 8: Department of Chemistry and Biochemistry, Montana State University-Bozeman, Bozeman, MT 59717; 9: Case Western Reserve University, Cleveland, OH; 10: University of Washington, Seattle, WA; 11: University of Texas at Austin, Austin, TX; 12: Case Western Reserve University, Cleveland, OH
  • Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
  • Received 30 December 2014 Accepted 17 February 2015 Published 21 August 2015
  • Michael J. Franklin, [email protected]
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  • Abstract:

    Bacteria have traditionally been studied as single-cell organisms. In laboratory settings, aerobic bacteria are usually cultured in aerated flasks, where the cells are considered essentially homogenous. However, in many natural environments, bacteria and other microorganisms grow in mixed communities, often associated with surfaces. Biofilms are comprised of surface-associated microorganisms, their extracellular matrix material, and environmental chemicals that have adsorbed to the bacteria or their matrix material. While this definition of a biofilm is fairly simple, biofilms are complex and dynamic. Our understanding of the activities of individual biofilm cells and whole biofilm systems has developed rapidly, due in part to advances in molecular, analytical, and imaging tools and the miniaturization of tools designed to characterize biofilms at the enzyme level, cellular level, and systems level.

  • Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms. Microbiol Spectrum 3(4):MB-0016-2014. doi:10.1128/microbiolspec.MB-0016-2014.


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Bacteria have traditionally been studied as single-cell organisms. In laboratory settings, aerobic bacteria are usually cultured in aerated flasks, where the cells are considered essentially homogenous. However, in many natural environments, bacteria and other microorganisms grow in mixed communities, often associated with surfaces. Biofilms are comprised of surface-associated microorganisms, their extracellular matrix material, and environmental chemicals that have adsorbed to the bacteria or their matrix material. While this definition of a biofilm is fairly simple, biofilms are complex and dynamic. Our understanding of the activities of individual biofilm cells and whole biofilm systems has developed rapidly, due in part to advances in molecular, analytical, and imaging tools and the miniaturization of tools designed to characterize biofilms at the enzyme level, cellular level, and systems level.

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Examples of methods for biofilm cultivation under static conditions. (A) Biofilm cultured at the air-water interface, forming a pellicle. Published with permission from reference 83 . (B) Biofilm cultured on a glass coupon under static conditions. Published with permission from reference 84 . (C) Example of biofilm growth as a colony biofilm. Published with permission from reference 84 .

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
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Examples of continuous-flow reactors for biofilm cultivation. (A) CDC reactor with medium inlet and outlet ports. Biofilms form on coupons arranged on removable Teflon rods. Published with permission from reference 88 . (B) Drip-flow reactor with medium inlet and outlet ports and air exchange ports. Biofilms form on removable slides. Published with permission from reference 89 . (C) Capillary flow cell for imaging biofilms. Published with permission from http://centerforgenomicsciences.org/research/biofilm_flow.html.

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
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Examples of microfluidics approaches applied to biofilm research. (A) Example of a microfluidics device for precise control of fluids. Published with permission from reference 110 . (B) Biofilm streamers forming within a microfluidics flow channel. Published with permission from reference 127 . Microdroplet biofilm reactor showing phenotypic switching of cells and simultaneous change in expression from cyan fluorescent protein to the yellow fluorescent protein Published with permission from reference 259 . (D) Schematic representation of microfluidics flow cell. Published with permission from reference 133 .

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
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Example of a biofilm vertical transect, showing GFP–gene expression heterogeneity. Areas were cut from different biofilm strata and captured for transcriptomics analyses. Published with permission from reference 61 .

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
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Gene expression heterogeneity, demonstrated by translational fusions of target proteins to the yellow fluorescent protein (YFP). (A) Translational fusion of the IbpA protein to YFP, showing uniform distribution of IbpA throughout the biofilm. Cells were counterstained with mCherry fluorescent protein (mCFP). (B) Translational fusion of Rmf protein to YFP, showing that most Rmf production occurs in cells at the top of the biofilm. Cells counterstained with mCFP (M.J. Franklin, unpublished data).

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
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(A) Images of biofilms, where the cells constitutively express the GFP. (B) Extracellular matrix material stained with Bodipy 630/650-X NHS from Life Technologies. (C) Combined image showing GFP-fluorescent bacteria and Bodipy-stained matrix (M.J. Franklin, unpublished data).

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
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Methods for cultivation of biofilms

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014
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Imaging strategies in biofilm studies

Source: microbiolspec August 2015 vol. 3 no. 4 doi:10.1128/microbiolspec.MB-0016-2014

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