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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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