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Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening

Nature Protocols volume 5, pages 1236–1254 (2010)Cite this article

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Abstract

Batch culture of biofilms on peg lids is a versatile method that can be used for microtiter determinations of biofilm antimicrobial susceptibility. In this paper, we describe a core protocol and a set of parameters (surface composition, the rate of rocking or orbital motion, temperature, cultivation time, inoculum size, atmospheric gases and nutritional medium) that can be adjusted to grow single- or multispecies biofilms on peg surfaces. Mature biofilms formed on peg lids can then be fitted into microtiter plates containing test agents. After a suitable exposure time, biofilm cells are disrupted into a recovery medium using sonication. Microbicidal endpoints can be determined qualitatively using optical density measurements or quantitatively using viable cell counting. Once equipment is calibrated and growth conditions are at an optimum, the procedure requires ∼5 h of work over 4–6 d. This efficient method allows antimicrobial agents and exposure conditions to be tested against biofilms on a high-throughput scale.

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Figure 1: A schematic of a peg lid biofilm reactor, using the Calgary Biofilm Device (CBD) as an example.
Figure 2: Flowchart and timeline for biofilm cultivation and antimicrobial susceptibility testing of biofilms growing on peg lids.
Figure 3: Measurement and calibration of the rocking table angle.
Figure 4: The basics: reading qualitative endpoints from patterns in recovery plates and interpreting biofilm survival data from kill curves.
Figure 5: Time-depending killing of P. aeruginosa ATCC 27853 biofilms exposed to the aminoglycoside gentamicin for 2 or 20 h.

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Acknowledgements

J.J.H. is supported by a post-doctoral fellowship from the Natural Sciences and Engineering Research Council (NSERC) of Canada. R.J.T. and H.C. are supported by NSERC discovery grants. The authors would like to thank W.D. Wade and S. Ravaioli for providing data on the calibration of the rocking table.

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Authors and Affiliations

  1. Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA

    Joe J Harrison

  2. Biofilm Research Group and Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

    Carol A Stremick, Raymond J Turner & Howard Ceri

  3. Innovotech Incorporated, Edmonton, Alberta, Canada

    Nick D Allan & Merle E Olson

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J.J.H. wrote the manuscript. All of the authors contributed extensively to protocol development and to revisions.

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Correspondence to Joe J Harrison or Howard Ceri.

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Competing interests

The authors declare competing financial interests. H.C. and M.E.O. are shareholders in Innovotech Inc. and N.D.A. and M.E.O. serve as the Laboratory Manager and Research Director for this company, respectively.

Supplementary information

Supplementary Table 1

Batch culture parameters and apparatus configurations for microbial attachment and/or biofilm growth on peg lids. (DOC 556 kb)

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Harrison, J., Stremick, C., Turner, R. et al. Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening. Nat Protoc 5, 1236–1254 (2010). https://doi.org/10.1038/nprot.2010.71

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