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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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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J.J.H. wrote the manuscript. All of the authors contributed extensively to protocol development and to revisions.
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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.
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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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DOI: https://doi.org/10.1038/nprot.2010.71
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