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Microtiter plate assays to assess antibiofilm activity against bacteria

Nature Protocols volume 16, pages 2615–2632 (2021)Cite this article

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Abstract

Bacterial biofilms demonstrate high broad-spectrum adaptive antibiotic resistance and cause two thirds of all infections, but there is a lack of approved antibiofilm agents. Unlike the standard minimal inhibitory concentration assay to assess antibacterial activity against planktonic cells, there is no standardized method to evaluate biofilm inhibition and/or eradication capacity of novel antibiofilm compounds. The protocol described here outlines simple and reproducible methods for assessing the biofilm inhibition and eradication capacities of novel antibiofilm agents against adherent bacterial biofilms grown in 96-well microtiter plates. It employs two inexpensive dyes: crystal violet to stain adhered biofilm biomass and 2,3,5-triphenyl tetrazolium chloride to quantify metabolism of the biofilm cells. The procedure is accessible to any laboratory with a plate reader, requires minimal technical expertise or training and takes 4 or 5 d to complete. Recommendations for how biofilm inhibition and eradication results should be interpreted and presented are also described.

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Fig. 1: Workflow and timeline estimates for the biofilm inhibition (gray boxes) and eradication (white boxes) assays for testing the antibiofilm effects of antibiotics and HDPs.
Fig. 2: Biofilm inhibition of various antimicrobials versus S. aureus USA300 LAC and P. aeruginosa PAO1.
Fig. 3: Biofilm eradication assay results for various antimicrobials versus S. aureus USA300 LAC and P. aeruginosa PAO1.
Fig. 4: Biofilm CFUs recovery for P. aeruginosa PAO1 and S. aureus USA300 LAC biofilms.

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The authors declare that all data generated or analyzed in this study are included in the article or in the accompanying supplementary information file. Source data are provided with this paper.

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Acknowledgements

Our own biofilm work was supported by the Canadian Institutes of Health Research (CIHR), funding reference number FDN-154287. REWH holds a Canada Research Chair in Health and Genomics and a UBC Killam Professorship.

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Author notes

  1. These authors contributed equally: Evan F. Haney, Michael J. Trimble.

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

    Evan F. Haney, Michael J. Trimble & Robert E. W. Hancock

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Contributions

E.F.H. and M.J.T. developed the protocols, collected and analyzed data, and wrote and edited the first draft of the manuscript. R.E.W.H. was involved in development of the protocol and extensively edited the manuscript.

Corresponding author

Correspondence to Robert E. W. Hancock.

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Competing interests

E.F.H. and R.E.W.H. have invented and filed for patent protection on related antibiofilm peptide sequences. This patent has been assigned to their employer, the University of British Columbia, and licensed to ABT Innovations, in which R.E.W.H. has an ownership position. M.J.T. declares no competing interests.

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Peer review information Nature Protocols thanks Tom Coenye and Henny C. van der Mei for their contribution to the peer review of this work.

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Key references using this protocol

Haney, E. F., Trimble, M. J., Cheng, J. T., Vallé, Q. & Hancock, R. E. Biomolecules 8, 29 (2018): https://doi.org/10.3390/biom8020029

Haney, E. F. et al. Sci. Rep. 8, 1871 (2018): https://doi.org/10.1038/s41598-018-19669-4

Haney, E. F., Barbosa, S. C., Baquir, B. & Hancock, R. E. W. J. Med. Chem. 62, 10294–10304 (2019): https://doi.org/10.1021/acs.jmedchem.9b01344

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Haney, E.F., Trimble, M.J. & Hancock, R.E.W. Microtiter plate assays to assess antibiofilm activity against bacteria. Nat Protoc 16, 2615–2632 (2021). https://doi.org/10.1038/s41596-021-00515-3

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