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A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing

Nature Protocols volume 3, pages 1494–1500 (2008)Cite this article

Abstract

The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.

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Figure 1
Figure 2: Colorimetric readings (OD490 values) from XTT-reduction assays of biofilms formed by C. albicans strain SC5314 in wells of microtiter plates.
Figure 3: Activity of different caspofungin concentrations against preformed biofilms of C. albicans SC5314.

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Acknowledgements

Work in the laboratory is funded by Grant number 5R21DE017294 from the National Institute of Dental & Craniofacial Research (to J.L.L.-R.). The content is solely our responsibility and does not necessarily represent the official views of the NIDCR, the NIAID or the NIH. A.R.T. was supported by the McNair Scholars Program at St Mary's University funded by the US Department of Education.

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Authors and Affiliations

  1. Department of Biology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, 78249, Texas, USA

    Christopher G Pierce, Priya Uppuluri, Amanda R Tristan, Floyd L Wormley Jr & Jose L Lopez-Ribot

  2. Section of Infection and Immunity, Glasgow Dental School and Hospital, 378 Sauchiehall Street, Glasgow, G2 3JZ, UK

    Eilidh Mowat & Gordon Ramage

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  1. Christopher G Pierce
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Correspondence to Jose L Lopez-Ribot.

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Pierce, C., Uppuluri, P., Tristan, A. et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 3, 1494–1500 (2008). https://doi.org/10.1038/nprot.2008.141

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