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In vitro growth and analysis of Candida biofilms

Nature Protocols volume 3, pages 1909–1924 (2008)Cite this article

Abstract

Evaluation of fungal biofilm formation can be performed using several techniques. In this protocol, we describe methods used to form Candida biofilms on three different medical device substrates (denture strips, catheter disks and contact lenses) to quantify them and to evaluate their architecture and drug susceptibility. Biofilm formation involves adhesion of fungal cells to pretreated substrates, followed by growth in medium. Formed biofilms are quantified by determining their metabolic activity and dry weight, whereas their gross morphology and architecture are evaluated using fluorescence microscopy, scanning electron microscopy and confocal scanning laser microscopy techniques. Susceptibility of biofilms is determined by comparing their metabolic activity in the presence of antifungal agents with that in their absence. The methods described here can be completed in a typical laboratory with minimum involvement of software. Evaluation of the growth of fungal biofilms and their analyses can be completed using the described methods in ∼15 d.

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Figure 1: Flow chart of steps involved in the protocol to form and analyze biofilms on different substrates.
Figure 2: Representative images showing gross morphology of C. albicans biofilm formed on different substrates.
Figure 3: Representative results obtained using the XTT and dry weight assays to quantify biofilms.
Figure 4: Fluorescence micrographs of C. albicans biofilms grown to different time points.
Figure 5: Schematic representation of C. albicans biofilm formation on different device substrates.
Figure 6: SEM images of C. albicans biofilms formed on denture strips, as described in Steps 8B(i–xxxviii).
Figure 7: Confocal scanning laser microscopy analysis of biofilms formed on different device substrates.
Figure 8: Antifungal susceptibility of different developmental phases of C. albicans denture biofilms.

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Acknowledgements

Results described in this article were generated in studies funded by the NIH (AI-35097-04, R01DE 13932-4), the Bristol Myers Squibb Foundation (Freedom to Discover Award to M.A.G.), Steris Corporation Award for Emerging/Nosocomial Infections (no. 1–88-8225), Center for AIDS Research at Case Western Reserve University (grant no. AI-36219), NCI (Confocal Core Facility, funded by grant no. P30 CA43703–12), the NIH-funded Skin Diseases Research Center at Case Western Reserve University (5P30-AR039750), Dermatology Foundation Janssen Pharmaceutical Research Fellowship (to P.K.M.) and American Heart Association (Scientist Development Grant 0335313N Award to P.K.M.). We appreciate the assistance of Nancy Isham for her critique and review of the manuscript.

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  1. Center for Medical Mycology and Department of Dermatology, University Hospitals Case Medical Center and Case Western Reserve University, 11100 Euclid Avenue, Cleveland, 44106, Ohio, USA

    Jyotsna Chandra, Pranab K Mukherjee & Mahmoud A Ghannoum

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  1. Jyotsna Chandra
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  3. Mahmoud A Ghannoum
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Correspondence to Mahmoud A Ghannoum.

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Chandra, J., Mukherjee, P. & Ghannoum, M. In vitro growth and analysis of Candida biofilms. Nat Protoc 3, 1909–1924 (2008). https://doi.org/10.1038/nprot.2008.192

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