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

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.

Figure 1

A flow chart diagram summarizing the different steps of the procedure.

Figure 2

Colorimetric readings (OD₄₉₀ values) from XTT-reduction assays of biofilms formed by C. albicans strain SC5314 in wells of microtiter plates. Values are for 10 independent biofilms formed in each of 8 different rows of the same 96 well microtiter plate.

Figure 3

Activity of different caspofungin concentrations against pre-formed biofilms of C. albicans SC5314. Values are expressed as average percent colorimetric readings for XTT-reduction assays as compared to control wells. Note that, as previously described, caspofungin loses efficacy at non-physiological high concentrations. This has been referred to as the “paradoxical” or “Eagle” effect.