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 since 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 (XTT) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately two days to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step towards the standardization of antifungal susceptibility testing of biofilms.

Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm

“…The tetrazolium salt XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt) (Sigma, St. Louis, MO) was used to measure the metabolic activity of A. fumigatus. Whereas XTT is a direct measure of the metabolic activity of cells, previous studies of A. fumigatus have indicated that XTT results are linear with mass and equated the XTT result with dry weight (19)(20)(21). Our confocal microscopy studies also address this point.…”

“…The 96-well assay is both sparing of reagents and allows more replicates to be studied concurrently, which increases the chances of statistical significance occurring when differences between groups are small, e.g., at the inflection point of a dose-response curve. An analogous 96-well assay used to assay antifungal drugs, and differing in specifics from ours, was previously described (19). The interpretations made here are based on 7 experiments on biofilm formation in the 12-well format and 9 in the 96-well format, which included 10AF as a reference control.…”

Plasticity of Candida albicans Biofilms

“…Aspects of this model were changed over time by other research groups. The plastic bottoms of the wells of multiwell tissue culture dishes, rather than serum-treated catheter discs, were employed as substrata for purposes of expediency, since tissue culture plastic proved to be adhesive (180)(181)(182). YNB medium was also replaced with different media to support planktonic growth and biofilm formation (38).…”

In Vitro Antibiofilm Activity of Eucarobustol E against Candida albicans

“…For detection of EE on mature biofilms, biofilms pregrown for 24 h were treated with different concentrations of EE and were incubated for 24 h at 37°C. After incubation, the supernatant was discarded and XTT (Sigma, USA) was added to the prewashed biofilms (69). The plate was incubated in the dark for 2 to 3 h at 37°C.…”