The use of new probes and stains for improved assessment of cell viability and extracellular polymeric substances in Candida albicans biofilms

Abstract

Phenotypic and genotypic cell differentiation is considered an important feature that confers enhanced antifungal resistance in candidal biofilms. Particular emphasis has been placed in this context on the viability of biofilm subpopulations, and their heterogeneity with regard to the production of extracellular polymeric substances (EPS). We therefore assessed the utility of two different labeled lectins Erythrina cristagalli (ECA) and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the viability of candidal biofilms, we further studied combination stains, SYTO9 and propidium iodide (PI). The latter combination has been successfully used to assess bacterial, but not fungal, viability although PI alone has been previously used to stain nuclei in fungal cells. Candida albicans biofilms were developed in a rotating disc biofilm reactor and observed in situ using confocal scanning laser microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable vital stains that may be used to investigate C. albicans biofilms. When used together with ConA, the lectin ECA optimized EPS visualization and revealed differential production of this material in mature candidal biofilms. The foregoing probes and stains and the methodology described should help better characterize C. albicans biofilms in terms of cell their viability, and EPS production.

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

“…1). We (and others) have previously shown that the XTT-reduction assay shows excellent correlation between cellular density and metabolic activity, thus providing a semiquantitative measurement of biofilm formation [22][23][24][25][26] . This colorimetric assay is non-invasive and non-destructive, requiring minimal postprocessing of samples as compared to other alternative methods (such as viable cell counts) and has the additional advantage that, in contrast to other methods such as crystal violet staining and dry mass measurements, it correlates with cell viability which is particularly useful for measuring the effects of drugs on biofilm cells.…”

“…In addition, alterations in the metabolic states during the different phases of biofilm formation may lead to fluctuations in the ability of cells within the biofilms to metabolize this dye 23,27 . Some more recent articles indicate that the use of other vital stains (SYTO9, propidium iodide), fluorogenic assays or bioluminescence may represent alternatives and have some practical advantages over the use of XTT 23,24,28 . This 96 well microtiter plate model for biofilm formation was originally developed for C. albicans and other Candida spp.…”

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms

“…In the presence of xanthochymol or garcinol, large propidium iodide-positive areas (red staining) were evident, characteristic of inviable cells with leaky membranes ( Fig. 2A and B) (28). Close examination of treated biofilms showed that both compounds were effective at killing hyphae present in the mature biofilm, whereas the yeast form cells were Syto-9 positive (green), indicating they were viable.…”

Cell Viability of Candida albicans Against the Antifungal Activity of Thymol

“…Silva et al (13) claim that it is expected that concentrations higher than the MIC could interfere on biofilms at metabolic levels, but it is not fully understood whether antifungal agents at concentrations higher than MIC could actually affect the biofilm architecture. According to Jin et al (14), many coloring substances may be used to allow quantification of microbial biofilms, such as the FUN-1 dye, widely used in the investigation of antifungal resistance and cell viability of C. biofilms. However, these authors pointed out that the FUN-1 could lead to overestimation of living cells, especially when cell density is high.…”