Assessment and Optimizations of Candida albicans In Vitro Biofilm Assays

Abstract

Candida albicans biofilms have a significant medical impact due to their rapid growth on implanted medical devices, their resistance to antifungal drugs, and their ability to seed disseminated infections. Biofilm assays performed in vitro allow for rapid, high-throughput screening of gene deletion libraries or antifungal compounds and typically serve as precursors to in vivo studies. Here, we compile and discuss the protocols for several recently published C. albicans in vitro biofilm assays. We also describe improved versions of these protocols as well as novel in vitro assays. Finally, we consider some of the advantages and disadvantages of these different types of assays.

Development and regulation of single- and multi-species Candida albicans biofilms

“…Candida albicans biofilms can form under a range of environmental conditions in vitro , such as different surface materials (for example, polystyrene plates or silicone squares), different growth media, different temperatures and different shaking or flow rates 151 . Whereas biofilms can be directly observed using some in vitro assays (for example, confocal laser-scanning microscopy and biofilm formation on silicone squares) 46,48 , other methods rely on correlated readouts for biofilm formation (for example, dry weight and optical density) 46,61,68,152 . Measurements of cell viability (for example, metabolic reduction of the tetrazolium salt reagent XTT) 153,154 and dye uptake (for example, crystal violet incorporation into the biofilm) 155 are also commonly used as readouts to evaluate biofilm formation in vitro .…”

Distinct roles of the 7-transmembrane receptor protein Rta3 in regulating the asymmetric distribution of phosphatidylcholine across the plasma membrane and biofilm formation inCandida albicans

“…In lieu of the transcriptional profiling data, we proposed that RTA3 may function in biofilm development by promoting adherence via BCR1 and its target adhesin genes. Therefore, we subjected the rta3 Δ/Δ mutant to two standard in vitro biofilm assays, an optical density assay and a silicone square assay (Lohse et al ., ). We observed no significant biofilm defects in the rta3 Δ/Δ strain versus the wild type in these two vitro biofilm assays (Figure S6a and S6b).…”

“…The internalized PE is represented as the percentile dithionite‐resistant NBD‐PE fluorescence from total NBD‐PE fluorescence. FIGURE S6 In vitro biofilm formation (a) In vitro biofilm assays were carried out in Spider medium by growing the biofilm directly on the bottom on the 96 well polystyrene plates, as described previously (Fox et al ., ; Lohse et al ., ). Briefly, strains were grown overnight in YEPD at 30 oC for 12‐14 h and diluted to an optical density at OD 600 of 0.5 in 200 μl Spider medium.…”

Visualization of Biofilm Formation in <em>Candida albicans</em> Using an Automated Microfluidic Device

“…The assay, however, is still an in vitro assay, and results will need to be validated in relevant animal models. In our experiences, the results of this microfluidic biofilm assay have had the best predictive value for in vivo biofilm assays compared with other in vitro assays we have assessed 14 .…”

“…In vitro biofilm assays, on the other hand, allow for the rapid, high-throughput assessment of antifungal compounds and mutant strains, and are much more cost-effective and ethical than biofilm assays carried out in animal models 9,10,11,12,13,14 . Here we describe an in vitro assay that we developed and optimized to observe biofilm formation temporally under laminar flow using a customizable microfluidic device 14,15 . The assay allows for the visualization of each stage of biofilm formation, including the initial adherence step, cell proliferation, biofilm maturation, and cell dispersion.…”