In Vitro Culturing and Screening of Candida albicans Biofilms

Abstract

Candida albicans is a normal member of the human microbiota that asymptomatically colonizes healthy individuals, however it is also an opportunistic pathogen that can cause severe infections, especially in immunocompromised individuals. The medical impact of C. albicans depends, in part, on its ability to form biofilms, communities of adhered cells encased in an extracellular matrix. Biofilms can form on both biotic and abiotic surfaces, such as tissues and implanted medical devices. Once formed, biofilms are highly resistant to antifungal agents and the host immune system, and can act as a protected reservoir to seed disseminated infections. Here, we present several in vitro biofilm protocols, including protocols that are optimized for high-throughput screening of mutant libraries and antifungal compounds. We also present protocols to examine specific stages of biofilm development and protocols to evaluate interspecies biofilms that C. albicans forms with interacting microbial partners. © 2018 by John Wiley & Sons, Inc.

WITHDRAWN: A modified crystal violet assay is comparable to XTT reduction assay for quantification of biofilm formation by Candida albicans

“…In this study, we introduced four modi cations stemmed from Deighton's work (2001) into the conventional CV assay described by Gulati et al (2018) [5,13]. We show that the modi ed CV assay is superior to the conventional CV assay and is at least comparable to XTT reduction assay in quanti cation of bio lm formation by C. albicans, supported by higher accuracy, higher reproducibility and other merits.…”

“…The bio lm CV assay described by Gulati et al (2018), referred to as the conventional CV assay in this study, was used as the basis for modi cations as it has been widely used by others to study Candida bio lms [4,5,8,[21][22][23][24]. After bio lms were developed, cell suspensions were aspirated from the microplate using a multichannel pipette, and each microwell was washed twice with 200 µl of PBS to remove non-adherent cells.…”

“…This characteristic applies to C. albicans clinical isolates and genetically-manipulated mutants, and has been accepted as an indicator of strain virulence [3,4]. Numerous assays have been developed to study bio lm formation of C. albicans [5]. Qualitative methods include scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) [4].…”

“…Qualitative methods include scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) [4]. Results from qualitative assays are complemented by quantitative methods such as the colony forming unit (CFU) assay, the bioluminescence assay, the 2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, dry weight measurement assay, and the crystal violet (CV) assay [5,6]. Each quantitative method has disadvantages, including not being amenable to high-throughput screening (CFU assay and dry weight measurement assay) [7], low-reproducibility (dry weight measurement assay and CV assay) [6,[8][9][10], unsatisfactory accuracy (XTT reduction assay) [11], long processing-time (CFU assay) [10], and high labor-intensity (CFU assay and ATP bioluminescence) [7].…”

“…The XTT reduction assay is a widely used quantitative method for bio lm formation of C. albicans [5]. The principle behind this assay is that tetrazolium XTT can be reduced to XTT formazan by the mitochondrial dehydrogenases of metabolically active yeast cells.…”