Conditions for Optimal Candida Biofilm Development in Microtiter Plates

Abstract

Development of Candida spp. biofilms on medical devices such as catheters and voice prosthesis has been recognized as an increasing clinical problem. Simple device removal is often impossible, while in addition, resulting candidal infections are difficult to resolve due to their increased resistance to many antifungal agents. Susceptibility studies of clinical isolates are generally performed according to the CLSI standard, which measures planktonic cell susceptibility, but similar standards have not been designed or applied to testing of cells growing within a biofilm. As consistent biofilms from many strains are more difficult to simultaneously obtain and analyze than are independent planktonic cultures, any standard assay must address these concerns. In the present chapter, optimized conditions that promote biofilm formation within individual wells of microtiter plates are described. In addition, the method has proven useful in preparing C. albicans biofilms for investigation by a variety of microscopic and molecular techniques.

In Vitro Culturing and Screening of Candida albicans Biofilms

“…The 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2 H -Tetrazolium-5-Carboxanilide (XTT) assay is a colorimetric assay that detects metabolic activity by measuring the reduction of the tetrazolium salt reagent XTT; alternative versions of this assay use the tetrazolium salt reagent MTT (Krom et al , 2009; Krom et al , 2007; Nett et al , 2011; Ramage et al , 2001). This assay can be performed at the end of the Standard Optical Density Assay (Basic Protocol 2) and be combined with the Inhibition and Disruption Optical Density Assays (Alternate Protocols 3 and 4).…”

Determination of biofilm production by Candida tropicalis isolated from hospitalized patients and its relation to cellular surface hydrophobicity, plastic adherence and filamentation ability

“…Adherence to host tissues and medical devices constitutes a first step in colonization and subsequent biofilm formation (BF). Yeast surface attachment occurs at different stages (Andes et al ., ; Kumamoto and Vinces, ) with immediate adhesion (IA) and late adhesion (LA), followed by a differentiation into germinated forms, microcolony formation (Krom et al ., ), and by exopolymeric matrix secretion (Baillie and Douglas, ; Chandra et al ., ). Non‐specific unions, principally attributed to hydrophobic forces, have been implicated in immediate adhesion (Gallardo‐Moreno et al ., ; Jabra‐Rizk et al ., ; Colling et al ., ).…”

Assessment and Optimizations of Candida albicans In Vitro Biofilm Assays

“…For example, a confocal scanning laser microscopy (CSLM) assay examines the architecture of a fluorescently dyed biofilm (or at least that of the parts of the biofilm that the dye can penetrate) (13,14), and a dry weight (biomass) assay (13,15,16,26) measures the mass of the biofilm. Other assays infer biofilm formation on the basis of measurements of cell viability {e.g., metabolic reduction of the tetrazolium salt reagent 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) (27,28) or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (29,30)} or by the uptake of dyes or stains (e.g., crystal violet incorporation into the biofilm [31]). Specialized assays have also been developed to examine specific stages of biofilm development; for example, the number of cells dispersed from an established biofilm can be monitored by CFU counts, hemocytometer counts, and/or optical density measurements (32,33).…”