Optimized candidal biofilm microtiter assay

Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

“…Different methods for assessing Candida biofilm formation have been reported in the literature (Krom et al 2007, Pierce et al 2008. In this study, colorimetric evaluation showed that the absorbance readings were proportional to cell density of the biofilm in agreement with Krom et al (2007) and Nuryastuti et al (2009).…”

“…It has been previously argued that metabolic activity of a biofilm is correlated with cell numbers and that higher levels of MTT reduction indicate higher numbers of cells (Krom et al 2007). In this study, we used a growth curve to show that MTT assay absorbance readings were proportional to the cellular density of the biofilm.…”

“…Standard cell suspensions [100 µL of a suspension containing 1 × 10 6 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 supplemented with Lglutamine and buffered with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma)] were seeded in selected wells and incubated for a series of time intervals (4,6,8,12,18,24,36,48,72,84 and 96 h) at 37ºC. A semi-quantitative measure of the formed biofilms was calculated using a 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-2-H-tetrazolium-5-carboxanilide (MTT) (Sigma) reduction assay (Berridge et al 2005, Krom et al 2007, Nuryastuti et al 2009). MTT solution (20 µL) [a stock solution containing 5 mg of MTT per mL of phosphate buffered saline (PBS)] was added to each well and the samples were incubated at 37ºC for 6 h. After staining, plates were washed three times with PBS.…”

Metal Ions May Suppress or Enhance Cellular Differentiation in Candida albicans and Candida tropicalis Biofilms

“…Note that the CBD, either used as supplied by the manufacturer or modified in a way that supported biofilm growth for C. tropicalis, could not be used to cultivate C. albicans biofilms (data not shown). The CBD is made of polystyrene, a hydrophobic substratum that may interfere with C. albicans surface adhesion (19). However, biofilm formation by C. tropicalis in the CBD followed a stepwise progression similar to that observed in microtiter plates.…”

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).…”