Three C. albicans strains were chosen for their varied abilities to form biofilms. The reference strain DAY185 produces extensive biofilms with abundant extracellular matrices and variable cell morphologies [19 (link)]. The two mutant strains lack adhesions, resulting in a partial biofilm defect for als3Δ/Δ and a more extensive biofilm defect in strain als1Δ/Δ als3Δ/Δ [8 ,20 (link)–22 (link)]. To evaluate biofilm formation, overnight cultures grown in yeast peptone dextrose (YPD) broth [8 ] at 30°C with orbital shaking at 200 rpm were enumerated by hemocytometer. Cells were resuspended in RPMI-MOPS [7 (link)] at 106 cells/ml and each well of a six-well polystyrene plate was inoculated with 1 ml of this suspension. After a 1 h adhesion period, the inoculum was removed and fresh media was applied. Biofilms were grown for 48 h at 37°C on an orbital shaker at 50 rpm. For comparative scanning electron microscopy (SEM), biofilms were formed on coverslips and processed and examined as previously described [23 (link)].
Protocols for the XTT and crystal violet assays have been described [7 (link),24 (link)]. For the crystal violet assay, 4% aqueous crystal violet was added for 45 min and 100 µl aliquots were taken from each well for absorbance measurement (595 nm). Candida DNA quantification was accomplished using a commercially available kit (Genomic DNA Wizard Kit – Promega) following cellular disruption by bead-beating [25 (link)]. DNA samples were also used for qPCR with ACT1 primers and probes [23 (link)] and Quantitect Probe qPCR kit (Qiagen) performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). The Protein Assay Kit (Pierce) was used to determine total cellular protein after biofilms were scraped from wells into 1 ml of ddH2O, sonicated for 20 min, disrupted by bead-beating, and boiled. Dry cell weight of each biofilm was determined following disruption, collection, and then dehydrated by vacuum centrifugation. Viable burdens were determined by plating serial dilutions following biofilm disruption, vortexing, and sonicated using a waterbath for 20 min to disperse cells [14 ].
Reproducibility was estimated by the standard deviation and coefficient of variation among replicates. At least six biofilms were grown for each strain and assay.
Protocols for the XTT and crystal violet assays have been described [7 (link),24 (link)]. For the crystal violet assay, 4% aqueous crystal violet was added for 45 min and 100 µl aliquots were taken from each well for absorbance measurement (595 nm). Candida DNA quantification was accomplished using a commercially available kit (Genomic DNA Wizard Kit – Promega) following cellular disruption by bead-beating [25 (link)]. DNA samples were also used for qPCR with ACT1 primers and probes [23 (link)] and Quantitect Probe qPCR kit (Qiagen) performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). The Protein Assay Kit (Pierce) was used to determine total cellular protein after biofilms were scraped from wells into 1 ml of ddH2O, sonicated for 20 min, disrupted by bead-beating, and boiled. Dry cell weight of each biofilm was determined following disruption, collection, and then dehydrated by vacuum centrifugation. Viable burdens were determined by plating serial dilutions following biofilm disruption, vortexing, and sonicated using a waterbath for 20 min to disperse cells [14 ].
Reproducibility was estimated by the standard deviation and coefficient of variation among replicates. At least six biofilms were grown for each strain and assay.
