Cells grown in the presence of TB displayed a staining similar to that of control cells (cf Numbers 5B,E). Furthermore, the aberrant cell wall morphology caused by growth in the presence of high concentrations of CFW was visualized in detail using the super-resolution microscopy technique 3D structured illumination (Figure ?(Figure6).6). demonstrating the potential of this approach for morphological investigations or testing assays. cell wall with TB, characterize its spectral properties and, by using confocal microscopy, demonstrate its potential for quantitative analysis of changes in cell wall morphology, as exemplified for growth under anaerobic conditions and in CFW-containing medium. Growth under anaerobic conditions is known to affect the candida cell and, specifically, the cell wall morphology (Aguilar-Uscanga and Francois, 2003). So far, only chemical and enzymatic methods have been used to investigate the changes caused by growth in the absence of oxygen. Likewise, CFW and the related compound Congo reddish are known to cause cell wall deformations at high concentrations (Vannini et al., 1983; Roncero and Durn, 1985), a property used to identify cell wall mutants (de Groot et al., 2001; Ram and Klis, 2006) and to test the involvement of proteins in cell wall synthesis (Kuznetsov et al., 2013). By confocal and super-resolution microscopy we display here that TB staining can be used to visualize fungal cell walls and to analyze variations in cell size and cell wall volume. Materials and methods Strains and growth conditions The wild-type strain W303-1 Vinflunine Tartrate (MAT was used in all experiments. Overnight cultures were cultivated at 28C in YPD medium (1% w/v candida draw out, 2% w/v peptone, and 2% w/v glucose) to a denseness of OD600 = 0.8. For growth on plates, HIF1A 2% agar was added to get solid press. Anaerobic conditions were generated by using an Anaerocult A system (Merck Milipore) and controlled by including an Anaerotest indication stripe (Merck-Millipore) with the tradition vial. When indicated, CFW (MP Biomedicals) and TB (Merck Millipore, for microscopy, order quantity: 111732) were added to the growth medium at a final concentration of 0.1 mg ml?1. This relatively high concentration was used in the context of the investigation of the dye’s effect on cell morphology but not for staining (observe below). Viability checks were performed with aliquots of the growth Vinflunine Tartrate cultures using prodium iodide staining (Sigma-Aldrich, final concentration 1 g ml?1). Staining process Yeast cells were collected by centrifugation (900 g, 2 min, 20C) and suspended in 200C300 l phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) to OD600 = 1. TB was added to the cells in the tube or directly on the slip at a final concentration of 10 g ml?1. Staining can be considered immediate, i.e., no incubation time is required. Additional washing methods after staining, including the resuspension in PBS and centrifugation repeated up to 3 times, were found to have minimal effect on the level of background fluorescence. Therefore, washing of cells after staining was generally omitted. Staining with CFW was performed similarly using a final concentration of 100 Vinflunine Tartrate g ml?1. Pontamine Fast Scarlet 4BS (Aldrich Rare Chemicals Library) was added to cell cultures 20 min before harvesting at concentrations up to 1 1 mg ml?1. Propidium iodide, utilized for vitality checks, was directly added to cells within the slip to a final concentration of 1 1 g ml?1. Before imaging, cells were allowed to settle for 1 min before applying the cover slip. Standard microscopy glass slides and cover slips were used. For the analysis of staining specificity, commercially available components of various cell wall parts were tested. Powders, specified as high purity from the supplier, of chitin from shrimp shells (Sigma, Product quantity C9752), glucan from baker’s candida (Sigma, G5011), xyloglucan from tamarind (Megazyme, 95% purity, P-XYGLN), beta glucans from barley (Megazyme, 95% purity, P-BGBM), esterified pectin from citrus (Sigma, 85% esterified, P9561), CM-cellulose (Megazyme, S-ACMC), Polygalacturonic.