Visualization of Sporulated Yeast Cells

Bright field and fluorescence microscopy were performed using a Leica DM4000B microscope equipped with a Leica DFC450 C digital CCD camera. Calcofluor White and Eosin Y staining was performed on cells that were sporulated in liquid SPM for 24 h as previously described (Lin et al. 2013 (link)). Cells were first harvested and washed in 1 ml McIlvaine’s buffer (0.2 M Na₂HPO₄/0.1 M citric acid, pH = 6.0). Staining was then performed using 30 µl Eosin Y disodium salt (5 mg/ml, Sigma) in 500 µl McIlvaine’s buffer for 10 min at room temperature in the dark. Cells were washed twice in McIlvaine’s buffer and resuspended in 200 µl McIlvaine’s buffer containing 1 µl of 1 mg/ml Calcofluor White solution (Sigma). Fluorescence of Calcofluor White and Eosin Y was examined using the DAPI and FITC filters, respectively. At least 200 cells were counted for the presence of internalized Eosin Y staining.