The spheroplast lysis assay

Cells were cultured in YPD broth at the initial inoculum density of 5 x 10⁴ cells/ml, for 24 h with shaking at 30ºC. There were two groups of samples: the control (no drug added) and samples treated with the C1 compound at the concentration of 32 μg/ml. The rate of spheroplasts lysis under the enzymatic digestion of the cell walls in hypotonic conditions was assessed according to the method described by Ovalle et al. [21]. After 24 h of culture, cells at the early stationary phase were harvested by centrifugation at 1000 x g for 5 min and washed three times in sterile deionized water. The harvested cells were suspended in TE buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA; pH 7.5) to obtain optical density between 0.6 and 0.7. Zymolyase^TM 100 T (AMS Biotechnology), i.e. lyophilized powder containing a mixture of protease and β-glucanase, was dissolved in a 1:1 glycerol/water solution to a concentration of 20 mg/ml and stored at -70°C. The working solution was prepared by diluting the stock solution 1:10 with TE buffer. A 100 μl aliquot of the Zymolyase working solution was added to 2 ml of the cell suspension in TE buffer (optical density between 0.6 and 0.7), vortexed, and kept in a thermostat at 25°C. The samples were vortexed for 30 s before each OD determination (600 nm, 10 min intervals) in the spectrophotometer (Agilent Cary 60 UV-Vis). The experiment was repeated three times with two replicates (n = 6). Basic statistics, multivariate analysis of variance (MANOVA), and a post-hoc Tukey (HSD) test were performed using StatSoft Statistica 12.5 software.