Alcian Blue Binding Assay

An Alcian Blue binding assay was used for the detection of cell wall integrity (Hobson et al., 2004). Overnight cell cultures were diluted to OD₆₀₀ = 0.2 and grown for 4 h at 30 or 37°C. Then robenidine was added to a final concentration of 8 and 16 μM. After 4 h, cells were stained with 30 μg/ml Alcian Blue (A3157-10G; Sigma/flu/Ald) for 10 min. Then the OD₆₂₀ of the supernatant was measured and the cell pellets were photographed. The amount of Alcian Blue binding was calculated by subtraction of unbound dye in the supernatant. The data was analyzed by a one-way ANOVA.