Fluorescence microscopy was performed as described previously (29) with several modifications. In short, control, 30-min ethanol-exposed and 24-h ethanol-exposed cultures (OD600 = 1.0) were 10 times diluted, incubated for 20 min on low-melting-point agarose-coated microscope slides containing 20 μg·ml−1 FM4-64 (Molecular Probes, Eugene, OR) and 0.5 μl·ml−1 Syto9 (Molecular Probes, Eugene, OR), and imaged by oil immersion fluorescence microscopy (BX51TRF fluorescence microscope; Olympus Corporation, Tokyo, Japan) at a 500-fold magnification.