Safe, rapid, and easy electro-orientation-based viability assay using a disposable insulating envelope within non-spherical yeast cells

Rapid and easy assays for fungal viability and susceptibility to antifungal drugs are important for controlling fungal infections and diseases. We developed an improved electro-orientation (EO) viability assay, in which opposite EO directions for living and dead non-spherical yeast cells in alternating current (AC) electric fields are observed under a light microscope. An envelope-type cell injection device was constructed with two 5-μm ultrathin insulating polyimide films to avoid direct cell–electrode contact via cross-contamination. The envelope, within a 1 µL cell sample, was sandwiched between a pair of indium-tin-oxide plate electrodes. After use, the disposable envelope within the cells could be peeled off and discarded, providing reusability of the electrodes while preventing biohazards. Furthermore, cell samples were prepared within 10 s by simply diluting the cell culture medium with distilled water to reduce the extracellular conductivity to 3 mS/m, without centrifugation. This resulted in almost no time lag between sampling and assessment. The antifungal efficacy of nystatin was evaluated by applying an AC voltage of 7 V at 10 MHz, for 5 s, using the envelope device. The improved EO method enabled safe, rapid, and easy viability assays with a single-use envelope and simplified sample preparation.