Development of a new coulter counter system: Measurement of the volume, internal conductivity, and dielectric breakdown voltage of a single guard cell protoplast ofVicia faba

Abstract

A new Coulter Counter is described which measures on single microscopic cells the volume, membrane breakdown voltage, and the underestimation of volume after breakdown, this last parameter reflecting in part the internal conductivity of the cells. The system requires very few cells for measurement and does not require the population to be normally distributed in volume or free of debris from the preparation of the cells. The new development is to apply a voltage ramp across a Coulter orifice through which a particle flows. A differential dual orifice system is used to reduce the magnitude of the amplified ramp signal to that of the signal produced by the particle in the presence of a DC voltage across the orifice. Breakdown is detected by an underestimation of the cell volume once a critical voltage across the cell membrane has been established. Measurements made on protoplasts isolated from guard ceils ofVicia faba with a mean volume of 1800 μm3 reveal a breakdown voltage of 1 V and an average volume underestimation of about 30% after breakdown. Variations in this underestimation reflect the different internal structure of the protoplasts in terms of the size and number of nonconducting compartments. Different stages of vacuole development exist in each preparation. The breakdown voltage of 1 V suggests that only one membrane breaks down and that the electromechanical properties of these protoplast membranes are similar to other cell systems, when the pulse length dependence of the breakdown voltage is taken into consideration.