Comparative real-time analysis of Saccharomyces cerevisiae cell viability, injury and death induced by ultrasound (20 kHz) and heat for the application of hurdle technology

Abstract

To design an appropriate hurdle technology using heat and ultrasound, the metabolic status of Saccharomyces cerevisiae in late exponential phase of growth was established using real-time fluorescent flow-cytometric analysis. The organism, suspended in physiological saline, was subjected for 5 min to either heat (55 or 60 °C) or ultrasound (20 kHz), or to a combination of both, and analysed for viable, membrane-injured and dead cells using thiazole orange (TO) and propidium iodide (PI). Analyses indicated that over a period of time, cells subjected to heat progressed from viable to membrane-injured, to death. In the heated preparations, the measured decline in viable cell population was principally due to membrane injury. When S. cerevisiae was treated with ultrasound (20 kHz, 124 μm), only viable and dead cell populations were detected. Scanning electron microscopy showed extensive boundary damage to cells after 1 min sonication. Ultrasound was rapidly microbicidal and cavitation-induced membrane damage was probably immediately lethal. The complete disintegration of cells undergoing sonication was slow and not the principal method of cell death. When S. cerevisiae was pre-treated with 1 min ultrasonication followed by immediate heating (55 or 60 °C), death rate at both temperatures tested was enhanced thus confirming synergism between the two hurdles. Real time fluorescent studies showed that ultrasound induced non-lethal intracellular injuries not associated with the membrane, making S. cerevisiae more susceptible to heat damage.