Measurement of the effects of acetic acid and extracellular pH on intracellular pH of nonfermenting, individual Saccharomyces cerevisiae cells by fluorescence microscopy

Abstract

The effects of acetic acid and extracellular pH (pH(ex)) on the intracellular pH (pH(i)) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer's wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pH(ex) values was controlled by perfusion with 2 g of total acetic acid per liter at pH(ex) 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pH(i) of individual S. cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pH(ex) 6.5, the pH(i) of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pH(ex) 3.5, 4.5, and 5.6, the pH(i) of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pH(i) remained constant. The decreases in pH(i) were dependent on the pH(ex); i.e., the decreases in pH(i) at pH(ex) 5.6 and 6.5 were significantly smaller than the decreases in pHi at pH(ex) 3.5 and 4.5.