MEASUREMENT OF THE EFFECTS OF ACETIC-ACID AND EXTRACELLULAR PH ON INTRACELLULAR PH OF NONFERMENTING, INDIVIDUAL SACCHAROMYCES-CEREVISIAE CELLS BY FLUORESCENCE MICROSCOPY

The effects of acetic acid and extracellular pH (pH(ex)) on the intrac ellular pH (pH(i)) of nonfermenting, individual Saccharomyces cerevisi ae cells were studied by using a new experimental setup comprising a f luorescence microscope and a perfusion system, S. cerevisiae cells gro wn in brewer's wort to the stationary phase were stained with fluoresc ein diacetate and transferred to a perfusion chamber, The extracellula r 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-pre cision pump, The pH(i) of individual S. cerevisiae cells during perfus ion was measured by fluorescence microscopy and ratio imaging, Potenti al artifacts, such as fading and efflux of fluorescein, could be negle cted within the experimental time used, At pH(ex) 6.5, the pH(i) of in dividual S. cerevisiae cells decreased as the extracellular concentrat ion of undissociated acetic acid increased from 0 to 0.035 g/liter, wh ereas at pH(ex) 3.5. 4.5, and 5.6, the pH(i) of individual S, cerevisi ae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter, At concentrations of un dissociated 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., t he decreases in pH(i) at pH(ex) 5.6 and 6.3 were significantly smaller than the decreases in pH(i) at pH(ex) 3.5 and 4.5.