Measurement of the effects of acetic acid and extracellular pH on intracellular pH of nonfermenting, individual Saccharomyces cerevisiae cells by fluorescence microscopy - PubMed (original) (raw)

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

L U Guldfeldt et al. Appl Environ Microbiol. 1998 Feb.

Abstract

The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) 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 pHex values was controlled by perfusion with 2 g of total acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pHi 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 pHex 6.5, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the pHi 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 pHi remained constant. The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at pHex 5.6 and 6.5 were significantly smaller than the decreases in pHi at pHex 3.5 and 4.5.

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Figures

FIG. 1

FIG. 1

Schematic diagrams showing the perfusion system (A) and the fluorescence microscope (B). CCD, charge-coupled device.

FIG. 2

FIG. 2

Relationship between the logarithm of the 490 nm/435 nm ratio and pH. The in vitro calibration curve was prepared by using 10 μM fluorescein in PBS adjusted to various pH values as described in Materials and Methods.

FIG. 3

FIG. 3

Characterization of the flow of perfusion solution through the perfusion chamber. The chamber was filled with PBS (pH 5.6). At time zero, perfusion with 5 μM fluorescein in PBS (pH 5.6) at a rate of 0.6 μl/s was initiated. Fluorescence intensity after excitation at 435 nm for 1,000 ms was measured as described in Materials and Methods.

FIG. 4

FIG. 4

Fluorescence intensity after excitation at 490 nm (A) and 435 nm (B), and pHi (C) of five representative S. cerevisiae cells during perfusion with PBS (pHex 4.5) at a rate of 0.6 μl/s. Fluorescent staining of cells and ratio imaging were performed as described in Materials and Methods.

FIG. 5

FIG. 5

Relationship between pHi of individual S. cerevisiae cells and extracellular concentration of undissociated acetic acid. The cells were perfused with 2 g of total acetic acid per liter in PBS at pHex 6.5 (A), pHex 5.6 (B), pHex 4.5 (C) and pHex 3.5 (D) at a rate of 0.6 μl/s. The pHi values presented are averages from 20 to 30 randomly selected cells. The calculated standard deviations are shown by error bars. The insets show the relationship between pHi and the full range of extracellular concentrations of undissociated acetic acid used in the experiments. The time intervals between data points are 20 s (A) (for clarity) and 10 s (B through D). Fluorescent staining of cells, ratio imaging, and calculation of the undissociated acetic acid concentrations during perfusion were performed as described in Materials and Methods.

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