Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality

Abstract

The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r 2 ‫؍‬ 0.898, n ‫؍‬ 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P ‫؍‬ 0.433), ICP via flow cytometry did (P ‫؍‬ 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population's vitality via a short measurement, which is suitable for routine analysis.Yeast plays an important role in the food industry. It is primarily used for making bread, beer, and wine, and the flavor and aromatic compounds it produces are characteristic of these fermented products. The physiological state of the yeast biomass influences the fermentation performance and thus the quality of the resulting product, e.g., of beer (1, 32). Food processors who depend on yeast health for consistent fermentations demand a method to measure yeast's physiological condition during yeast growth, that is, its vitality. The term "vitality" refers to the health of living biomass where high vitality results in a fast fermentation with minimal undesired by-products, while low vitality results in sluggish or poorly attenuating fermentation. In contrast, the term "viability" only distinguishes between dead and alive cells.

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

“…Such understanding enables the optimization of industrial processes (4,8,35) or the exploitation of pH homeostasis machinery as a therapeutic target. pH i has been measured through a wide range of monitoring techniques, from cell loading with pH-selective fluorescent dyes (4,(7)(8)(9)(10)(11) to the use of green fluorescent protein-based sensors (12)(13)(14)(15). These approaches considerably simplify measurement, especially when sensors are expressed in stably transformed cells.…”

“…The primary drawback of this technique is the need to post-process images to evaluate the subpopulation content. Ratiometric flow cytometry combines high-throughput analysis at the single-cell level with visualization of pH i distribution within yeast populations (4,7,8). The combination of genetically encoded ratiometric pH probes and flow cytometry is an effective approach.…”

“…A good alternative to spectroscopy and microscopy is flow cytometry, which combines rapid online analysis and the acquisition of multi-parameter results at the singlecell level for each cell in a population. This method also enables cell sorting (4,7,8). A good demonstration of the effectiveness of flow cytometry is that provided by Valli et al (4).…”

“…Several research groups (4,7,8,17) correlated the acidification of cytosol to inhibited yeast growth. Cells in stationary phase maintain a lower pH i than do those in exponential growth phase (8), and the pH homeostasis in the former is more sensitive to extracellular perturbations (7). We analyzed yeasts grown to a stationary phase by ratiometric flow cytometry (Fig.…”

“…In addition to pH-sensitive fluorescent dyes (e.g., carboxy SNARF-4F [4,7], 5,6-carboxyfluorescein [8][9][10], and 9-aminoacridine [11]), noninvasive pH i measurement techniques have been developed that apply recombinant pH-sensitive fluorescent proteins, pHluorin (12), RaVC (13), pHRed (14), and pHluorin2 (15). All pHluorin-based probes have a characteristic bimodal excitation spectrum, with a dose-dependent decrease in excitation at 395 nm and an increase in excitation at 475 nm with increasing acidity.…”