Flow‐Cytometric Determination of the Respiratory Activity in Growing Saccharomyces cerevisiae Populations

Abstract

Flow cytometry allows one to measure relevant physical or chemical properties on a single cell, yielding the distribution of these properties in the cell population. Typically, flow cytometry has been used to determine DNA or protein distributions, but it could be extended to the determination of other relevant parameters, such as intracellular pH, membrane potential, intracellular Ca2+ concentration, mitochondrial activity, etc. In the present work, we used flow cytometry to determine the respiratory activity in intact Saccharomyces cerevisiae cells after staining with the cationic lipophilic dye rhodamine 123 (Rhl23). We found a good correlation between the Rh123 fluorescence distribution in yeast populations and the degree of respiratory activity, which can be varied by changing the carbon source used for yeast growth. In addition, we developed a vital staining procedure which allows one to measure fast changes in the respiratory activity. We used this technique to follow the kinetics of glucose repression and to measure the apparent Km for the substrate. Our results demonstrate that flow cytometry is a fast and very sensitive method to evaluate the respiratory activity in yeast cells and is also suitable for the determination of rapid changes in yeast metabolism. Biotech‐nological implications of this study are also discussed.

Assessment of viability of microorganisms employing fluorescence techniques

“…In general, the cytoplasmic membrane potential of microorganisms is determined by the nernstian distribution of small lipophilic charged molecules, such as tetraphenylphosphonium (TPP + ) ions (66,71), and the earlier mentioned fluorescent probes. In S. cerevisiae a good correlation was found between the specific accumulation of Rhodamine 123 in mitochondria, determined by FCM, and the respiratory activity of the cells (26,100). Kaprelyants and Kell (55) showed that by use of FCM, Micrococcus luteus cells could be divided in viable, non-viable, and non-viable but resuscitable cell populations.…”

Single-Cell Sorting of Microorganisms by Flow or Slide-Based (Including Laser Scanning) Cytometry

“…One reason for a check of the viability during cell selection is to be sure that dead cells are not selected [53]. The lipophilic cationic fluorochromes rhodamine 123 and DiOC 6 (3) (3,3'-dihexyloxacarbocyanine iodide) [54] also are used for viability tests.…”

“…They can be incorporated by living cells of Saccharomyces cerevisiae, in which they accumulate in the mitochondria in different amounts depending on the level of the mitochondrial transmembrane potential driven by oxidative phosphorylation. These fluorochromes can be used for selective staining of mitochondria or as indicators of respiratory activity or viability in FCM [53,54] and confocal microscopy [54]. Rhodamine 123 and bis-oxonol (also known as DiBAC4 (3); which is anionic, and stains DNA, as do ethidium bromide and propidium iodide) generally are excluded by intact plasma membranes, and their uptake is sometimes used as an indicator of the death of bacteria and bacterioplankton [55][56][57].…”

Population profiles of a commercial yeast strain in the course of brewing

“…Individual yeast cells have been physiologically characterised by a number of authors, notably Alberghina, Mariani, Martegani, and Vanoni (1983), Block, Eitzman, Wangensteen, and Srienc (1990), Hutter and Eipel (1978), Porro, Smeraldi, Martegani, Ranzi, and Alberghina (1994), Scheper, Hoffmann, and Sch€ ugerl (1987) and Srienc (1999). However, few results have been published to date which can be used for controlling population dynamics, and even fewer which could be applied to industrial processes (M€ uller, Hutter, Bley, Petzold, & Babel, 1997;M€ uller, L€ osche, Schmidt, & Babel, 2001).…”