Cells of the budding yeast Saccharomyces cerevisiae undergo a process akin to differentiation during prolonged culture without medium replenishment. Various methods have been used to separate and determine the potential role and fate of the different cell species. We have stratified chronologically-aged yeast cultures into cells of different sizes, using centrifugal elutriation, and characterized these subpopulations physiologically. We distinguish two extreme cell types, very small (XS) and very large (L) cells. L cells display higher viability based on two separate criteria. They respire much more actively, but produce lower levels of reactive oxygen species (ROS). L cells are capable of dividing, albeit slowly, giving rise to XS cells which do not divide. L cells are more resistant to osmotic stress and they have higher trehalose content, a storage carbohydrate often connected to stress resistance. Depletion of trehalose by deletion of TPS2 does not affect the vital characteristics of L cells, but it improves some of these characteristics in XS cells. Therefore, we propose that the response of L and XS cells to the trehalose produced in the former differs in a way that lowers the vitality of the latter. We compare our XS- and L-fraction cell characteristics with those of cells isolated from stationary cultures by others based on density. This comparison suggests that the cells have some similarities but also differences that may prove useful in addressing whether it is the segregation or the response to trehalose that may play the predominant role in cell division from stationary culture.

During a 10-day culture ageing, cells of the wild-type Saccharomyces cerevisiae strain JC 482 retain their viability, while mitochondrial function and morphology change. Cell routine and uncoupled respiration rates increase to a maximum on day 4 and then decline to near zero. The decline, which occurs also in mitochondria isolated from cells of different age, is not due to increasing proportion of petites. Rhodamine 123 fluorescence intensity reporting on mitochondrial membrane potential appears to drop slightly for 4 days and then more sharply at the time when respiration rate also decreases. The MitoTracker Green fluorescent signal related to the mitochondrial content per cell also decreases. The branched tubular mitochondrial network of 1-day-old cells dissolves into short fragments; during the first 4 days, this fragmentation is associated with increasing function of mitochondria, while later on, it accompanies functional decline, which is also indicated by the decreasing ratio of Rhodamine 123 fluorescence to MitoTracker Green fluorescence. As shown by cell counting, microscopy and flow cytometry, the cell size distribution in the population broadens, and the population thus becomes more heterogeneous. The changes in respiration rate, mitochondrial membrane potential, mass and structure point to changes in the mitochondrial status during ageing.