Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation.

The centrifugal elutriator has been used as a baby machine by loading the chamber with a population of mixed-generation daughter cells and allowing this population to grow, divide and age under continuous washing-out of newborn daughter cells. Clear peaks in the number of elutriated cells were reproducibly obtained for at least ten generations. The parent cells growing in the chamber continued to divide at the steady-state generation time of 95-100 min, showing no change in cycle time during aging. The washed-out daughter cells increased in volume during the first five generations from their steady-state value of 17 micro3 to a maximum of 34 micro3. As to be expected, the generation times of these large daughters, determined in a synchronous batch culture, were shorter (130 min) than that of the steady-state daughters (240 min), even when derived from 15-generation parents. No indication for a volume increase of daughter cells without bud was observed when a population was allowed to grow in the chamber without washing-out the smaller daughter cells. The 15-generation parent population, recovered from the chamber, had an average volume of 80 micro3 and consisted of: (i) 71% cells with more than ten scars, (ii) 13% cells with one to nine scars, and (iii) 17% daughter cells. The production of senescent cells by undisturbed growth in the elutriator chamber has been prolonged to 29 generations. The method is therefore suitable to examine what factors determine the life span of budding yeast.