INFLUENCE OF VIABLE CELLS ON THE RESUSCITATION OF DORMANT CELLS IN MICROCOCCUS-LUTEUS CULTURES HELD IN AN EXTENDED STATIONARY-PHASE - THE POPULATION EFFECT

A high proportion of Micrococcus luteus cells in cultures which had be en starved for 3 to 6 months lost the ability to grow and form colonie s on agar plates but could be resuscitated from their dormancy by incu bation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Ke ll, Appl. Environ. Microbiol. 59:3187-3196, 1993). In the present work , such cultures were studied by both flow cytometry and conventional m icrobiological methods and were found to contain various numbers of vi able cells. Pretreatment of such cultures with penicillin G, and subse quent dilution, was used to vary this number. When the initial number of colony-forming cells per 30-ml flask was approximately nine (+/-fiv e) or more, resuscitation of 10 to 40% of the cells, and thus culture growth, was observed. The lag period before the appearance of a popula tion of cells showing significant accumulation of the fluorescent dye rhodamine 123 (i.e., of cells with measurable membrane energization) d ecreased from 70 to 27 h when the number of viable cells was increased from 30 to 10(5) per flask, while the lag period before an observable increase in the number of colony-forming cells occurred was almost co nstant (at some 20 h). Provided there were more than nine (+/-five) in itially viable cells per flask, the number of initially viable cells d id not affect the final percentage of resuscitable cells in the cultur e. The lag period could be ascribed in part to the time taken to resto re the membrane permeability barrier of starved cells during resuscita tion, as revealed by flow cytometric assessment of the uptake of the n ormally membrane-impermeant fluorescent DNA stain PO-PRO-3 {4 ,3-oxazo le)-2-methylidene]-1-(3'-trimethylammonium propyl)-pyridinium diiodide ). Although cell populations which contained fewer than nine +/-five v iable cells per flask failed to grow, 4 to 20% of the cells (of 1.2 x 10(6)) were able to accumulate rhodamine 123 after 80 to 100 h of incu bation, showing the ability of a significant number of the cells in th e population at least to display ''metabolic resuscitation.'' Resuscit ation and cell growth under such conditions were favored by the use of a 1:1 mixture of fresh lactate medium and supernatant from late-logar ithmic-phase M. luteus cultures as the resuscitation medium. We conclu de that the presence of a small fraction of viable cells at the onset of resuscitation facilitates the recovery of the majority of the remai ning (dormant) cells. The cell density dependence of the kinetics, or population effect, suggests that this recovery is due to the excretion of some factor(s) which promoted the transition of cells from a state in which they are incapable of growth and division to one in which th ey are capable of colony formation.