ESTIMATION OF DORMANT MICROCOCCUS-LUTEUS CELLS BY PENICILLIN LYSIS AND BY RESUSCITATION IN CELL-FREE SPENT CULTURE-MEDIUM AT HIGH DILUTION

Micrococcus luteus starved for 2-7 months in spent medium following gr owth to stationary phase in batch culture exhibited a culturability (a s estimated by direct plating on nutrient agar plates) of < 0.001%. Ho wever, following a lag, some 70% of the cells could be lysed upon inoc ulation into and cultivation in fresh lactate minimal medium containin g penicillin, showing the capability of a significant portion of the c ells at least to enlarge (and thus potentially to resuscitate). When t he viable cell count was estimated using the most probable number meth od, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, an d little different from the viable cell count as assessed by plating o n solid; media. However, the apparent viability of these populations e videnced with the most probable number method was 1000-100000-fold gre ater when samples were diluted into liquid media containing supernatan ts taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under condition s in which the growth of a limited number of viable cells during resus citation is excluded, that a significant portion of the apparently non -viable cell population in an extended stationary phase is dormant, an d not dead.