QUANTITATIVE-ANALYSIS OF THE PHYSIOLOGICAL HETEROGENEITY WITHIN STARVED CULTURES OF MICROCOCCUS-LUTEUS BY FLOW-CYTOMETRY AND CELL SORTING

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). We used flow cytome try and cell sorting to study populations of bacteria that had been st arved for 5 months. These cells could be stained by the fluorescent li pophilic ration rhodamine 123, but such staining was almost independen t of metabolically generated energy in that it was not affected by unc ouplers. Two populations could be distinguished, one with a lower degr ee of rhodamine fluorescence (a degree of fluorescence referred to as region A and containing approximately 80% of the cells) and one with a more elevated degree of fluorescence (region B, approximately 20% of the cells). Subsequent incubation of starved cells in fresh medium in the presence of the antibiotic chloramphenicol (to which M. luteus is sensitive) resulted in the transient appearance of cells actively accu mulating rhodamine 123 (and fluorescing in region B) and of larger cel ls exhibiting a yet-greater degree of fluorescence (region C). These m ore fluorescent cells accounted for as much as 50% of the total popula tion, under conditions in which the viable and total counts were const ant. Thus, metabolic resuscitation of at least one-half of the cells t akes place under conditions in which cryptic growth cannot play any ro le. Sorting experiments revealed that the great majority of the viable cells in the starved population are concentrated in regions B and C a nd that the extent of rhodamine staining under conditions of starvatio n therefore reflects the physiological state of the cells, Physical se paration of these cells from cells in region A resulted in an increase (of approximately 25-fold) in the viability of cells in regions B and C and of the population as a whole. Resuscitation of dormant cells in a most-probable-number assay in the presence of supernatant taken fro m growing M. luteus revealed the resuscitation of cells from regions B and C but not from region A. It is suggested that initially dormant ( resuscitable) cells are concentrated in regions B and C.