The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.

Cell buoyant densities were determined in exponentially growing cultures of Escherichia coli B/r NC32 and E. coli K-12 PAT84 by equilibrium centrifugation in Percoll gradients. Distributions within density bands were measured as viable cells or total numbers of cells. At all growth rates, buoyant densities had narrow normal distributions with essentially the same value for the coefficient of variation, 0.15%. When the density distributions were determined in Ficoll gradients, they were more than twice as broad, but this increased variability was associated with the binding of Ficoll to the bacteria. Mean cell volumes and cell lengths were independent of cell densities in Percoll bands, within experimental errors, both in slowly and in rapidly growing cultures. Buoyant densities of cells separated by size, and therefore by age, in sucrose gradients also were observed to be independent of age. The results make unlikely any stepwise change in mean buoyant density of 0.1% or more during the cycle. These results also make it unlikely that signaling functions for cell division or for other cell cycle events are provided by density variations. How does cell buoyant density vary during the growth and division cycle? That question may be of fundamental importance in understanding the nature of cell growth and its controls. Rosenberger et al. (8) postulated that density fluctuations may have a signaling function in cell division or in other critical steps of the cell division cycle. Such physical models of control would become less tenable if density fluctuations were very small.A knowledge of density variation is important to cell cycle studies in another way. Cell mass, usually the major variable in such studies, is not readily determinable. Cell volume, however, is relatively easily measured with modem cell size analyzer equipment, and cell mass can then be determined if the density is known.Widely different results have been obtained for variation in bacterial buoyant densities during the cell cycle. Baldwin and Wegener (1) reported that density changes during the cycle in the bacterium Lineola longa allowed selection of synchronous cultures from Ficoll equilibrium gradients. Poole (7) employed gradients of colloidal silica and polyvinylpyrrolidine and observed large variations, ca. 5%, in buoyant densities of cells from exponentially growing t Permanent address: cultures of Escherichia coli K-12. Woldringh et al. (10), using Percoll gradients, found that density variations in E. coli B/r were <1%. Earlier, Koch and Blumberg (3) had also concluded from unpublished data that density fluctuations were <1% in E. coli.Despite the inconsistencies among these estimates, the results established qualitatively that buoyant density variation is small in exponentially growing cultures of E. coli. In this paper we report the quantitative determination of even smaller values for buoyant density variation in this bacterium, and we also provide evidence that density is independent of age during the cell cycle. MATERIALS AND METHODSBa...

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines. Recent reports have indicated that the buoyant density of bacterial cells measured by equilibrium density banding in gradients of Percoll or similar colloids is constant both at different stages of the cell cycle (4, 6) and at widely different cell growth rates (6; unpublished observations). Buoyant densities of mammalian cells are also independent of the phase of the cell cycle (M.

The buoyant density ofEscherichia coli was shown to be related to the osmolarity of the growth medium. This was true whether the osmolarity was adjusted with either NaCl or sucrose. When cells were grown at one osmolarity and shocked to another osmolarity, their buoyant density adjusted to nearly suit the new osmolarity. When cells were subjected to hyperosmotic shock, they became denser than expected. When cells were subjected to hypoosmotic shock they occasionally undershot the new projected density, but the undershoot was not as dramatic as the overshoot seen with hyperosmotic shocks. Shrinkage and swelling of the cells in response to osmotic shocks could account for the change in their buoyant density. The changes in cell size after osmotic shocks were measured by two independent methods. The first method measured cell size with a Coulter Counter, and the second method measured cell size by stereologic analysis of Nomarski light micrographs. Both methods gave qualitatively similar results and showed the cells to be flexible. The maximum swelling recorded was 23% of the original cell volume, while the maximum shrinkage observed was 33%.

The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored. At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml. As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml. From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities. The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml. Both strains of E. coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9. (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM. To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly. From this study, it was determined that both Ca 2؉ and Mg 2؉ could stimulate growth below 40 mosM. With Ca 2؉ -and Mg 2؉-supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9. Strain ML3 would only haltingly grow at 15 mosM. Four attempts were made to grow both ML3 and ML308 at 5 mosM. In three of the experiments, ML308 grew, while strain ML3 grew in one experiment. While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently. Therefore, from this study, we believe that E. coli might be defined as a euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.Interest in osmotic regulation of bacteria has had a long and colorful history. Early works of Mitchell (for his review of this early work, see reference 14) led to important discoveries. More recently, interest has been centered more on the physiology of osmotic regulation than the energetics (for a review of more recent work, see reference 8). Epstein and Schultz (10) were able to prove that K ϩ and the counterion glutamate Ϫ played a major role in cytoplasmic osmotic regulation in media of 500 mosM and below (also see a review by Epstein [9]), while the work of Cayley et al. (5, 6) and others (13) has shown that in Escherichia coli betaine and/or proline seems to function as an osmoprotectant for cell growth at higher osmolarities. Some years ago, it was found that the buoyant density of the bacterial cell appeared to be closely related to the osmolarity of the growth medium (2). In those studies, Luria-Bertani medium (LB) supplemented wi...