The relationship between yeast cell size and cell division in Candida albicans

The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.     

Increase in cell mass during the division cycle of Escherichia coli B/rA.

Increase in the mean cell mass of undivided cells was determined during the division cycle of Escherichia coli B/rA. Cell buoyant densities during the division cycle were determined after cells from an exponentially growing culture were separated by size. The buoyant densities of these cells were essentially independent of cell age, with a mean value of 1.094 g ml-1. Mean cell volume and buoyant density were also determined during synchronous growth in two different media, which provided doubling times of 40 and 25 min. Cell volume and mass increased linearly at both growth rates, as buoyant density did not vary significantly. The results are consistent with only one of the three major models of cell growth, linear growth, which specifies that the rate of increase in cell mass is constant throughout the division cycle.     

Electrochemical Polarization-Induced Changes in the Growth of Individual Cells and Biofilms of Pseudomonas fluorescens (ATCC 17552)

The effect of surface electrochemical polarization on the growth of cells of Pseudomonas fluorescens (ATCC 17552) on gold electrodes has been examined. Potentials positive or negative to the potential of zero charge (PZC) of gold were applied, and these resulted in changes in cell morphology, size at cell division, time to division, and biofilm structure. At −0.2 V (Ag/AgCl-3 M NaCl), cells elongated at a rate of up to 0.19 μm min⁻¹, rendering daughter cells that reached up to 3.8 μm immediately after division. The doubling time for the entire population, estimated from the increment in the fraction of surface covered by bacteria, was 82 ± 7 min. Eight-hour-old biofilms at −0.2 V were composed of large cells distributed in expanded mushroom-like microcolonies that protruded several micrometers in the solution. A different behavior was observed under positive polarization. At an applied potential of 0.5 V, the doubling time of the population was 103 ± 8 min, cells elongated at a lower rate (up to 0.08 μm min⁻¹), rendering shorter daughters (2.5 ± 0.5 μm) after division, although the duplication times were virtually the same at all potentials. Biofilms grown under this positive potential were composed of short cells distributed in a large number of compact microcolonies. These were flatter than those grown at −0.2 V or at the PZC and were pyramidal in shape. Polarization effects on cell growth and biofilm structure resembled those previously reported as produced by changes in the nutritional level of the culture medium.     

Relationship between changes in buoyant density and formation of new sites of cell wall growth in cultures of streptococci (Enterococcus hirae ATCC 9790) undergoing a nutritional shift-up.

When the glutamate concentration of cultures of Enterococcus hirae was raised from 20 to 300 micrograms/ml, the mass doubling time decreased from ca. 85 to 45 min in 9 min, but balanced growth was not reestablished for 30 to 40 min. During the unbalanced period of growth, RNA and protein synthesis proceeded more rapidly than did peptidoglycan synthesis, buoyant density increased from ca. 1.1024 to 1.1075 g/ml, and the rate of formation of new cell wall growth sites transitorily accelerated above the new growth rate. When studied as a function of cell size, all cultures showed buoyant density to decrease around cell separation, increase as cells increased in size, and then plateau when cells reached large volumes. Greater increases in buoyant density as a function of cell size were seen after shift-up, with the greatest increases observed at 15 to 20 min after shift-up, when the rate of formation of new sites was also maximal. In a population of cells examined by electron microscopy 15 min after shift-up, buoyant density and the frequency of cells with new sites increased as old sites approached the size of two poles. These data were consistent with a model whereby buoyant density increases in the terminal stages of the cell cycle when the surface grows slower than the cytoplasm. The greater the difference in the rates of inside to outside growth, the greater the increase in buoyant density and the more frequently new sites will be initiated.     

Nucleoside phosphotransferase activity through the growth and cell cycle of Tetrahymena pyriformis GL-I

Tetrahymena pyriformis GL-I were synchronized by three different techniques and nucleoside phosphotransferase activity measured through the different cell cycles obtained. In cells that were starved and then refed, activity did not increase until 75 min after refeeding. This increase in activity occurred well before nuclear DNA synthesis and was not blocked by hydroxyurea. In cells synchronized by the induction technique of one heat shock per generation and the selection technique of differential density labelling, enzyme activity increased continuously over the cell cycle but did not double. However, during early logarithmic growth nucleoside phosphotransferase activity more than doubled over one cell cycle time while late in log growth phase less than a doubling was observed. Cycloheximide and mixed extract experiments suggest that the patterns of activity observed reflect the patterns of enzyme synthesis. These results are discussed with respect to the pattern of activity observed for thymidine kinase in other organisms.     

Microgravity Alters the Physiological Characteristics of Escherichia coli O157:H7 ATCC 35150, ATCC 43889, and ATCC 43895 under Different Nutrient Conditions

The aim of this study is to provide understanding of microgravity effects on important food-borne bacteria, Escherichia coli O157:H7 ATCC 35150, ATCC 43889, and ATCC 43895, cultured in nutrient-rich or minimal medium. Physiological characteristics, such as growth (measured by optical density and plating), cell morphology, and pH, were monitored under low-shear modeled microgravity (LSMMG; space conditions) and normal gravity (NG; Earth conditions). In nutrient-rich medium, all strains except ATCC 35150 showed significantly higher optical density after 6 h of culture under LSMMG conditions than under NG conditions (P < 0.05). LSMMG-cultured cells were approximately 1.8 times larger than NG-cultured cells at 24 h; therefore, it was assumed that the increase in optical density was due to the size of individual cells rather than an increase in the cell population. The higher pH of the NG cultures relative to that of the LSMMG cultures suggests that nitrogen metabolism was slower in the latter. After 24 h of culturing in minimal media, LSMMG-cultured cells had an optical density 1.3 times higher than that of NG-cultured cells; thus, the higher optical density in the LSMMG cultures may be due to an increase in both cell size and number. Since bacteria actively grew under LSMMG conditions in minimal medium despite the lower pH, it is of some concern that LSMMG-cultured E. coli O157:H7 may be able to adapt well to acidic environments. These changes may be caused by changes in nutrient metabolism under LSMMG conditions, although this needs to be demonstrated in future studies.     

Electrochemical polarization-induced changes in the growth of individual cells and biofilms of Pseudomonas fluorescens (ATCC 17552)

The effect of surface electrochemical polarization on the growth of cells of Pseudomonas fluorescens (ATCC 17552) on gold electrodes has been examined. Potentials positive or negative to the potential of zero charge (PZC) of gold were applied, and these resulted in changes in cell morphology, size at cell division, time to division, and biofilm structure. At -0.2 V (Ag/AgCl-3 M NaCl), cells elongated at a rate of up to 0.19 microm min(-1), rendering daughter cells that reached up to 3.8 microm immediately after division. The doubling time for the entire population, estimated from the increment in the fraction of surface covered by bacteria, was 82 +/- 7 min. Eight-hour-old biofilms at -0.2 V were composed of large cells distributed in expanded mushroom-like microcolonies that protruded several micrometers in the solution. A different behavior was observed under positive polarization. At an applied potential of 0.5 V, the doubling time of the population was 103 +/- 8 min, cells elongated at a lower rate (up to 0.08 microm min(-1)), rendering shorter daughters (2.5 +/- 0.5 microm) after division, although the duplication times were virtually the same at all potentials. Biofilms grown under this positive potential were composed of short cells distributed in a large number of compact microcolonies. These were flatter than those grown at -0.2 V or at the PZC and were pyramidal in shape. Polarization effects on cell growth and biofilm structure resembled those previously reported as produced by changes in the nutritional level of the culture medium.     

Analysis of respiration during germination and enlargement of spores of Bacillus megaterium and of the fungus Myrothecium verrucaria

1. There are certainly two, and probably three, stages in the development of B. megaterium from the spore to inception of cell division. The rapid increase in rate of respiration during the initial 10 minutes on glucose-peptone-yeast extract medium coincides with decrease in optical density and with increase in stainability. From about 10 to 100 minutes, the rate increases linearly, coinciding with swelling of the spores and ending at approximately the time of rupture of the spore case. From about 100 to 180 minutes, there is a second and steeper linear increase in respiration rate coinciding with cell elongation. These physiological and morphological phenomena are discussed as criteria for germination. 2. The rate of respiration of M. verrucaria spores also increases linearly up to about 300 minutes in sucrose-yeast extract medium. No breaks in the curves are observed during formation of the germ tubes. 3. Oxygen uptake follows the parabolic curve See PDF for Equation within the limits of experimental error for both types of spores. 4. It is postulated that metabolism during these stages of linear increase may be regulated by processes occurring at cellular or intracellular surfaces or by synthesis of a limiting enzyme at constant rate.     

The rate of development of a microsporidan in moth cell culture.

A microsporidan parasite of the forest tent caterpillar Malacosoma disstria infected cells and replicated in vitro in a line from the moth Heliothis zea. After spore germination, the incidence of infected cells increased with time until leveling off with sporulation. During the first 24 hr, there was a static number of parasites, followed by a 2-day logarithmic growth phase during which the population doubled five to six times. The growth rate was 9 to 11 hr per population doubling. Sporulation commenced on day 3, and 40 to 50 spores were recovered from each infected cell. The life cycle was completed within 6 days, culminating in spores that were infectious for cultured cells. The antibiotic fumagillin at a dose of 1 ppm in the culture medium was microsporida-static.     

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD₇₅₀) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD₇₅₀ measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.     

Influence of environmental stress on distributions of times to first division in Escherichia coli populations, as determined by digital-image analysis of individual cells

The distributions of times to first cell division were determined for populations of Escherichia coli stationary-phase cells inoculated onto agar media. This was accomplished by using automated analysis of digital images of individual cells growing on agar and calculation of the "box area ratio." Using approximately 300 cells per experiment, the mean time to first division and standard deviation for cells grown in liquid medium at 37 degrees C and inoculated on agar and incubated at 20 degrees C were determined as 3.0 h and 0.7 h, respectively. Distributions were observed to tail toward the higher values, but no definitive model distribution was identified. Both preinoculation stress by heating cultures at 50 degrees C and postinoculation stress by growth in the presence of higher concentrations of NaCl increased mean times to first division. Both stresses also resulted in an increase in the spread of the distributions that was proportional to the mean division time, the coefficient of variation being constant at approximately 0.2 in all cases. The "relative division time," which is the time to first division for individual cells expressed in terms of the cell size doubling time, was used as measure of the "work to be done" to prepare for cell division. Relative division times were greater for heat-stressed cells than for those growing under osmotic stress.     

Metal ion content of Escherichia coli versus cell age.

The potassium, calcium, magnesium, and zinc ion content of cells in exponential and synchronously growing cultures of Escherichia coli B/r was determined with an X-ray fluorescence spectrometer and an atomic absorption spectrophotometer. Cellular potassium, calcium, and magnesium content increased smoothly during the cell cycle, but cellular zinc showed a steplike increase about 10 to 15 min after cell division in a culture having a doubling time of 47 min. The possible role of cellular zinc in the control of cell division is discussed.     

Morphological analysis of nuclear separation and cell division during the life cycle of Escherichia coli.

Quantitative electron microscope observations were performed on Escherichia coli B/r after balanced growth with doubling times (tau) of 32 and 60 min. The experimental approach allowed the timing of morphological events during the cell cycle by classifying serially sectioned cells according to length. Visible separation of the nucleoplasm was found to coincide with the time of termination of chromosome replication as predicted by the Cooper-Helmstetter model. The duration of the process of constrictive cell division (10 min) appeared to be independent of the growth rate for tau equals 60 min or less but to increase with increase doubling time in more slowly growing cells. Physiological division, i.e., compartmentalization prior to physical separation of the cells, was only observed to occur in the last minute of the cell cycle. The morphological results indicate that cell elongation continues during the division process in cells with tau equals 32 min, but fails to continue in cells with tau equals 60 min.     

Cell cycle length, cell size, and proliferation rate in hydra stem cells

We have analyzed the cell cycle parameters of interstitial cells in Hydra oligactis. Three subpopulations of cells with short, medium, and long cell cycles were identified. Short-cycle cells are stem cells; medium-cycle cells are precursors to nematocyte differentiation; long-cycle cells are precursors to gamete differentiation. We have also determined the effect of different cell densities on the population doubling time, cell cycle length, and cell size of interstitial cells. Our results indicate that decreasing the interstitial cell density from 0.35 to 0.1 interstitial cells/epithelial cell (1) shortens the population doubling time from 4 to 1.8 days, (2) increases the [3H]thymidine labeling index from 0.5 to 0.75 and shifts the nuclear DNA distribution from G2 to S phase cells, and (3) decreases the length of G2 in stem cells from 6 to 3 hr. The shortened cell cycle is correlated with a significant decrease in the size of interstitial stem cells. Coincident with the shortened cell cycle and increased growth rate there is an increase in stem cell self-renewal and a decrease in stem cell differentiation.     

Differential growth rates of Candida utilis mother and daughter cells under phased cultivation.

The yeast Candida utilis was continuously synchronized by the phased method of cultivation with the nitrogen source as the growth-limiting nutrient. The doubling time (phasing period) of cells was 6 h. Both cell number and deoxyribonucleic acid synthesis showed a characteristic stepwise increase during the phased growth. The time of bud emergence coincided with the time of initiation of deoxyribonucleic acid synthesis. Size distribution studies combined with microscopic analysis showed that the cells expanded only during the unbudded phase of growth. Usually the cells stopped increasing in size about 30 min before bud emergence, and the arrest of the increase in cell volume coincided with the exhaustion of nitron from the medium. There was no net change in the volume of cells during the bud expansion phase of growth, suggesting that as the bud expanded, the volume of the mother portion of the cell decreased. After division the cells expanded slightly. The postdivision expansion of cells, unlike the growth before bud initiation, occurred in the absence of the growth-limiting nutrient. The newly formed daughter cells were smaller than the mother cells and expanded at a faster rate, so that both types of cells reached maximum size at the same time. Possible reasons for the different rates of expansion of mother and daughter cells are discussed.     

Selection of synchronous bacterial cultures by density sedimentation.

A procedure previously used to select synchronous cultures of Chlorella was found to produce similar results with the bacterium Lineola longa (Bacillus macroides). A midlog culture of L. longa was layered onto a 31-42% dialyzed Ficoll gradient and ceitruged at 51 000 3 g. The culture sedimented into a broad band in 30 min. Continued centrifugation failed to cause further migration. Cells taken from the top of the band and reinoculated into the broth in which they had previously grown, pH adjusted to 7.0, grew without a lag, doubled in optical density at the same rate as midlog cultures, and divided synchronously. Coulter counter sizing of these cells showed a doubling in volume just before division followed by a halving of volume after division. The major advantages of this method are the low osmolarity of Ficoll and the large volume of cells that can be separated.     

Rapid Swelling of a CHO-K1 Aspartate/Glutamate Transport Mutant in Hypo-osmotic Medium

Two Chinese hamster ovary cell (CHO-K1) mutants selected for defective glutamate transport via system X⁻ _AG are also highly permeable to small neutral molecules. Light microscopy demonstrated that exposure of one of these mutants, Ed-A1, to hypo-osmotic medium led to extremely rapid swelling, presumably due to increased water flux. When placed in 20% saline, Ed-A1 cells swelled to three times their original volume within 15 sec, a sixfold larger increase than parental CHO-K1. In spite of this rapid volume increase, mutant and wild-type cells remained viable for 20 min in dilute saline. A regulatory volume decrease in Ed-A1, and the continual swelling of CHO-K1, resulted in the two cells achieving equal size after 5 min in 20% saline. The time course of these volume changes permitted analysis of large numbers of cells by a hydrodynamic technique, steric field flow fractionation (FFF). Steric FFF demonstrated the expected inhibition of osmotic swelling of human erythrocytes by the mercurial, p-chloromercuribenzenesulfonic acid (PCMBS). However, PCMBS increased the apparent swelling rate of Ed-A1 and CHO-K1, suggesting that an aquaporin-like molecule is not responsible for any significant fraction of the water fluxes into either line. PCMBS also strongly inhibited aspartate transport by system X⁻ _AG. By taking advantage of their different swelling rates in hypotonic medium, steric FFF can separate mixtures of CHO-K1 and Ed-A1. Received: 2 August 1996/Revised: 25 October 1996     

Control of wall band splitting in Streptococcus faecalis

Computer reconstructions of 659 and 1325 whole mounted, shadowed cells, randomly chosen from cultures of Streptococcus faecalis undergoing balanced growth and doubling in mass every 83 min and 30 min, respectively, were used to analyse the cell cycle. The size limits and duration of phases of the cell cycle were estimated by applying a method previously described by the authors, details of which are given here to allow others to use the method. Deeply constricted cells whose primary septal radius, Rs, was less than or equal to 0.18 micron were considered as belonging to an E-phase ending the cell cycle. The statistical parameters of these E-phase cells were used to calculate the mean and coefficient of variation of dividing cells. These latter values, in turn, predicted the moments of the total population well enough so that the method's assumptions were judged adequately satisfied. Therefore, the method was considered applicable to other phases and sub-phases of the cell cycle of these two cultures. The E-phase cells were further classified as having either 0, 1 or 2 secondary growth zones, allowing us to calculate the percentage of newborn cells without growth zones. In the slow-growing cells, 69% of the cells arose with no growth zone. On the other hand, in more rapidly growing cells 16% of the cells or less arose with no growth zone. Our calculations showed that they could exist without a growth zone for only 2 and 0.1 min, respectively. We also classified cells as possessing a 'birth site' if the volume between the two daughter bands was greater than 0, but less than 0.06 micron3. From the statistical properties of such cells with new growth zones, the mean pole time, W, was estimated. We also estimated W from the size of cells in E-phase. The major conclusion is that the pole time is only slightly greater than the mass doubling time at both growth rates. Since DNA synthesis in S. faecalis takes longer (C = 50 to 52 min) than the mass doubling time in rich medium (30 min), a new round of chromosome replication must be initiated before the old round of synthesis is completed (dichotomous replication). Consequently, wall band splitting and initiation of chromosome replication do not occur simultaneously. It was also concluded that the cell initiates wall band splitting, resulting in pole formation and cell division, when the growth zones cannot function rapidly enough to allow the increase of surface area required to accommodate continuing production of cytoplasm.     

Replication of Deoxyribonucleic Acid During the Division Cycle of Salmonella typhimurium

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.