Development of an optical method for monitoring protoplast formation from cultured plant cells

The size distribution of cell aggregates during protoplast isolation from Catharanthus roseus and Nicotiana tabacum was measured by a Coulter counter. It was observed that a gradual reduction in the size of cell aggregates occured during protoplast formation. A previously developed specialized spectrophotometer for the photometric measurement of plant cell concentration was used for continuous monitoring of the reduction in the size distribution of cell aggregates during protoplast formation. This made it possible to use changes in optical density (O.D.) to distinguish the three stages in protoplast formation—plasmolysis, maceration and cell wall digestion. During the processes of maceration and cell wall digestion, the O.D. decreased and reached a steady value at the end of each process. Consequently, changes in the O.D. could be used to determine precisely the end of each process. The cell wall digestion process was described by a simple first order reaction model and the rate of protoplast formation (cell wall digestion) was quantitatively evaluated from the rate constant (k) of this reaction. By using the values of k, the optimal enzymatic reaction conditions for isolating protoplasts from C. roseus and N. tabacum cells were determined.     

Determination of the Volume and Surface Area of Tetrahymena pyriformis,and their Relationship to Endocytosis*

Methods are described for the determination of the mean cellular volume and surface area of Tetrahymena pyriformis GL by direct microscopic measurement or by Coulter counter. The results are compared and discussed. It is suggested that the latter is the more accurate method of estimation. Fed cells showed a bimodal size distribution by Coulter counter determination and had a mean volume of 10,000–11,000 μM³, whereas cells which had been starved for 1 or 2 days showed a unimodal distribution and had mean cellular volumes of ～ 1,600 and 1,200 μ³ respectively. The corresponding mean surface areas were ～ 1,900, 550 and 450 μM² respectively. The discrepancy between the results of the 2 methods of estimation was greater with starved than with fed cells because of the greater asymmetry of the former. Continued cell division during the early part of the starvation period had a considerable reducing effect upon the mean cellular volume, but other unidentified factors were also important in producing the observed diminution in volume. Comparison of the mean surface areas of starved cells with previously recorded rates of membranous utilization in endocytosis upon refeeding indicate that insufficient cell membrane was present to maintain the rates of vacuole formation observed.     

Coulter counter cell size determination of protoplasts fromArabidopsis thaliana PIP1b aquaporin antisense lines under iso- and hypo-osmotic conditions

Summary Water uptake ofArabidopsis thaliana protoplasts was measured after transfer into hypo-osmotic conditions. The time-dependent swelling of protoplast populations was monitored by a Coulter counter device. In order to ascertain the contribution of the plasma membrane intrinsic protein 1b (PIP1b) to the membrane's water permeability, protoplasts of five different plant lines that were transformed with a PIP1b antisense construct were compared to controls. The size distribution of 5 independent protoplast preparations provided similar results for control and antisense lines under iso-osmolar conditions. After transfer into hypo-osmotic conditions, a time difference for the swelling of protoplasts from the different sources was observed. The sizes of control protoplasts changed in less than 20 s, which indicates high water influx rates. In contrast, the protoplast populations obtained from 5 different antisense plants took about 75 s to reach a steady-state cell size distribution. The difference in time by a factor of about 3 confirms the significance of the aquaporin PIP1b for the water permeability of plant plasma membranes and the cellular water transport.     

Cell water permeability and cryotolerance of Saccharomyces cerevisiae

Electron particle sizing (Coulter counter) was used to measure cell and protoplast volumes of Saccharomyces cerevisiae grown under different conditions designed to increase its cryotolerance. Membrane water permeabilities were estimated from those measurements. A relationship was obtained between the lower water permeability of yeast grown under microaerobic batch conditions and its weaker cryotolerance in water (cooling rate of 39·6°C/min), as compared to fed-batch cells. For the latter, cell water permeability was not related to the observed differences in survival for frozen-thawed cells grown under strong or partial (with temporary limitation of dissolved oxygen in growth media) aerobic conditions.     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 Ω - cm for the intracellular resistivity of intact red blood cells is consistent with the literature.     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 omega X cm for the intracellular resistivity of intact red blood cells is consistent with the literature.     

Changes in the incorporation of carbon derived from glucose into cellular pools during the cell cycle of Saccharomyces cerevisiae

The rate of incorporation of 14C derived from [U-14C]glucose into cells of Saccharomyces cerevisiae X2180(1B) was investigated as a function of the cell cycle. After pulse-labelling of exponentially growing populations, centrifugal elutriation was used to isolate various cell fractions of increasing cell size, representing successive stages of the cell cycle. The total amount of 14C incorporated per cell was found to increase continuously during the cell cycle along with cellular protein content and Coulter counter cell volume. This pattern supports the model of exponential cell growth. In order to evaluate changes in intracellular carbon flow during the cell cycle, chemical extraction procedures were used to obtain four cellular fractions enriched in either low-molecular-mass components, lipid material, polysaccharides or proteins. The distribution of 14C among these cellular fractions varied during successive stages of the cell cycle, indicating cell-cycle-dependent fluctuations in intracellular carbon flow. During the G1 phase the flow of 14C into the low-molecular-mass pool increased markedly; concurrently, the rate of incorporation into the polysaccharide-enriched pool decreased.     

Development of a sample preparation method for fungal proteomics

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.     

Protein factors in extracts of ribosomes isolated from L-929 cells during various phases of the cell cycle

Centrifugal elutriation has been utilized in order to separate cultures of L-929 fibroblasts into subpopulations containing cells at different stages of the cell cycle. The subpopulations were characterized by Coulter counter volume determination, [3H]thymidine label into DNA and flow cytometry. When a population of early G1 cells was returned to roller culture it was shown to progress through the cell cycle in a synchronous manner. Ribosomal factor extracts were prepared from cells at various phases during the cell cycle. The amounts of protein in the extracts varied greatly, being lowest in early G1 phase and showing a peak during S phase. Polyacrylamide gel electrophoresis demonstrated that there were differences in the protein species present in the extracts. Some proteins were present in the same amounts throughout the cell cycle, whereas others appeared to show a form of cyclical behaviour.     

Characterization of aggregate size in Taxus suspension cell culture

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 μm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R ² > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.     

Methods for the determination of adipose cell size in man and animals

Four methods for the sizing of adipose cells in small samples of human or animal adipose tissue are compared. These methods depend on the preparation of cell suspensions by incubation of the tissue with collagenase or by prolonged fixation with osmium tetroxide and separation of the fixed cells. A Coulter electronic counter was used to count and size the suspended cells and a Zeiss particle size analyzer for the sizing of cells in photomicrographs. The use of the Coulter counter to count cells in a suspension derived from a known amount of tissue and subjected to osmium tetroxide fixation is recommended for accuracy and general applicability to adipose cells of all sizes in man and animals.     

Electrostatic properties of cells estimated by absorption and fluorescence spectroscopy

A colloid titration method was used to determine the surface charge of cells of a human colon adenocarcinoma cell line WiDr; 6.2±0.8×10⁸ charges per cell were found. The apparent surface charge density was calculated using the cell surface area estimated by a Coulter counter. Alternatively, the lower limit of the cell surface area was estimated by visible microscopy. The same procedure was applied for human skin fibroblasts, resulting in the value 9.4±1.1×10⁸ charges per cell. This is significantly higher (p<0.05) than that of WiDr cells, presumably because of the different size of the cells. According to the estimations using the Coulter counter, the median diameter was higher in the case of skin fibroblasts. Fluorimetric titration of the fluorescent probe U-6 was used to estimate the interfacial potential of the WiDr cells. A shift of the titration curve of the U-6 probe toward higher pH values compared to that in pure buffer solutions was found in the presence of the WiDr cells. From the displacement of the midpoints of the titration curves, the interfacial potential of the WiDr cells was found to be about−35.8 mV. Incubation of the cells at two different pH values (7.4 and 6.8) did not result in any significant modification of the electrostatic properties of the cells under the experimental conditions of the present study. Electron microscopy revealed a distinct difference in the surface morphology of the WiDr cells compared to human skin fibroblasts. Numerous microvilli present on the surface of WiDr cells indicated marked uncertainties in cell surface area estimations. This gives large uncertainties in the real surface charge densities of cells.     

Contributions of photosynthetic and non‐photosynthetic cell types to leaf respiration in Vicia faba L. and their responses to growth temperature

In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non‐photosynthetic (epidermal) cells from fully expanded leaves of warm‐developed (WD) and cold‐developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non‐photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.     

The Erythrocyte Ghost Is a Perfect Osmometer

The osmotic swelling of intact erythrocytes in hypotonic solutions was measured using microhematocrit tubes, Van Allen tubes, and a calibrated Coulter counter. In agreement with earlier workers the intact cells did not behave as perfect osmometers, the cells swelling less than predicted by the Boyle-van't Hoff law. Erythrocyte ghosts were prepared from fresh intact erythrocytes by one-step hemolysis in 0.25% NaCl at an extremely dilute concentration of cells and the membranes were sealed at 37°. The ghosts were mixed with NaCl solutions of different osmolarities and the MCV (mean cell volume) of the shrunken cells immediately monitored by a calibrated Coulter counter. It was found that the MCV values of the shrunken ghosts were accurately predicted by the Boyle-van't Hoff law. These results indicate that these erythrocyte ghosts behaved as perfect osmometers.     

Comparison of coulter volumes with radiometrically determined intracellular water volumes for cultured cells

Summary During methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells, proliferation is inhibited, morphologic and biochemical changes occur, and giant, often multinucleated, cells form. We have used the increase in cell volume as a marker of the mature syncytiotrophoblastlike phenotype. Uninduced and differentiated BeWo cells are not spherical, and theoretical considerations suggested that deviations in shape could result in significant errors in Coulter volume. To determine if the values obtained by electrical pulse sizing reflected the actual mass of BeWo cells, we have evaluated the relationship between Coulter volumes and intracellular water volumes obtained using a shape-independent estimate for eight cell types. A close correlation (r ²=0.97) was found, indicating that cell volume changes in populations of irregularly shaped cells can be accurately measured using a Coulter instrument. Supported by an operating grant from the National Cancer Institute of Canada. N.S.B. was a recipient of a studentship award from the Alberta Heritage Foundation for Medical Research. C.E.C. is a Senior Research Scientist of the National Cancer Institute of Canada. The McEachern Laboratory is a research facility of the Faculty of Medicine, University of Alberta, and the Cross Cancer Institute, Edmonton, Alberta.     

Study on effect of diluent osmotic pressure on yeast cell strength and cell size using a novel micromanipulation technique

A new micromanipulation technique which has previously been used to measure the mechanical properties of single animal cells has now been applied to yeast cells. In this study this technique was used to measure yeast cell strength and cell size across a 2l batch fermentation. Alternatively the cell size could also be determined using a Coulter counter while cell measurement was diluted with a conducting fluid (Isoton II). For the cell strength, it was found that the osmotic pressure of diluents did affect cell strength. However, it was also found that there was no significant effect of osmotic pressure of diluents on cell size whether a Coulter counter or micromanipulation was used for measurement. Micromanipulation has been shown to be a powerful technique for measuring the mechanical properties of yeast cells and it will be very useful for studying their behaviour in cell disruption equipment, e.g. high-pressure homogenizers.     

A Growth Curve of Cell Numbers In the Neural Retina of Embryonic Chicks

ABSTRACT The growth of cell numbers in a normal embryonic population of ncural retinal cells is described. the numbers were estimated from a time shortly after the neural retina first becomes recognizable to a time when numbers of retinal cells have become steady. Cell numbers were estimated in preparations of an entire neural retina dispersed into a suspension of single nuclei which were then counted in a Coulter counter. the growth curve of the In numbers of cells has three phases of growth: an exponential phase during which there is steady-state exponential growth, a differentiative phase during which cell proliferation ceases and an end phase when no further change in cell numbers can be detected. the variances of the In numbers of cells were highest during the exponential phase. the variances decreased during the differentiative phase and were at their lowest during the end phase. For variances to decrease requires mechanisms which control the final numbers of cells in the neural retina very precisely. the implications of mechanisms which operate by controlling cell lineages are explored.     

Elutriation and Coulter Counts of Tetrahymena pyriformis Grown in Peanut and Cottonseed Meal Media

Growth of Tetrahymena pyriformis W has been used to evaluate nutritional quality of peanut and cottonseed meals. An efficient elutriation method is described for separating cells of this organism from particulate matter left in the substrate (enriched with basal medium) after 4 days of incubation. After elutriation the cells can be counted with a Coulter counter by using calibration procedures which are presented. Elutriation and Coulter counting provide a rapid and efficient method of measuring the growth response of T. pyriformis W. Utility of the method is demonstrated by agreement between Coulter counts and visual counts of the cells and by demonstration of a linear response of cell numbers to substrate nitrogen.     

Measurement of cell volume of phototrophic bacteria in pure cultures and natural samples: phase contrast, epifluorescence and particle sizing

Abstract Cell volumes of different purple phototrophic bacteria were measured using several techniques: Coulter counter, phase contrast and epifluorescence microscopy. Volumes of Chromatium warmingii, C. minutissimum, Thiocapsa roseopersicina , and Thiocystis gelatinosa were measured as the organisms were accumulating sulfur. Cell volumes of Rhodobacter capsulatus were measured under different growth conditions including both anaerobically in the light and aerobically in the dark. Size distributions were flatter and more irregular by phase contrast microscopy than by Coulter counter. This latter technique could not be used in many cases, however, because phototrophic bacteria associate to form chains and aggregates of cells. In addition, Coulter counter measurements for organisms with capsules gave volumes intermediate between the volume of the cell and the volume of the capsule, as measured by phase contrast microscopy. Epifluorescence gave similar results to phase contrast if organic solvents were not used in the preparation of samples. Finally, cell volume of two phototrophic bacteria was shown to change both with depth and with season in a natural system.     

MULTINUCLEATE GIANT CELL FORMATION IN A PACHYPSYLLA GALL ON CELTIS

Dundon , Thomas R. (Saint Louis U., Saint Louis, Mo.) Multinucleate giant cell formation in a Pachypsylla gall on Celtis . Amer. Jour. Bot. 49(7): 800–805. Illus. 1962.—Cytological antecedents of the multinucleate giant cell in gall tissue of Pachypsylla mamma on leaves of hackberry, Celtis occidentalis, have been presented. The nymph's stylet punctured the epidermis and terminated in a single mesophyll cell where it remained fixed until a multinucleate giant cell formed there. An intracellular cytoplasmic reaction occurred at the tip of the stylet which was attributable either to the secretory or the sucking activity of the nymph. The walls of cells surrounding the tip of the stylet gradually became less distinct as demonstrated by a decreasing affinity for cellulose stain. Later, cellulolytic effects were easily visible as large breaches formed in the walls of cells grouped around the stylet. The cellulolytic process spread centrifugally from the tip of the stylet until complete dissolution of all but the outermost walls of 6–8 cells was accomplished, leaving a typically shaped multinucleate giant cell. In one instance, nuclei from adjacent cells were observed partially drawn through apertures in the cell walls into the protoplast of an incompletely formed giant cell containing the stylet. This nuclear movement was attributed to a centripetal flow of plant juice caused by the sucking activities of the nymph. These findings do not support an earlier theory that the multinucleate giant cell was a product of plant growth, namely, enlargement of a single mesophyll cell and multiple karyokinesis or amitosis.