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.     

The density of protein precipitates and its effect on centrifugal sedimentation

Isoelectric soya-protein precipitate densities were measured for mean particle sizes ranging from 3.4-65 mum by gradient centrifugation, centrifugation in water-immiscible solvents, tracerdilution, gravity sedimentation of isolated particles. Coulter counter volume determination, and a comparison of Coulter counter and centrifugal sedimentation size distributions. The immiscible system and tracer dilution methods were both found to be unreliable due to experimental uncertainties. The Coulter counter volume measurement indicated the existence of a density-size relationship with the aggregate density decreasing as the size increased. Comparison with sedimentation measurements showed that the Coulter counter measures 80% of the total aggregate volume for 6-mum particles. The relation between aggregate density (rho(a), kg m (-3)) and size (d, mum) was measured for isoelectric soya protein and casein precipitated by ammonium sulfate, using a comparison of the Coulter counter size distribution and centrifugal sedimentation. The functions were described for soya by \documentclass{article}\pagestyle{empty}\begin{document}$$ \rho _a - 1004 = 246d;{ - 0.408} $$\end{document} and for casein by \documentclass{article}\pagestyle{empty}\begin{document}$$ \rho _a - 1136 = 31d;{ - 0.441} $$\end{document} The gradient centrifugation method measured the buoyant density of hydrated protein precipitate which was independent of size, and is consistent with an aggregate structure consisting of primary particles. However, the aggregate structure was not described for all sizes by the theoretical cubic packing of hard-sphere primary particles, nor by the successive random addition of primary particles. The density-size functions indicated up to a fivefold difference in Stokes settling velocities compared to those calculated assuming a constant density difference.     

UNDERSTANDING AND INTERPRETING HEMATOCRIT MEASUREMENTS IN PINNIPEDS

Hematocrit data are relatively easily obtained from blood samples of pinnipeds but differences in methodology and variable physiological conditions of the subjects can significantly alter their values. This two-fold problem makes comparative data and modeling efforts difficult. To quantify the difficulty of obtaining accurate and representative hematocrit values in pinnipeds, hematocrit was measured by both microcentrifugation and Coulter counter methods in a range of pinnipeds under a variety of physiological and handling conditions. The data show that the Coulter counter hematocrit values were 4%-15% higher than those measured by microcentrifugation. In addition, blood samples from restrained animals showed consistently elevated hematocrit values relative to resting subjects. A significant difference was also found between hematocrit values from pups and adults. Finally, hematocrit was shown to decline over the course of isofluorane anesthesia. Taken together, these results suggest that laboratory methodology, developmental state, and animal handling techniques can significantly alter hematocrit values in pinnipeds. Thus, modeling efforts that require representative hematocrit values, such as calculations of total blood oxygen stores, can be markedly impacted by variations in hematocrit measurement techniques and sampling regimes.     

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.     

Evaluation of a new blood cell counter with sheath flow system

A blood cell counter with a sheath flow system has been developed to eliminate the drawback of the Coulter type blood cell counter, namely, signal distortion caused by the cells passing through the electric aperture gate. With the new system, signal distortion was low and a near-normal distribution curve for erythrocytes and resin particles was obtained. The counter has a computer program for determining the red cell distribution width, which represents an actual size difference at 20% of the relative frequency of the distribution curve. This is independent of mean cell volume, and is considered to be of clinical importance. We examined the values in 2,300 healthy subjects and patients with various hematological disorders.     

Investigation of hydrodynamic focusing in a microfluidic coulter counter device

The Coulter technique enables rapid analysis of particles or cells suspended in a fluid stream. In this technique, the cells are suspended in an electrically conductive solution, which is hydrodynamically focused by nonconducting sheath flows. The cells produce a characteristic voltage signal when they interrupt an electrical path. The population and size of the cells can be obtained through analyzing the voltage signal. In a microfluidic Coulter counter device, the hydrodynamic focusing technique is used to position the conducting sample stream and the cells and also to separate close cells to generate distinct signals for each cell and avoid signal jam. The performance of hydrodynamic focusing depends on the relative flow ratio between the sample stream and sheath stream. We use a numerical approach to study the hydrodynamic focusing in a microfluidic Coulter counter device. In this approach, the flow field is described by solving the incompressible Navier-Stokes equations. The sample stream concentration is modeled by an advection-diffusion equation. The motion of the cells is governed by the Newton-Euler equations of motion. Particle motion through the flow field is handled using an overlapping grid technique. A numerical model for studying a microfluidic Coulter counter has been validated. Using the model, the impact of relative flow rate on the performance of hydrodynamic focusing was studied. Our numerical results show that the position of the sample stream can be controlled by adjusting the relative flow rate. Our simulations also show that particles can be focused into the stream and initially close particles can be separated by the hydrodynamic focusing. From our study, we conclude that hydrodynamic focusing provides an effective way to control the position of the sample stream and cells and it also can be used to separate cells to avoid signal jam.     

Continuous and long-term volume measurements with a commercial Coulter counter

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter.     

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.     

Comparative determination of the rates of Kluyveromyces bulgaricus protoplast formation by the coulter counter technique and spectrophotometry

Kluyveromyces bulgaricus protoplast formation was studied by the determination of the remaining intact cells with the Coulter counter, the decrease of the absorbance of the cell suspension upon dilution into an hypoosmotic medium and the liberation of intracellular proteins after protoplast lysis. Due to the scattering of light by cells and ghosts, spectrophotometric readings were not enough specific and led to the underestimation of the reaction rates (about 40% less). The Coulter counter technique and the determination of the liberation of intracellular proteins gave the same rates over a large range of cell concentrations and in the presence of various cell lytic enzymes. The importance of corrected etermination of the time course of the reaction was illustrated by the good correlation obtained between protoplast formation and ATP turnover.     

Isoelectric precipitation of soy protein. II. Kinetics of protein aggregate growth and breakage

Protein aggregate growth and breakage in agitated suspensions are modeled. The model includes growth of particles by a turbulent collision mechanism and breakage by a hydrodynamic shear mechanism. In the model, breakage results in the splitting of the particles into several small fragments. The model parameters are a growth rate constant and a breakage rate constant. Aggregate size distributions were measured with a Coulter counter and the data interpreted using a population balance that governs the steady-state particle size distribution in a continuous stirred tank reactor. Effects of changes in the operating variables pH, concentration, mean residence time, ionic strength, and mixing power input on the model kinetic parameters are investigated.     

Sizing of Bdellovibrio During Growth

A technique for the counting and relative sizing of host-independent bdellovibrio during growth, with the aid of a modified Coulter counter, is described.     

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.     

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.     

Transcellular Ion Flow in Escherichia coli B and Electrical Sizing of Bacterias

Dielectric breakdown of cell membranes and, in response, transcellular ion flows were measured in Escherichia coli B 163 and B 525 using a Coulter counter as the detector with a hydrodynamic jet focusing close to the orifice of the counter. Plotting the relative pulse height for compensated amplification of a certain size of the cells against increasing detector current, a rather sharp bend within the linear function was found, which did not occur when measuring fixed cells or polystyrene latex. The start current for transcellular ion flow causing the change of the slope is different for the potassium-deficient mutant B 525 in comparison with the wild-type B 163, indicating a change in the membrane structure of B 525 by mutation and demonstrating the sensitivity of the method for studying slight changes in membrane structure in general. The theoretical size distributions for two current values in the range of transcellular ion flow were constructed from the true size distribution at low detector currents, assuming an idealized sharp changeover of the bacterial conductivity from zero to one-third of the electrolyte conductivity.     

Cholesterol crystal formation and growth in model bile solutions

Cholesterol monohydrate crystal formation was studied in supersaturated model bile solutions, containing unlabeled cholesterol, sodium cholate and soybean phosphatidylcholine, and tracer amounts of [3H]cholesterol. Solutions were either seeded with cholesterol crystals to initiate growth, or not seeded to allow self-nucleation and subsequent crystal growth to occur. Crystal growth at 37 degrees C was measured by two methods. First, radioactive cholesterol crystals were isolated by filtration, and the mass of cholesterol that had precipitated was calculated. In unseeded solutions, there was a long lag period before crystal growth was detected. This lag time was decreased by increases in the cholesterol concentration, temperature, and lipid concentration. In seeded solutions, crystal growth also was dependent on the cholesterol concentration, temperature, and lipid concentration. The second method used to measure crystal growth involved the Coulter Counter. At 37 degrees C, reproducible results were not obtained using unseeded solutions due to blocking of the counter aperture with large crystals. In seeded solutions, crystal growth could be measured as an increase in total particle volume. However, comparison of growth rate estimates from the Coulter Counter with those obtained radiochemically revealed poor agreement between the two methods. It is probable that the Coulter Counter is inaccurate in measuring the volume of cholesterol monohydrate crystals due to their anisometric shape.     

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.     

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.     

Evaluation of leukocyte number by using an automated blood cell counter and a traditional hematological method in animals irradiated with gamma rays

Female mice were irradiated with a single whole body dose of 7 Gy of gamma-rays. Leucocyte numbers were monitored in the peripheral blood using automated blood cell counter Coulter counter and a traditional hematological method with a light microscope in the Bürker chamber. Reticulocyte numbers, RNA blood concentration, spleen weight and morphological changes in spleen and bone marrow were also studied. In the period between 15th-19th days after irradiation the numbers of leucocytes obtained by CC counting were manifold higher than those obtained by microscope counting. Since this period is characterised by a steep increase in the reticulocyte number and RNA concentration in blood as well as by increased weight of spleen as the result of marked regeneration of extramedullar erythropoiesis, leukocytes as well as reticulocytes are assumed to be additionally registered by the automated counter CC in this period, probably due to a higher resistance of reticulocytes to the lysing agent Zapoglobine.     

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.