ELECTRIC IMPEDANCE OF INJURED AND SENSITIZED RED BLOOD CORPUSCLES

On the basis of previous work on the electrical properties of hemolyzed red cells, it might be supposed that the variation of the capacity with frequency at low frequencies is an indication of membrane permeability. To test this, rabbit red cells were subjected to treatment with lecithin, tannic acid, glucose, saponin, amboceptor, and colloidal silicic acid, each in sub-lytic doses. No change in any of the electrical properties of any of the suspensions could be detected. The result may mean that the form of the frequency variation is an extremely insensitive measure of permeability and other membrane changes, and capable only of disclosing the very great changes associated with hemolysis, or it may mean that the change in the frequency variation at low frequencies has nothing to do with permeability.     

THE COLLOIDAL BEHAVIOR OF SERUM GLOBULIN

1. The globulin prepared from ox serum by dilution and precipitation with carbon dioxide has been found, by electrometric titration experiments, to behave like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of globulin chloride, phosphate, or acetate and a solution of the corresponding acid, free from protein, separated from the globulin by a collodion membrane, was found to be influenced by hydrogen ion concentration and salt concentration in the way predicted by Donnan''s theory of membrane equilibrium. In experiments with sodium globulinate and sodium hydroxide it was found that the potential difference could be similarly explained. 3. The osmotic pressure of such solutions could be qualitatively accounted for by the Donnan theory, but exhibited a discrepancy which is explicable by analogy with certain experiments of Loeb on gelatin. 4. The application of Loeb''s theory of colloidal behavior, which had previously been found to hold in the case of gelatin, casein, egg albumin, and edestin, has thus been extended to another protein, serum globulin.     

A nondestructive method for measuring electrical conductivity of intracellular matter of tissue in situ

Summary A nondestructive method has been developed for measuring electrical conductivity of intracellular matter. The method is based on easily measurable electric parameters of cells in suspension. Experiments were made using suspensions of erythrocytes in a physiological salt solution. Results confirmed suitability of the method for determining conductance of the cytoplasm and were in close agreement with those reported by other authors. The method allows the determination of intracellular conductivity in tissue as well.     

THE FLOCCULATION OF BACTERIA BY PROTEINS

1. The effect of adding pure proteins to bacterial suspensions at different H ion concentrations has been studied. 2. The zone of flocculation of protein-treated bacteria bears a significant relationship to the isoelectric point of the protein used. With the higher concentration of protein, agglutination occurs at or near the isoelectric point of that protein; at reactions acid to this, the bacteria carry a positive charge and are not agglutinated. With diminishing concentration of protein, the zone of flocculation shifts toward and goes beyond that characteristic of the untreated bacteria. This occurs both in the presence and absence of salts. 3. A diversity of other suspensions, such as sols of gold, mastic, cellulose nitrate, cellulose acetate, Fe(OH)₃, oil emulsions, and erythrocytes, have been found by ourselves and others to exhibit a similar altered stability when treated with proteins in the same way.     

PRELIMINARY STUDIES OF THE GASTROINTESTINAL TRACT WITH COLLOIDAL BARIUM

A stable colloidal suspension of barium sulfate has been developed and tested in roentgen examination of the gastrointestinal tract. The new material is rather distinctive in radiographic appearance and can usually be differentiated from simple barium-water mixtures by inspection of roentgenograms of the opacified stomach and small intestine. It usually affords a satisfactory demonstration of the mucosal folds of the stomach and duodenal bulb and is considerably more resistant to flocculation and precipitation by retained gastric secretions. In the small intestine, it has little tendency to undergo flocculation and fragmentation, and permits visualization of fine mucosal configurations with unusual clarity. Its motility is about the same as that of conventional suspensions. Air contrast colon examinations with the colloidal preparation exhibit a very uniform, opaque, and stable coating of the bowel wall and are more consistently satisfactory than when simple barium-water mixtures are used.     

Determination of the content of nonfilterable cells in erythrocyte suspensions as a function of the medium osmolality

The results of most filtration assays for deformability of erythrocytes do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple filtration method for determination of the percentage of nonfilterable cells in erythrocyte suspension using membrane filters with mean pore diameter of 3.1 microns. This method makes it possible to detect even minor abnormal subpopulations in erythrocyte suspensions. The flow rate of buffer depends on the number of free pores of a filter. The plot of the number of pores clogged by nonfilterable cells vs the total number of erythrocytes that were allowed to pass through the filter had a linear portion, with a slope representing the relative content, Z%, of nonfilterable cells in the suspension. We determined Z% for various medium osmolalities u and used the data to derive the distribution of erythrocytes in ucr (ucr is the maximum value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations). The distribution of ucr in suspension of normal erythrocytes has a maximum of about 200 mOsm/kg and a half-width of about 20 mOsm/kg. The distributions of ucr are altered in normal erythrocyte suspensions at decreased pH values, in cryopreserved and ATP-depleted erythrocyte suspensions and in erythrocytes from a xerocytosis patient.     

Double-decker chemotaxis: no evidence for photonic stimulation of directed locomotion by human blood polymorphonuclear leukocytes

The study was carried out under direct videomicroscopic control to ascertain whether electromagnetic forces (photons) can initiate directed cell motility of human polymorphonuclear neutrophils (PMN). Cell suspensions containing a mixture of randomly motile white blood cells and erythrocytes (red cells) were placed in a double-decked preparation created by a glass slide and two cover slips and sealed by paraffin. Erythrocytes in the upper or lower chamber were destroyed by a single burst from a narrow ruby laser beam. Directed locomotion of PMN toward the erythrocyte debris occurred exclusively in the chamber in which the erythrocytes had been destroyed. Only random PMN locomotion was observed in the adjacent chamber. The results indicate that in this experimental model, electromagnetic forces do not initiate directed locomotion.     

A flow system for the study of shear forces upon cultured endothelial cells

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.     

THE COLLOIDAL BEHAVIOR OF EDESTIN

1. It has been shown by titration experiments that the globulin edestin behaves like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of edestin chloride or acetate separated by a collodion membrane from an acid solution free from protein was found to be influenced by salt concentration and hydrogen ion concentration in the way predicted by Donnan''s theory of membrane equilibrium. 3. The osmotic pressure of such edestin-acid salt solutions was found to be influenced by salt concentration and by hydrogen ion concentration in the same way as is the potential difference. 4. The colloidal behavior of edestin is thus completely analogous to that observed by Loeb with gelatin, casein, and egg albumin, and may be explained by Loeb''s theory of colloidal behavior, which is based on the idea that proteins react stoichiometrically as amphoteric electrolytes and on Donnan''s theory of membrane equilibrium.     

Electron microscopy of free-floating cells: thin-layering technique, and selection of individual cells for ultramicrotomy.

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily be relocated by the aid of the pre-embedding reference micrographs.     

Bioimpedance spectroscopy parameters of suspensions of young and old erythrocytes

The bioimpedance spectroscopy (BIS) parameters of the suspensions of young and old erythrocytes were studied. The separation of the erythrocytes by age was made by density gradient. The BIS parameters: extracellular (Re) and intracellular (Ri) fluid resistance, characteristic frequency (Fchar), cell membranes capacitance (Cm) and Alpha parameter of concentrate suspensions of young and old erythrocytes were measured on the BIA analyzer ABC-01 "Medass" in the frequency range 5-500 kHz. It was found that Re (300.4 +/- 30.0 Ohm and 261.2 +/- 21.8 Ohm for old and young respectively, p < 0.05), Ri (86.6 +/- 9.1 Ohm and 73.4 +/- 7.3 Ohm for old and young respectively, p < 0.001) and Alpha (0.305 +/- 0.003 and 0.302 +/- 0.001 for old and young respectively, p < 0.05) of the old erythrocytes suspensions were higher, than of the young one, and Fchar (308.3 +/- 42.0 kHz and 347.4 +/- 48.0 kHz for old and young respectively, p <0.05) and Cm (99.3 +/- 10.1 pF and 112.8 +/- 6.3 pF for old and young respectively, p < 0.01) of the old erythrocytes were lower, than of the young one. The found differences between electrical properties of the suspensions of young and old erythrocytes were obviously determined by the alterations of the red blood cells during aging (growth of intracellular hemoglobin concentration, erythrocytes rapprochement because of diminishing of surface negative charge, increase of red blood cell sphericity and cell membrane permeability for ions). Thus the BIS parameters are related to the erythrocyte aging.     

A semi-empirical model for flow of blood and other particulate suspensions through narrow tubes

A semi-empirical model applicable to the flow of blood and other particulate suspensions through narrow tubes has been developed. It envisages a central core of blood surrounded by a wall layer of reduced hematocrit. With the help of this model the wall layer thickness and extent of plug flow may be calculated using pressure drop, flow rate and hematocrit reduction data. It has been found from the available data in the literature that for a given sample of blood the extent of plug flow increases with decreasing tube diameter. Also for a flow through a given tube it increases with hematocrit. The wall layer thickness is found to decrease with increase in blood hematocrit. A comparison between the results of rigid particulate suspensions and blood reveals that the thicker wall layer and smaller plug flow radius in the case of blood may be attributed to the deformability of the erythrocytes.     

Electron Microscopy of Free-Floating Cells: Thin-Layering Technique, and Selection of Individual Cells for Ultramicrotomy

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily he relocated by the aid of the pre-embedding reference micrographs.     

Observation of Aspherical Particle Rotation in Poiseuille Flow via the Resistance Pulse Technique: I. Application to Human Erythrocytes

The resistance pulse detector (Coulter counter) has been widely applied to the problem of determining the volumes of insulating particles in electrolyte solutions. This technique is based on the simple relationship, ΔR/R = fv/V, between the fractional resistance change ΔR/R and the ratio of particle volume v to pore volume V. The proportionality constant f is a function of particle shape and orientation. Direct observation of the expected resistance anisotropies for aspherical particles is reported here. As predicted by simple hydrodynamic theory each individual resistance pulse has a periodically varying amplitude as it traverses a long pore in the shear field of Poiseuille flow. Characteristics of the particle motion allow improved volume distribution determinations by properly accounting for the shape factor. Application is made to normal human erythrocytes and a gaussian volume distribution with a coefficient of variation ~ 19% is found. The electrical shape effect for erythrocytes is consistent with an oblate ellipsoidal particle with a diameter-to-thickness ratio of 4. Analysis of the data indicates that the convergent entrance flow orients the cells so that they enter the pore with their axis of symmetry perpendicular to the pore axis.     

THE USE OF STORED SUSPENSIONS OF ESCHERICHIA COLI I IN THE EVALUATION OF BACTERICIDAL ACTION

SUMMARY: Suspensions of Escherichia coli I, consisting of washed cells suspended in a phosphate buffer solution, maintained a higher viability and resistance to phenol than suspensions either of unwashed cells or of washed cells suspended in water. When stored for 5 weeks at room temperature, variations in their extinction times on exposure to aqueous phenol solutions were not significantly greater than variations with suspensions freshly prepared for each determination. Loss of resistance of a stored suspension to phenol, roxenol, lysol and potassium laurate was roughly parallel. Conditions of culture of the bacteria influenced the survival of suspensions, but the results indicated that pronounced differences may only be found in suspensions prepared from young cultures. The use of stored suspensions in the routine evaluation of bactericides is recommended.     

THE NUCLEAR DNA CONTENT OF LARGE VENTRAL SPINAL NEURONS

Abstract— A nuclear fraction has been obtained from large spinal neurons previously isolated from bovine ventral spinal cord in bulk suspensions. The fraction contained an average of 5.3 ± 0.9 pg DNA/nucleus, indicating a high incidence of diploid nuclei. This conclusion was confirmed by distribution analysis of DNA in propidium iodide-stained nuclei examined by flow microfluorometry. That technique showed that at least 90% of the nuclei from large spinal neurons are diploid. Mixed, mostly non-neuronal nuclei derived from many types of cells in the ventral spinal cord contained an average of 5.9 ± 0.6 pg DNA/nucleus, 19% of which possibly possess more than diploid amounts of DNA. The uniform DNA content in nuclei of large spinal neurons and most other types of cells in the ventral spinal cord contrasts sharply with a wide variation (av 26-fold) in the nuclear volumes of the same cells.     

The study of the bioenergetic characteristics of the red blood cells of Black Sea fish: the common stingray (<Emphasis Type="Italic">Dasyatis pastinaca</Emphasis> L.) and black scorpionfish (<Emphasis Type="Italic">Scorpaena porcus</Emphasis> L.)

The oxygen consumption rate in red blood cell suspensions of two Black Sea fish species, a cartilaginous fish, the common stingray (Dasyatis pastinaca L.) and the teleost black scorpionfish (Scorpaena porcus L.) has been studied. The proposed stimulants of activators and inhibitors of the mitochondria electron transport chain had very predictable responses, indicating that mitochondria in fish erythrocytes have a classical set of respiratory enzymes. Despite the fact that the basic respiratory activity of common stingray erythrocytes was greater than those of the scorpionfish, the responses of common stingray red blood cells to the exposure during investigation of the respiratory activity of the mitochondria have an inverse relationship. The oxygen consumption rates in suspensions of scorpionfish erythrocytes in response to the stimulant were higher according to both the amplitude and the duration of the response. Investigations have shown the high energy potential of the red blood cell mitochondria of the scorpionfish and stingray. This may be the energy basis for maintaining the high intracellular concentrations of ATP required not only to keep an adequate level of intracellular metabolism, but also to provide a special mode of blood flow through the capillary beds.     

THE EFFECT OF NISIN ON THE GROWTH OF CELLS AND SPORES OF CLOSTRIDIUM WELCHII IN GELATINE

SUMMARY: The action of various concentrations of nisin on the development of cells and spores from untreated and heated suspensions of Clostridium welchii added to gelatine has been investigated, using a tube colony count technique. The development of vegetative cells was prevented by the presence of 2 Reading Units (R.U.) of nisin/ml of the final culture medium, although the effective concentration may have been 6 R.U./ml. Approximately 40 R.U./ml prevented colony formation from spores. Gelatine containing nisin was dried. A considerable proportion of the activity of the antibiotic was still present after storage for 5 weeks.     

Electrical injury mechanisms: electrical breakdown of cell membranes

Electric fields are capable of damaging cells through both thermal and nonthermal mechanisms. While joule heating is generally recognized to mediate tissue injury in electrical trauma, the possible role of electrical breakdown of cell membranes has not been thoroughly considered. Evidence is presented suggestive that in many instances of electrical trauma the local electrical field is of sufficient magnitude to cause electrical breakdown of cell membranes and cell lysis. In theory, large cells such as muscle and nerve cells are more vulnerable to electrical breakdown. To illustrate the significance of cell size and orientation, a geometrically simple model of an elongated cell is analyzed.