INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

THE CELL JUNCTION IN A LAMELLIBRANCH GILL CILIATED EPITHELIUM : Localization of Pyroantimonate Precipitate

The junctional complex in the gill epithelium of the freshwater mussel (Elliptio complanatus) consists of an intermediary junction followed by a 2–3 µ long septate junction. Homologous and heterologous cell pairs are connected by this junction. After fixation with 1% OsO₄ containing 1% potassium pyroantimonate, electron microscopy of the gill reveals deposits of electron-opaque precipitate, specifically and consistently localized along cellular membranes. In both junctional and nonjunctional membrane regions, the precipitate usefully outlines the convolutions without obliterating the 150 A intercellular space, which suggests the rarity or absence of either vertebrate-type gap or tight junctions along the entire cell border. The precipitate appears on the cytoplasmic side of the limiting unit membranes of frontal (F), laterofrontal (LF), intermediate (I), lateral (L), and postlateral (PL) cells. The membrane surfaces of certain vesicles of the smooth endoplasmic reticulum, of multivesicular bodies, and of mitochondrial cristae contain precipitate, as does the nucleolus. In other portions of the cell, precipitate is largely absent. The amount of over-all deposition is variable and depends on the treatment of the tissue prior to fixation. Deposition is usually enhanced by pretreatment with 40 mM NaCl as opposed to 40 mM KCl, which suggests that the precipitate is in part sodium pyroantimonate. Treatment with 0.2 mM ouabain does not enhance deposition. Regional differentiation of cell membranes with respect to their ability to precipitate pyroantimonate is found in at least three instances: (a) between the ciliary membranes and other portions of the cell membrane: the precipitate terminates abruptly at the ciliary base, (b) between the LF and I cell borders: the precipitate is asymmetric, favoring the LF side of the junction, and (c) between the septate junctional membrane and adjacent membrane: the precipitate occurs periodically throughout the septate junction region with the periodicity corresponding to the spacing of the septa. This suggests that different regions of the cell membrane may have differing ion permeability properties and, in particular, that the septa may be the regions of high ion permeability in the septate junction.     

EFFECTS OF CALCIUM-CONTAINING FIXATION SOLUTIONS ON CHOLINERGIC SYNAPTIC VESICLES

Calcium (Ca)-containing fixation solutions applied to slices of electric organ of the electric ray, Narcine brasiliensis, have been shown to have three distinct ultrastructural effects on cholinergic synaptic vesicles of the nerve terminals. (a) An electron-dense particle (EDS) is observed within the vesicle; the particle is seen in unosmicated, unstained tissues and can be removed from thin sections by Ca-chelating agents. It is concluded that the EDS represents Ca bound by the vesicle. It is suggested that the bound ATP of the vesicle provides anionic Ca binding sites. (b) The vesicle membrane tends to ‘crinkle’ or collapse depending on the concentration of the other components of the fixative solution. The ‘crinkling’ or collapse are largely reversed by a wash step in the absence of Ca. (c) The presence of Ca results in the appearance of a population of vesicles which form characteristic fusions or ‘tight’ junctions with the terminal membrane. This appears to be morphological evidence for the proposal, which has been frequently put forward, that Ca facilitates such a fusion before discharge of vesicle-bound transmitter. With the discovery that the use of Ca-containing fixatives leads to the demonstration of a subpopulation of synaptic vesicles fused to the terminal membrane, we are led to propose that this is the ultrastructural location of the newly synthesized acetylcholine which has been shown by others to be preferentially released by stimulation.     

Osmotic behavior of liposomes of phosphatidylcholine and phosphatidylsulfocholine as a function of lipid concentration

A relationship between the initial rate of liposome swelling, d(1/A)/dt and the reciprocal of the lipid concentration of the liposomes has been derived and then utilized to describe the osmotic swelling behavior of serially diluted liposomes and chloroplasts exposed to hypertonic urea solutions. The slopes of plots of d(1/A)/dt vs. the reciprocal of the lipid concentration of liposomes were not affected by differences in the initial absorbance of phosphatidylcholine-sterol bilayers, and were used to assess the ability of sterols to reduce the initial rates of urea permeation through dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline state. Multilamellar liposomes and sonicated vesicles were prepared from dimyristoylphosphatidylsulfocholine (DMPSC), in which the quaternary ammonium group of choline is replaced by -S⁺(CH₃)₂. Cholesterol reduced the initial rate of osmotic urea penetration into liposomes and the rate of 6-carboxyfluorescein efflux from vesicles at 35°C. The effect of cholesterol on bilayers of phosphatidylsulfocholine and phosphatidylcholine was very similar, suggesting that no strict structural requirements need be met in the choline moiety for lecithin-cholesterol interaction. The sulfonium analog could thus functionally replace phosphatidylcholine in natural membranes.     

Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

Background and Aims

Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium.

Methods

Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration.

Key Results

The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock.

Conclusions

It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.     

MEASUREMENTS OF SOME CELLULAR CHANGES DURING THE FIXATION OF AMPHIBIAN ERYTHROCYTES WITH OSMIUM TETROXIDE SOLUTIONS

On average, 15 per cent of the total haemoglobin present in the blood of the newt Triturus cristatus was extracted during 45 minutes of fixation in Palade-Caulfield fixative. This extraction was reduced with fixatives buffered at pH 6.2 instead of pH 7.4. The addition of Ca⁺⁺ ions to a final concentration of 0.01 M in the fixative completely suppressed haemoglobin extraction. The effect of the pH, and the presence or absence of Ca⁺⁺ ions in the fixative, on the rate of haemoglobin extraction has been determined. During Palade-Caulfield fixation the average projected area of newt erythrocytes increased by 37 per cent, and after dehydration and embedding in Epon the average area was 25 per cent greater than that of the unfixed cell. Fixatives buffered at pH 6.2 and containing 0.01 M Ca⁺⁺ ions caused cellular shrinkage, with the average projected area decreasing by 10 per cent in the fixative. This shrinkage continued during dehydration, and the final average area of the erythrocytes in Epon was 26 per cent less than that of the unfixed cells. Similar measurements with erythrocytes of Amphiuma tridactylum showed that after Palade-Caulfield fixation the average cellular area was increased by 45 per cent, and after dehydration and embedding in Araldite it was 36 per cent greater than that of the unfixed cell. The average nuclear area increased by 35 per cent during fixation but after embedding it was 26 per cent greater than that of the unfixed nuclei. With a fixative at pH 6.2 containing 0.01 M Ca⁺⁺ ions, both the nucleus and the whole cell shrank during fixation. The nuclear area decreased by 20 per cent and the cellular area by 22 per cent. After dehydration and embedding in Araldite, the average nuclear area had decreased by 35 per cent and the cellular area by 40 per cent. It has been shown that OsO₄ fixation lowers the isoelectric points of haemoglobins and other proteins. This finding has been used in the interpretation of the observed cellular changes resulting from fixation.     

Membranous cytochrome c oxidase. A freeze-fracture electron microscopic analysis

Suspensions of membranous cytochrome c oxidase prepared from beef heart mitochondria by Triton extraction were ultra-rapidly cooled (in excess of 10,000 deg.C/s) and analyzed using freeze-fracture and freeze-fracture-etch techniques. The preparations contained non-crystalline and crystalline vesicles as isolated vesicles, vesicles inside other vesicles and stacks of vesicles. In non-crystalline vesicles the particles (about 100 Å diameter) are probably formed by the deviation of hydrophobic fracture planes of the membranes around the large transmembrane enzymes. The intramembrane particles thus formed are compared to particles (about 80 Å diameter) in a vesicle reconstituted from purified enzyme and lipid. Crystalline membranous cytochrome c oxidase vesicles display an unusual fracture pattern in which adjacent crystalline surfaces are separated from each other and from the surrounding ice by fracture steps that are approximately the thickness of a single membrane (100 to 120 Å). In addition adjacent crystalline fracture surfaces have similar low-relief textures, both of which differ significantly from the hydrophobic surfaces normally exposed in membrane fractures. This fracture morphology is interpreted in terms of fractures along hydrophilic surfaces of the membranes. Images of etched crystalline vesicles provide support for this interpretation because etching exposes no new surfaces. It is concluded that the crystalline lattices are derived from the portions of enzymes that protrude from the membrane bilayers and that the interdigitation of the enzymes on the inside surfaces of the vesicles or between vesicles determines the appearance of the crystalline surfaces. The arrangement of the tails of the y-shaped molecules on the cytoplasmic sides of the crystalline membranes can be visualized in micrographs directly and in reconstructions of filtered images. The more complex pattern of arms protruding on the matrix side is obscured by the unidirectional shadowing. Fragmentation of the crystalline membranes during fracturing is indicated by particles sometimes present at the edges of fractured membranes and by deep, irregular pits observed in crystalline surfaces. Particles resting on some crystalline surfaces may be fragments of crystalline membranes removed during fracturing. In other crystalline membranes non-protein is removed during fracturing, leaving globular particles embedded in the lattice, which measure about 118 Å diameter. Comparing these particles to the 3-dimensional arrangement of protein described in the accompanying paper (Frey et al., 1982) suggests that such particles are composed of 2 dimers paired along the a-axis. Intramembrane and fragmentation particles of similar size may also have this protein composition.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

Glutaraldehyde fixation preserves the permeability properties of the ADH-induced water channels

Summary Unidirectional and net water movements were determined, in frog urinary bladders, before and after glutraldehyde fixation. Experiments were performed in three experimental conditions: 1) in nonstimulated preparations, 2) after the action of antidiuretic hormone (ADH) and 3) in nonstimulated preparations to which amphotericin B was incorporated from the luminal bath. As previously observed for net water fluxes, the increase in the unidirectional water movement induced by ADH was well preserved by glutaraldehyde fixation. After correction for the effects of unstirred layers and nonosmotic pathways, the observed correlation between the ADH-induced increases in the osmotic (Pf) and diffusional (Pd) permeability coefficients was not modified by the fixative action (before glutaraldehyde: slope 11.19,r:0.87±0.07;n=12; after glutaraldehyde: slope 10.67,r:0.86±0.04,n=39). In the case of amphotericin B, Pf/Pd=3.08 (r: 0.83±0.08), a value similar to that observed in lipid bilayers or in nonfixed toad urinary bladders. It is concluded that 1) The experimental approach previously employed to study water channels in artificial lipid membranes and in amphibian urinary bladders, can be applied to the glutaraldehyde-fixed frog urinary bladder. 2) Glutaraldehyde fixation does not modify the permeability properties of the ADH-induced water channels. 3) Any contribution of exo-endocytic processes or cell regulatory mechanisms to the observed permeability parameters can probably be excluded. 4) Glutaraldehyde-fixed preparations are a good model to characterize these water pathways.     

PHYSICOCHEMICAL EFFECTS OF ALDEHYDES ON THE HUMAN ERYTHROCYTE

The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K⁺ leakage during fixation. The concentrations of K⁺ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.     

Lipid-protein interactions in mitochondria. The effect of protein interaction on susceptibility of phospholipids to phospholipase A2 and C hydrolysis.

Phospholipase A₂ (Naja naja) and phospholipase C (from either Clostridium welchii or Bacillus cereus) have been tested on phospholipid dispersions and natural or reconstituted membranes; notwithstanding the different substrate specificities, the different enzymes gave comparable behaviors, suggesting that the results were the expression of sterical features in the lipid bilayers, i.e., availability of the phospholipids to enzymatic attack. The hydrolysis of phospholipids (Asolectin) in sonic protein-free vesicles is hindered by ionic interaction with basic proteins (cytochrome c or lysozyme). On the other hand binding of Asolectin to lipid-depleted mitochondria to obtain reconstituted mitochondria does not prevent phospholipase action on the phospholipids; similarly, phospholipids are hydrolyzed at maximal rates in natural membranes (mitochondria or submitochondrial particles). Surprisingly, ionic interaction of RM or natural membranes with basic proteins does not prevent phospholipase hydrolysis of the membrane phospholipids. The interpretation of this phenomenon may be related to the heterogeneity of phospholipid distribution in protein-containing membranes.     

THE FINE STRUCTURAL ORGANIZATION OF NERVE FIBERS, SHEATHS, AND GLIAL CELLS IN THE PRAWN, PALAEMONETES VULGARIS

In view of reports that the nerve fibers of the sea prawn conduct impulses more rapidly than other invertebrate nerves and look like myelinated vertebrate nerves in the light microscope, prawn nerve fibers were studied with the electron microscope. Their sheaths are found to have a consistent and unique structure that is unlike vertebrate myelin in four respects: (1) The sheath is composed of 10 to 50 thin (200- to 1000-A) layers or laminae; each lamina is a cellular process that contains cytoplasm and wraps concentrically around the axon. The laminae do not connect to form a spiral; in fact, no cytoplasmic continuity has been demonstrated among them. (2) Nuclei of sheath cells occur only in the innermost lamina of the sheath; thus, they lie between the sheath and the axon rather than outside the sheath as in vertebrate myelinated fibers. (3) In regions in which the structural integrity of the sheath is most prominent, radially oriented stacks of desmosomes are formed between adjacent laminae. (4) An ~200-A extracellular gap occurs around the axon and between the innermost sheath laminae, but it is separated from surrounding extracellular spaces by gap closure between the outer sheath laminae, as the membranes of adjacent laminae adhere to form external compound membranes (ECM's). Sheaths are interrupted periodically to form nodes, analogous to vertebrate nodes of Ranvier, where a new type of glial cell called the "nodal cell" loosely enmeshes the axon and intermittently forms tight junctions (ECM's) with it. This nodal cell, in turn, forms tight junctions with other glial cells which ramify widely within the cord, suggesting the possibility of functional axon-glia interaction.     

Permeability properties of peroxisomal membranes from yeasts

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH_I complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments. Generation of a membrane potential () was also not observed when RCLH_I complex was introduced into vesicles of purified peroxisomal membranes. The significance of the observed permeability of isolated yeast peroxisomal membranes to small molecules with respect to current and future in vitro import studies is discussed.Abbreviations CL cardiolinin - PE phosphatidylethanolamine - PC phosphatidylcholine - MES 2-(N-Morpholino)ethanesulfonic acid - R₁₈ octadecyl Rhodamine B Chloride - SUVs small unilamellar vesicles - RCLH_I-complex reaction center-light-harvesting complex I - BR bacteriorhodopsin - DCCD N,N-dicyclohexylcarbodiimide     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

Influence of calcium and magnesium containing fixatives of the ultrastructure of parathyroids

The effect of Ca²⁺ and Mg²⁺ on feline parathyroid cells during perfusion fixation with glutaraldehyde and subsequent immersion in OsO₄ was investigated. Both Ca²⁺ and Mg²⁺ may exert a stabilizing or destabilizing effect on cell membranes and on elements of the cytoskeleton. The effect depends (1) on the ion concentration, (2) on the buffer concentration and (3) on the fixative. Stabilization due to Ca²⁺ or Mg²⁺ during glutaraldehyde fixation is not altered during subsequent osmication but both cations may cause destabilization during osmication in tissue prefixed without cations. Ca²⁺ and Mg²⁺ also reduce cell volume in combination with low osmolar buffer but they prevent cells from excessive shrinkage due to high osmolar buffers. Ca²⁺ and Mg²⁺ alone or in combination reduce swelling of RER, extraction of cellular material and loss of subcellular compartments, such as secretory granules, under optimal conditions. Ca²⁺, however, provokes formation of dark (shrunken) and light (swollen) cells accompanied by loss of subcellular components when used in low concentration during osmication. Low concentrations of Mg²⁺ added to glutaraldehyde exert similar effects. Stabilization of membranes is assumed to be due to the binding capacity of Ca²⁺ and Mg²⁺ to both phospholipids and proteins. The influence of Ca²⁺ and Mg²⁺ to changes in cell volume is considered likely to be the result of ionic interaction in the cytoplasmic gel, the maintenance of cell volume being a matter of equilibrium between the swelling pressure of the cytoplasmic gel and osmotic pressure of the fixative solution.     

Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.This work was supported in part by funding from the USDA-SBIR program, 4Cs Breeding Technologies, and the Louisiana Sea Grant College Program.     

A methodological study for the quantification and the control of the physico-chemical processes occurring during cell fixation

Summary Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation the fixative is always offered (1) in excess of the cell sample, and the process of fixation is influenced by (2) chemical impurities of the fixative fluid.Both factors influence the succeeding dyeing of cells. In order to avoid these uncontrolled criteria, a new technology for controlled cell fixation has been developed, whereby freshly prepared formaldehyde and methanol gas in an inert gas-flow of helium was applied to thin membranes by aid of a capillary flow-in technique.The instrumental equipment consists of (1) an ultra-high vacuum flowapparatus with a total-pressure measuring unit, (2) a gas-supply device, (3) a mass spectrometer including a pump system, and (4) a Teflon and/or glass-gas chamber for the treatment of synthetic (Hostaphan foils) or biological membranes (mesenterium) with formaldehyde as the fixative gas.The amount of offered, adsorbed, absorbed, diffused, and desorbed fixative gas could be absolutely estimated after the saturation of the membranes with an on-line operating inert mass spectrometer of the Omegatron type.The gas treatment of the Hostaphan foils with formaldehyde showed that nearly all adsorbed gas molecules could be desorbed. In contrast to native membranes the greatest proportion of the gas molecules adhered to the biological surface, and only a small quantity were desorbable. Physisorption or physisorption and chemisorption occured depending on the adsorber surface property.A monolayer of formaldehyde of 5·10¹⁴ to 1·10¹⁵ molecules per 10¹⁶ Ų surface area can be postulated on the basis of these preliminary results. This value corresponds to a mass of about 5·10^–8g CH₂O. It resulted in an area-coverage ratio of CH₂O molecules per cell of 10⁹:1.The membrane surface facing the gas side always amounted to 1 cm². A fixative gas concentration of 10⁶ molecules/cm³, and therefore a degree of coverage of <1/1000 monolayer can be estimated absolutely. For a precise determination of the degree of fixation, further experiments and the evaluation of additional physico-chemical parameters are necessary.     

Studies on reaction and binding of monoamines after fixation and processing for electron microscopy with special reference to fixation with potassium permanganate

Summary In the present paper certain properties of potassium permanganate (KMnO₄), a fixative used for electron microscopical investigations, have been studied in model test tube experiments and on tissues. Evidence was obtained that KMnO₄ reacts with different types of biogenic monoamines resulting in a formation of a precipitate. In addition, also various monoamine analogues, precursors and metabolites reacts with KMnO₄. The reaction taking place may be an oxidation-reduction-reaction in which KMnO₄ is reduced, probably mainly to manganese dioxide by hydroxyl groups of the amines and related compounds. This is corroborated by the fact that no reaction takes place between KMnO₄ and -phenylethylamine or amphetamine, two substances, which lack hydroxyl groups.Using labelled monoamines evidence was obtained that the amine partly is retained within the precipitate formed after the reaction with KMnO₄ and also in tissues fixed with KMnO₄, indicating a possibility to perform autoradiographic studies on KMnO₄ fixed tissue.Electron microscopic studies on tissues fixed under various conditions revealed that fixation with low concentrations (0.6 and 1.0%) of KMnO₄ and at high temperatures (about 20° C) leads to inferior results as to general morphology and as to the visualization of intraneuronal amine stores.Different types of permanganates were tested as fixatives. These results show that fixation with permanganates with monovalent metallic ions (K⁺, Li⁺ and Na⁺) give good results of comparable quality, whereas fixation with zinc permanganate results in seriously destroyed tissues. However, tissue fixed with calcium permanganate reveals very distinct membranes. Furthermore, evidence was obtained that fixation with high concentrations of LiMnO₄ (6 and 9%) and NaMnO₄ (6 and 9%) was more sensitive as to the demonstration of monoamines at the ultrastructural level as compared to 3% KMnO₄. Thus, with e.g. 6 and 9% LiMnO₄ small granular vesicles could be seen in slices from the caudate nucleus after incubation with -methyl-dopamine. This was not possible when using 3% KMnO₄ as a fixative.     

Inhibition of photosynthetic carbon dioxide fixation in isolated spinach chloroplasts exposed to reduced osmotic potentials

Reduced osmotic potentials inhibited the rate of CO₂ fixation by isolated intact spinach (Spinacia oleracea) chloroplasts. This inhibition was observed immediately after transfer of chloroplasts from a solution containing 0.33 m sorbitol to higher sorbitol concentrations, and the depressed rate remained constant. The inhibited CO₂ fixation could not be attributed to a decreased rate of photosynthetic electron transport, since NADP reduction was unaffected by subjecting the chloroplasts to low potentials. It could also not result from restricted permeability to CO₂, as CO₂ concentrations had no effect on the relative inhibition induced by the lowered potential.     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.