Acetone Dehydration Research Articles

Brain extracellular space and membrane morphology variations with preparative procedures.

ABSTRACT When considering extracellular space and plasma membrane structure in the brain, duration of postfixation in osmium tetroxide and embedding procedure are equally as important as the dehydrating agent employed. The use of ethanol instead of acetone for dehydration does not adequately explain the finding of a 5–30 nm space instead of apposed membranes. In this investigation it was found that postfixation for 3 h stabilized the tissue sufficiently so that ethanol or acetone dehydration followed by Vestopal W, Epon 812, or Araldite 502 embedding produced similar results, and apposed, triple-layered plasma membranes were prevalent. In contrast, when postfixed for a shorter time, namely 90, 45 or 30 min, ethanol dehydration followed by Epon embedding resulted in the common ‘20-ntn ‘extracellular space and only partially triple-layered plasma membranes. The same tissue dehydrated with acetone and Vestopal embedded looked much like the equivalent 3-h postfixed material, i.e. it had apposed, triple-layered plasma membranes. When acetone dehydration and Epon embedding, or ethanol dehydration and Vestopal embedding were used with tissue of short postfixation time, the results were comparable. Apposed membranes interspersed with erratic amounts of extracellular space were common and plasma membranes tended to show an indistinct outer leaflet. Taken together the results indicate that the presence of apposed triple-layered plasma membranes is at least as valid as the common finding of a 20-nm space and partially tri-laminar membranes.

Phosphotungstic acid-chromic acid as a selective electron-dense stain for plasma membranes of plant cells.

A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO3) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO4 postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO3 mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.

A simple freeze-substitution method for the study of ultrastructure of plant tissues

A simple method for freeze-substitution is described. Substitution takes three days in a solution of osmium tetroxide in acetone and the material is subsequently embedded in araldite. The fixation obtained is comparable to that given by glutaraldehyde. The method is being used to study the ultrastructure of maize root tips, phloem and yeast. As fixation of the material only occurs after dehydration in acetone, the preservation of the various membrane systems of the cell is different from that seen in conventionally fixed cells. As the exact time of fixation by freezing is known, the method is also useful for the study of cells in which biochemical reactions occurring during conventional fixation result in ultrastructural changes.

Organization of the cytosomal membranes of molluscan neurons under normal and anaerobic conditions as revealed by electron microscopy

Cytosomes (yellow pigment granules) of nerve cells of Anodonta cygnea have been investigated. The prolonged anoxia (5-8 days) induced a marked alteration of cytosomes involving the mass proliferation of membranes inside them at the expense of the homogeneous lipid droplets and granular matrix. These membranes are mainly of lipid character as revealed by the method of single glutaraldehyde fixation and subsequent acetone dehydration. Some of the normal cytosomes contain acid phosphatase activity being much more intense and of more general occurrence during prolonged anoxia. The functional significance of the structural alterations in the electron transport mechanisms is discussed.

Effect of Temperature on Ectotherm Tissue Ultrastructure

Environmental temperature variations result in physiological compensations in the metabolism of ectotherms. These compensatory mechanisms are manifested at various organizational levels within the organism. Compensatory changes at the cellular level constitute the most fundamental mechanisms involved during the acclimation process. The metabolism of various tissues during temperature acclimation has been the subject of numerous investigations (Prosser, 1967). Hochachka (1967) demonstrated increased activities of various metabolic pathways including the pentose shunt, lipogenesis and glycogenesis during cold temperature compensation. Limited information is available concerning the ultrastructure correlates of tissues in response to temperature variations and temperature extremes. This paper reports some preliminary results concerning the effect of temperature on the ultrastructure of various tissues of ectotherms.Heart, liver, skeletal muscle, and brain tissue were excised from heat killed sidewinders, Crotalus cerastes, as were tissues from C. cerastes acclimated to 20 and 30 C. Prefixation was carried out in 3%, cacodylate buffered glutaraldehyde followed by postfixation in 1%, osmium tetroxide. Acetone dehydration and epon embedment were according to standard procedure.

Osmium binding to the surface coat of intestinal microvilli in the cat under various conditions.

AbstractThe appearance of the carbohydrate‐rich surface coat covering the the intestinal epithelium of the cat varies with different methods of fixation for electron microscopy. A fibrillar surface coat covers the external surfaces of the microvilli after fixation in glutaraldehyde and postfixation in phosphate‐buffered OsO4; its density is enhanced by heavy metal staining. In tissue fixed in glutaraldehyde alone or followed by dehydration in acetone and postfixation in OsO4 in carbon tetrachloride, a structureless zone less dense than Epon occurs where the surface coat is usually visualized; heavy metal staining does not impart density to the zone. The absence of structure and low density of the zone are not due to extraction of the surface coat by dehydrating agents or by carbon tetrachloride. The data suggest that osmium binds to the surface coat from phosphate buffer, but not from carbon tetrachloride. Also OsO4 in phosphate buffer, but not OsO4 in carbon tetrachloride, facilitates heavy metal staining of the surface coat. Acidic carbohydrates appear to be important for the differential binding of osmium observed in this study. The need for caution in interpreting low density areas of electron micrographs as extracted structures and high density areas as “osmiophilic” structures is discussed.

The Ultrastructure of Muscular Cirrhosis of the Human Lung

Muscular cirrhosis of the lung is an uncommon form of idiopathic fibrosis in which excessive amounts of collagen and non-striated muscle form in the alveolar septa interfering with the transport of oxygen and carbon dioxide across the alveolar membrane.The patient in this case was a man aged 56 years, who had had progressive shortness of breath for three years. Thoracotomy was performed and the lingular lobe biopsied. The tissue was fixed in 3% phosphate buffered gluteraldehyde, and after dehydration in acetone, embedded in Vestopal. Thin sections were stained with uranyl magnesium acetate.

The quantitative retention of cholesterol in mouse liver prepared for electron microscopy by fixation in a digitonin-containing aldehyde solution.

A major methodological problem in the intracellular localization of cholesterol is the nearly complete extraction of sterols during routine dehydration and embedding procedures for electron microscopy. Cholesterol digitonide (a sterol complex with digitonin), however, is qualitatively insoluble in these solvents. Mouse liver has been prepared as follows: (a) Flickinger's aldehyde fixative, 20 hr; (b) Flickinger's fixative containing 0.2% digitonin, 24 hr; (c) cacodylate wash, 24 hr; (d) 1% OsO(4), 2 hr; (e) acetone dehydration; and (f) Epon 812 infiltration under vacuum, 28 hr. After the last step, an analysis of the tissue for sterol content under optimal analytical conditions demonstrates a retention of 99% of the unesterified cholesterol present in unfixed mouse liver. Liver prepared in an identical manner except for omission of digitonin is essentially devoid of sterols. Cholesterol isolated chromatographically from liver processed as outlined above has been identified unequivocally by mass spectrometry. Liver from step (f) also has been polymerized, thin-sectioned, and examined in the electron microscope. A remarkable quality of fine-structural preservation is obtained. The major alteration encountered is the presence of small cylindrical "spicules," often occurring as tightly packed concentric lamellae, at membrane surfaces.

Correlated Responses of Blood Coagulation and Prothrombin Times to Selection for Body Weight

Concomitant changes in unselected traits resulting from selection for a particular trait are considered correlated responses. The theory of correlated responses and computational procedures are discussed in Falconer (1960). Reported here are correlated responses of blood coagulation and plasma prothrombin times in F9 and F10 generation White Rock females selected for high and low body wieght at eight weeks of age (Siegel, 1962).Chicks, in both generations, were removed from the hatcher on the 22nd day of incubation, pedigreed, and reared in floor pens. Body weights were obtained at eight weeks of age and coagulation and prothrombin times were measured at 18 weeks by the methods of Quick (1959). Thromboplastin preparations were made from brain tissue of chickens by the acetone dehydration method (Quick, 1959).A completely randomized design was used for comparisons between lines within generations. All data for the calculation of realized genetic correlations (Falconer, 1954) were from…

Scanning electron microscopy of normal and pathological human erythrocytes.

A few drops of human blood were fixed in a relatively large volume of glutaraldehyde of various concentrations. After dehydration in acetone the blood cells were dried on glass slides in a warm breeze, coated with carbon and gold and observed under the scanning electron microscope.1. The deformations of erythrocytes caused by the osmotic effect of the fixative were examined. Large differences in osmotic resistance were shown among individual cells especially in hypotonic fixation. Glutaraldehyde concentration of 1% (in a 0.1M phosphate buffer) was found to be optimal for the preservation of the natural shape of normal erythrocytes.2. After this fixation the normal erythrocytes were represented by smooth-surfaced biconcave disks occasionally mingled with uniconcave ones. Some erythrocytes showed one or several wart-like elevations in their cell surface. These are believed to correspond to the segresome-like appendage seen in the transmission electron microscopy of sectioned erythrocytes (MIYOSHI and MATSUKURA, 1969).3. As pathological materials, the erythrocytes in elliptocytosis, hereditary spherocytosis and aplastic anemia were observed and their morphological abnormalities were described. Small conic pits were found on the surface of the erythrocytes in spherocytosis.

Comparison of Cholesterol Extraction from Tissues During Processing for Electron Microscopic Radioautography

Radioautographic studies of lipid metabolism with the electron microscope have been limited by the large losses of lipid which occur during the usual dehydration and infiltration procedures. In this study different methods of processing tissue for electron microscopic radioautography have been compared with respect to the extraction of cholesterol-1, 2-3H from labeled mouse liver and nerve.In all methods primary fixation had been in 10% formalin for several months followed by washing overnight. Post-fixation for one hour in Dalton's chrome osmium and staining in 1% uranyl acetate in 10% formalin resulted in small but constant losses. Four methods were assessed: 1) graded acetone dehydration with propylene oxide and epon-araldite mixture infiltration, 2) exposure to 0.5% digitonin in 50% ethanol for 1 hour before post-fixing in osmium followed by procedure 1, 3) Durcupan embedding, and 4) limited dehydration followed by embedding in an epon:araldite mixture (modified Idelman).

Fixation of the central nervous system by perfusion with aldehydes and its effect on the extracellular space as seen by electron microscopy.

Abstract Rat brains fixed by perfusion with glutaraldehyde and prepared for electron microscopy under certain conditions showed an abundance of very closely apposed or completely fused surface membranes in some areas. This apparent lack of extracellular space was most consistently observed in the lateral geniculate body and the superior colliculus and less consistently in the cerebellum and parietal cortex. Lowering the temperature of the perfusate from body temperature (38–39 °C) to room temperature (19–20 °C) and/or lowering the pressure at which it was delivered (from 140 to 70 cm of water) led to the appearance of more extracellular space. A striking change in the amount of extracellular space was observed when ethanol was used for dehydrating the samples instead of acetone. When ethanol was employed a space of approximately too A was seen between most cellular elements, whereas acetone dehydration led to an abundance of closely apposed or fused surface membranes. It is suggested that if glutaraldehyde acts by cross-linking proteins in apposing membranes, this reaction is more effectively completed in the presence of acetone and that this may be due to depolymerization of glutaraldehyde by the solvent. It is also suggested that the variation in the amount of extracellular space seen in brains fixed by perfusion with aldehydes may reflect real differences in distances between membranes at the time of fixation.

THE FINE STRUCTURE OF THE ELECTRIC ORGAN OF TORPEDO MARMORATA.

The fine structure of the electric organ of the fish Torpedo marmorata has been examined after osmium tetroxide or potassium permanganate fixation, acetone dehydration, and Araldite embedment. This organ consists of stacks of electroplaques which possess a dorsal noninnervated and a ventral richly innervated surface. Both surfaces are covered with a thin basement membrane. A tubular membranous network whose lumen is continuous with the extracellular space occupies the dorsal third of the electroplaque. Nerve endings, separated from the ventral surface of the electroplaque by a thin basement membrane, contain synaptic vesicles (diameter 300 to 1200 A), mitochondria, and electron-opaque granules (diameter 300 A). Projections from the nerve endings occupy the lumina of the finger-like invaginations of the ventral surface. The cytoplasm of the electroplaques contains the usual organelles. A "cellular cuff" surrounds most of the nerve fibers in the intercellular space, and is separated from the nerve fibre and its Schwann cell by a space containing connective tissue fibrils. The connective tissue fibrils and fibroblasts in the intercellular space are primarily associated with the dorsal surface of the electroplaque.