Showing papers on "Glutaraldehyde published in 1974"
TL;DR: Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue preservation, a task which has not been successfully accomplished by conventional fixatives.
Abstract: A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue successfully accomplished by conventional
3,819 citations
TL;DR: The procedure is a simple, rapid staining technique suitable for photomicrography and tissue orientation for electron microscopy, and considerable variation was found in methylene blue-azure II staining times for different embedding media.
Abstract: One micron-thick sections of tissues fixed in glutaraldehyde, or in glutaraldehyde followed by osmium tetroxide, and embedded in a variety of plastic resins were stained in a methylene blue-azure II solution at 65 C, then counterstained in 0.05% basic fuchsin in 2.5% ethanol at room temperature (24 C). Considerable variation was found in methylene blue-azure II staining times for different embedding media. Aged Epon-812 required less staining time than freshly polymerized blocks of Epon-812. The procedure is a simple, rapid staining technique suitable for photomicrography and tissue orientation for electron microscopy.
499 citations
TL;DR: The acrosome morphology, as assessed by phase-contrast microscopy, was not altered by fixing boar spermatozoa with 0.1 to 2% glutaraldehyde in comparison to spermatozosa inhibited with NaF.
168 citations
95 citations
TL;DR: The results indicate that rapid membrane permeability changes of EATC follow fixative action, consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation.
Abstract: 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.
89 citations
TL;DR: In this paper, the polymerization rate of monomeric glutaraldehyde was determined at different temperatures and pHs during a 5-month period, and the role of temperature and pH was investigated.
Abstract: During a 5-month period the polymerization rate of monomeric glutaraldehyde was determined at different temperatures and pHs. To elucidate the role of temperature, the authors used a 20.8% aqueous solution of glutaraldehyde at the following temperatures: −14°, 4°, 20°, 25°, 30°, 40°, 50°, and 60° C. With increasing temperature the rate of polymerization rose exponentially. To elucidate the role of pH the authors used 4% glutaraldehyde solutions in Teorell and Stenhagen's universal buffer of the following pHs: 3.0, 5.0, 6.5, 7.5, 8.5, 10.0, and 12.0 and in phosphate and cacodylate buffers pH 7.4. On both sides of the neutral point the polymerization rate showed a marked rise, so that the pH dependence could be depicted as an exponential curve. Moreover, a marked difference in the polymerization rate was found for the various buffers at the same pH. It was not possible to demonstrate a significant decrease of the osmolar concentration within the experimental range gradually as the polymerization increased.
87 citations
01 Jan 1974
TL;DR: In this paper, a cross-linking assay was developed based on the assumption that protein aggregates formed by intermolecular cross-links would be water-insoluble.
Abstract: Diimidoesters are bifunctional reagents which react with the c-amino group of lysine, forming intermolecular cross-links with minimal alteration of the native properties of proteins. To determine the optimal conditions for tissue fixation with diimidoesters, a cross-linking assay was developed based on the assumption that protein aggregates formed by intermolecular cross-links would be water-insoluble. With this assay method, the proportion of insoluble protein in fresh liver was 16%, while optimal conditions for cross-linking with the diimidoester, dimethylsuberimidate (DMS) (16-20 mg/ml, 0.02 M Ca� + and 0.15 M Tris-HC1, pH 9.5), rendered 92.1% insoluble and permitted only 3.3% of the protein to diffuse into the fixative solution. The smaller diimidoesters, dimethyladipimate and dimethylmalonimidate, rendered 74 and 16% of the protein insoluble, respectively. Fixation with various aldehydes produced insoluble fractions varying from 74% to over 90%. Ultrastructurally, hepatocytes of DMS-fixed liver and glutaraldehyde-fixed liver were similar except that with the former fixative the Golgi saccules and smooth endoplasmic reticulum were dilated and mitochondrial matrices exhibited an increased electron density. Furthermore, the appearance of glutaraldehyde- and DMS-fixed liver was more readily correlated with the degree of cross-linking than with the pH of the fixative solution per se. The results of this study indicate that DMS is a potentially useful fixative for light and electron microscopy.
86 citations
TL;DR: DMS is a potentially useful fixative for light and electron microscopy and the appearance of glutaraldehyde- and DMS-fixed liver was more readily correlated with the degree of cross-linking than with the pH of the fixative solution per se.
Abstract: Diimidoesters are bifunctional reagents which react with the e-amino group of lysine, forming intermolecular cross-links with minimal alteration of the native properties of proteins. To determine t...
86 citations
TL;DR: It is demonstrated that fixation of tissues and cells with 1% paraformaldehyde does not seriously affect tissue and membrane-bound antigens and addition of glutaraldehyde in very low concentrations improved the morphological conditions required for immunoelectron microscopical studies.
61 citations
TL;DR: The preparation of various cross-linked acrylic copolymers in bead-form, suitable for the immobilization of enzymes, is described, and attempts were made to optimize immobilization conditions.
59 citations
TL;DR: Conjugates with molar ratios of antibody or Fab fragment to peroxidase being one, seem more effective, although no significant differences were noted between per oxidase conjugates of whole antibody molecules and Fab fragments.
TL;DR: Fixation with PFA under specified conditions did not diminish the fluorescence intensity of cells stained by direct or indirect immunofluorescence and decreased only slightly the reaction of anti-immunoglobulin with cell-bound fixed antibody.
TL;DR: It was found that on increasing the hydrophobicity of the matrix by copolymerization of methylacrylate with acrylamide, the apparent Michaelis-Menten constant for the more hydrophobic substrate n- butanol decreased by a factor of four, whereas that for ethanol was essentially unaffected.
TL;DR: Evidence is presented which confirms that cholera toxoids obtained by reaction of purified toxin with Formalin possess the ability to partially reactivate both in vivo and in vitro, and suggests a relationship between complete and irreversible elimination of toxicity and the formation of higher-molecular-weight toxoids.
Abstract: Evidence is presented which confirms that cholera toxoids obtained by reaction of purified toxin with Formalin possess the ability to partially reactivate both in vivo and in vitro. At the same time, conditions are presented for the preparation of stable, antigenic cholera toxoids by reaction of purified toxin with glutaraldehyde. Treatment of purified cholera toxin with approximately 200 mol of glutaraldehyde per mol of toxin at pH 7.8 reproducibly resulted in the preparation of toxoids which: (i) possessed less than 20 bluing doses per 100 μg; (ii) did not reactivate in vivo or in vitro; (iii) precipitated with, and neutralized antitoxin; (iv) elevated prolonged serum antitoxin in immunized rabbits; (v) protected immunized guinea pigs against toxin skin challenge; and (vi) lent themselves to enhanced antigenicity by means of an in situ adjuvant system which may be suitable for man. Acrylamide gel electrophoresis and molecular sieve chromatography of a series of glutaraldehyde-derived toxoids suggested that the reaction products consisted of monomeric and polymeric species and that the proportion of higher-molecular-weight species was determined by the relative concentrations of toxin and glutaraldehyde. The results suggested a relationship between complete and irreversible elimination of toxicity and the formation of higher-molecular-weight toxoids.
TL;DR: The incidence of systemic reactions was reduced by the chemical treatment to negligible levels, permitting the therapeutic‐schedule to be reduced to three doses without impairment of clinical effectiveness.
Abstract: Summary
Grass pollen extract was reacted with glutaraldehyde, and used in the form of a lyrosine adsorbate for the treatment of hay fever. The incidence of systemic reactions was reduced by the chemical treatment to negligible levels, permitting the therapeutic-schedule to be reduced to three doses without impairment of clinical effectiveness. The levels of pollen-specific IgG antibody induced by the chemically modified material were similar to those induced by pollen-tyrosine or by alum-precipitated pyridine extract. The increase in IgE antibody induced by glutaraldehyde-pollen-tyrosine was lower than that induced by pollen-tyrosine: only the falter achieved significance.
TL;DR: Low concentrations of glutaraldehyde inhibited or prevented colony formation by Escherichia coli, Bacillus subtilis and B. pumilis in agar, and inhibited germination of spores of the Bacillus spp.
Abstract: Summary. Low concentrations (0.025–0.125%) of glutaraldehyde inhibited or prevented colony formation by Escherichia coli, Bacillus subtilis and B. pumilis in agar, and inhibited germination of spores of the Bacillus spp. in L-alanine plus D-glucose. Higher concentrations (2%) of glutaraldehyde at pH 8.5 were sporicidal. Pre-treatment of spores with glutaraldehyde lessened release of dipicolinic acid when the spores were subsequently heated at 100°, but not at 121°. Spores treated with glutaraldehyde and then with 0.5 M thioglycollic acid in 6 M urea at 70° were less sensitive to lysis by hydrogen peroxide than spores which had not been exposed to glutaraldehyde. Glutaraldehyde was less effective in preventing peroxide induced lysis if added to spores which had been previously exposed to thioglycollic acid plus urea at 70°. The mechanism of the sporicidal activity of glutaraldehyde is discussed in relation to these findings.
TL;DR: The acid-catalyzed condensation of either 2-ethoxy-3,4-dihydro-2H-pyran or pentanedial (glutaraldehyde) with ammonium-15N chloride in the presence of methylene blue is a convenient one-step synthesis of pyridine-15 N.
Abstract: The acid-catalyzed condensation of either 2-ethoxy-3,4-dihydro-2H-pyran or pentanedial (glutaraldehyde) with ammonium-15N chloride in the presence of methylene blue is a convenient one-step synthesis of pyridine-15N.
TL;DR: In this paper, a polymerizable mixture of glutataldehyde and urea was used to embed skeletal muscle and myocardium in various ways to decrease electrolyte disturbances, which appeared preferable to conventional techniques for electron microscope X-ray microanalysis and histochemical studies.
Patent•
28 Aug 1974
TL;DR: A water insoluble glucose isomerase product is prepared from microorganism cells by concentrating and homogenizing the cells to form a homogenized cell concentrate containing ruptured cells.
Abstract: A water insoluble glucose isomerase product is prepared from microorganism cells exhibiting glucose isomerase activity by concentrating and homogenizing the microorganism cells to form a homogenized cell concentrate containing ruptured cells, reacting the homogenized concentrate with glutaraldehyde to form a coherent solid product and removing water and shaping the coherent product into a divided form.
TL;DR: Mixtures of glutaraldehyde and osmium tetroxide do not preserve the cytoplasmic non-membranous lamellae characteristic of the hamster oocyte, and cause vesiculation of the endoplasmic reticulum, and appear to have no advantage for fixing ovarian tissue.
Abstract: For fixation of ovarian oocytes and granulosa cells the percentage concentration of glutaraldehyde in the fixative solution makes little difference between 2 and 4 per cent as long as the osmolarity of the buffer falls between 195–225 milliosmoles. With 1 per cent glutaraldehyde, fixation is erratic. Buffer osmolarities of 95–115 mOsm result in swelling and leaching of cytoplasm regardless of glutaraldehyde concentration. Post-osmication reduces leaching but does not prevent swelling. Increase in osmolarity of the non-fixative portion of the solution to either isotonic or hypertonic levels with buffer ions, glucose or sucrose results in shrinkage which is reversed during post-osmication. Certain features remain constant regardless of fixative concentration or osmolarity: a) The presence among the pale granulosa cells in multilaminar follicles of small numbers of extremely dark cells, b) the irregular outlines of granulosa cell nuclei and of oocyte nuclei in follicles with two or more layers of granulosa cells, c) the typical features of atretic oocytes in multilaminar follicles. Mixtures of glutaraldehyde and osmium tetroxide do not preserve the cytoplasmic non-membranous lamellae characteristic of the hamster oocyte, and cause vesiculation of the endoplasmic reticulum. They appear to have no advantage for fixing ovarian tissue.
TL;DR: All antigens were rendered non-reactive to their corresponding antibodies after single treatment with formaldehyde or glutaraldehyde, but the antigenicity of at least some antigen combining with antinuclear antibodies could be restored with 30% sucrose solution.
Abstract: The effects of formaldehyde and glutaraldehyde alone and in combination with sucrose on antigens reactive with pemphigus antibodies, bullous pemphigoid and antinuclear antibodies were studied. All antigens were rendered non-reactive to their corresponding antibodies after single treatment with formaldehyde or glutaraldehyde. However, the antigenicity of at least some antigens combining with antinuclear antibodies could be restored with 30% sucrose solution. On the other hand, the antigens for pemphigus or bullous pemphigoid antibodies could not be recovered by the sucrose treatment.
TL;DR: It was demonstrated that the shrinkage or dilatation of the tissue depended more upon the osmotic concentration of the buffer than of the fixative.
Patent•
01 Mar 1974
TL;DR: In this article, a buffered aqueous solution of glutaraldehyde, antigen or antigen-protein conjugate and formaldehyde is used to separate sensitized erythrocytes and then they can be lyophilized and become stable at room temperature.
Abstract: Stable preparations of sensitized erythrocytes are provided by simultaneously admixing washed erythrocytes in a buffered aqueous solution of glutaraldehyde, antigen or antigen-protein conjugate and formaldehyde. After washing, the separated sensitized erythrocytes can be lyophilized and thereby become stable at room temperature.
TL;DR: In this article, the electron microscopic X-ray analyser (EMMA-4) was used to investigate the calcium concentration and intracellular localization of dog myocardial tissues.
Abstract: Calcium concentration and intracellular localization were investigated using the electron microscopic X-ray analyser (EMMA-4) in preparations of dog myocardial tissues fixed either in glutaraldehyde, osmium tetroxide or potassium pyroantimonate plus osmium tetroxide.
TL;DR: No change in the transverse fascicular area of rat peroneal nerve occurred when the duration of fixation in 2% glutaraldehyde was increased from 1 to 12 hr, and shrinkage in endoneurial area was inferred from the development of clefts.
01 Jan 1974
TL;DR: It is concluded from this study that membrane structure, as revealed in thin sections as well as in freeze-etch replicas, is consistent with Singer9s ‘fluid lipid-crystal protein’ model.
Abstract: The structure of the basal labyrinth in kidney cells of freshwater sticklebacks was studied in ultrathin sections (after fixation with permanganate, osmium tetroxide, and combinations of glutaraldehyde with osmium tetroxide) and in freeze-etch replicas (after pretreatment with glutaraldehyde and/or glycerol, or without pretreatment). The structure of the basal labyrinth in sticklebacks, and probably in other teleost species, differs essentially from the type of labyrinth found in kidney cells of mammals like the rat. In the latter animals, the space enclosed by the membranes of the labyrinth is intercellular. In the stickleback the labyrinth consists of an intracellular system of branched membranes lining narrow saccular spaces. These spaces communicate with the exterior of the cells by means of small pores, located in the lateral and basal parts of the outer cell membranes. All chemical fixation procedures used introduced specific structural artifacts. It is concluded that the structure of the basal labyrinth is relatively well preserved after fixation with potassium permanganate, with a mixture of glutaraldehyde and osmium tetroxide, or with osmium tetroxide when applied for 10 min only. The unit-membrane structure was, however, absent after all procedures involving osmium tetroxide. In freeze-etch replicas determinations were made of the numbers of small particles covering the surfaces and fracture faces of the membranes of the basal labyrinth and of the outer cell membranes. The numbers per unit area of surface proved to be markedly constant and specific for each of the four faces of both types of membranes. Specific differences were found between the particle densities of the outer cell membranes and the membranes of the basal labyrinth. This finding points to functional differences between these types of membranes. Particle densities were not influenced by pre-incubation with glycerol. After fixation with glutaraldehyde, the particles adhering to the outer and inner surfaces had decreased in number. It is concluded from this study that membrane structure, as revealed in thin sections as well as in freeze-etch replicas, is consistent with Singer's 'fluid lipid-crystal protein' model.
TL;DR: In this paper, the structure of the basal labyrinth in kidney cells of freshwater sticklebacks was studied in ultrathin sections and in freeze-etch replicas (after pretreatment with glutaraldehyde and/or glycerol, or without pretreatment).
Abstract: The structure of the basal labyrinth in kidney cells of freshwater sticklebacks was studied in ultrathin sections (after fixation with permanganate, osmium tetroxide, and combinations of glutaraldehyde with osmium tetroxide) and in freeze-etch replicas (after pretreatment with glutaraldehyde and/or glycerol, or without pretreatment). The structure of the basal labyrinth in sticklebacks, and probably in other teleost species, differs essentially from the type of labyrinth found in kidney cells of mammals like the rat. In the latter animals, the space enclosed by the membranes of the labyrinth is intercellular. In the stickleback the labyrinth consists of an intracellular system of branched membranes lining narrow saccular spaces. These spaces communicate with the exterior of the cells by means of small pores, located in the lateral and basal parts of the outer cell membranes. All chemical fixation procedures used introduced specific structural artifacts. It is concluded that the structure of the basal labyrinth is relatively well preserved after fixation with potassium permanganate, with a mixture of glutaraldehyde and osmium tetroxide, or with osmium tetroxide when applied for 10 min only. The unit-membrane structure was, however, absent after all procedures involving osmium tetroxide. In freeze-etch replicas determinations were made of the numbers of small particles covering the surfaces and fracture faces of the membranes of the basal labyrinth and of the outer cell membranes. The numbers per unit area of surface proved to be markedly constant and specific for each of the four faces of both types of membranes. Specific differences were found between the particle densities of the outer cell membranes and the membranes of the basal labyrinth. This finding points to functional differences between these types of membranes. Particle densities were not influenced by pre-incubation with glycerol. After fixation with glutaraldehyde, the particles adhering to the outer and inner surfaces had decreased in number. It is concluded from this study that membrane structure, as revealed in thin sections as well as in freeze-etch replicas, is consistent with Singer9s ‘fluid lipid-crystal protein’ model.
TL;DR: Treatment of spores with freshly prepared glutaraldehyde solutions at temperature above 40 C reduced the effect of pH on sporicidal activity and alkaline solutions stored at 4, 20, and 37 C showed comparatively few changes in their properties.
Abstract: Freshly prepared 2% acid and alkaline glutaraldehyde solutions were stored at 4, 20, and 37 C. At intervals, samples were removed and changes in pH, ultraviolet spectrum, and sporicidal activity (against Bacillus pumilus spores) were recorded. Alkaline solutions stored at 4 C showed little changes in these properties, whereas such solutions stored at 37 C became turbid and showed a decrease in pH, marked changes in ultraviolet spectrum, and an almost complete loss of sporicidal activity. Intermediate results were obtained with alkaline solutions stored at 20 C. In contrast, acid 2% glutaraldehyde solutions (initial pH 3.5) showed comparatively few changes in their properties. Treatment of spores with freshly prepared glutaraldehyde solutions (0.5%) at temperature above 40 C reduced the effect of pH on sporicidal activity.
TL;DR: Both formaldehyde and glutaraldehyde caused a decrease in MAO activity, as expected, but the decrease was more pronounced at higher fixative concentrations and was also greater at 25#{176}C than at 0#{ 176}C.
Abstract: A brief, mild prefixation of samples in formaldehyde or glutaraldehyde is usually required for cytochemical and immunologic staining for electron microscopy . In spite of the risk of inactivation of enzymic or antigenic activities, this prefixation is necessary for preservation of the fine structure and retention of sufficient biologic functions of the molecules concerned to allow histochemical studies. As a continuation of our research (7-9) on the binding and localization of monoamine oxidase (monoamine-oxygen oxidoreductase, EC 1.4.3.4) (MAO) in the mitochondrial membrane by histochemical and immunologic staining, we have studied the effect of two commonly used fixatives, formaldehyde and glutaraldehyde, on MAO activity. Adult Sprague-Dawley rats weighing 200-250 g were killed by a blow on the head and the liver was removed. Mitochondria were prepared in 0.25 M sucrose solution by the method of Thompson, Coleman and Finean (10). The mitochondria so prepared had a specific activity of about 8 mMmoles tyramine oxidized/mm/mg of protein under the conditions described below. Monoamine oxidase activity: MAO activity was measured with a Clark-type oxygen electrode (Rank Brothers, Bottisham, Cambridge, England) as described by Tipton (11) at 30#{176}C. The reaction mixture (2.4 ml) contained 200 tmoles potassium phosphate buffer (pH 7.2), 100 units bovine liver catalase (Sigma Chemical Company, St. Louis, Mo.), 20 tmoles of semicarbazid (neutralized), 2 Mmoles of KCN and enzyme preparation. The reaction was started by the addition of 0.1 ml 0.025 M tyramine-HC1. Fixation procedure: Mitochondria were spun down at 10,000 x g for 15 mm at 4#{176}C and the pellets were resuspended in various concentrations of formaldehyde or glutaraldehyde in 0.1 M potassium phosphate buffer (pH 7.2). Formaldehyde solutions were prepared freshly by depolymerizing paraformaldehyde in the same buffer at 60-70#{176}C prior to use. Glutaraldehyde (25%) was obtained from the British Drug Houses (Poole, England) and its concentration was determined by the method of Frigerio and Shaw (2). The concentration was found to be 23%. Incubations were carried out at temperatures and times as indicated in Figures 1-4. As shown in Figures 1-4, both formaldehyde and glutaraldehyde caused a decrease in MAO activity. The decrease, as expected, was more pronounced at higher fixative concentrations. The decrease was also greater at 25#{176}C than at 0#{176}C. Most of the decrease in enzyme activity occurred during the first 5 mm of incubation and the losses varying from 0 to 70%, depending on choice and concentrations of the fixatives and fixation temperature. After this initial decrease, the loss of activity during the following 55 mm was slow. Less than 20% of the remaining activity was lost at 0#{176}C in both fixatives at all concentrations tested (Figs. 1 and 3). At 25#{176}C, the loss of MAO activity in glutaraldehyde after initial 5 mm was of the same magnitude as at 0#{176}C (Fig. 4). In formaldehyde, however, a considerable greater loss of MAO activity occurred at 25#{176}C. When the fixation was performed at 0#{176}C in 0.7% of either fixative, 70-80% of the original MAO activity