Showing papers on "Glutaraldehyde published in 1981"

Covalent stabilization of alginate gel for the entrapment of living whole cells

Abstract: Three methods were developed for preparing alginate gels containing cells that are stable in phosphate containing media. In Method I preformed alginate beads containing entrapped cells were treated with polyethyl eneimine followed by glutaraldehyde. In Method II alginate sol was treated with a carbodiimide and N-hydroxysuccinimide (to form active esters), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. In Method III alginate so] was treated with periodate (to form aldehyde groups), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine.

Spectral properties of fluorescence induced by glutaraldehyde fixation.

Abstract: Following fixation with glutaraldehyde, tissues or gelatin films fluoresce. This fluorescence can be enhanced more than thirtyfold by several minutes exposure to near ultraviolet light. Longer wavelengths produce a smaller effect. The enhanced fluorescence is maximally excited at 540 nm (half-band width about 45 nm), and fluorescence emission peaks at 560 nm. The rate of photoenhancement is independent of temperature in the 12--30 degrees C range. Photoenhancement is greater at alkaline than acid pH; the pH dependence involves a single acid binding group with pK = 7.3. These effects are not observed following treatment with paraformaldehyde or prior reduction with boranedimethylamine. Schiff base linkages between the bifunctional cross-linking reagent and free amino groups therefore seem to be involved. The effects of pH and wavelength on the photoenhancement of fluorescence are best accounted for by a kinetic scheme that includes both photogeneration of fluorophore from an ultraviolet-absorbing precursor, and its subsequent photodestruction by visible light.

The use of a simple method to avoid cell shrinkage during SEM preparation.

Abstract: SUMMARY In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO4-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO4, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.

The fixation, dehydration, drying and coating of cultured cells for SEM

Abstract: Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrance preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30-40 kV. Without treatment with OsO4 a thicker metal coating is required.

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

Abstract: The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96%+1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin, Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde+ 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections as regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.

FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.

Abstract: Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of stained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.

Preparation of small and soft specimen for scanning electron microscopy: Investigation of mouse optic nerve

Abstract: We have previously reported details of the dimensional changes taking place during the processing of soft tissue specimens for scanning electron microscopy. Mouse embryo limbs were used for many of these measurements and the present paper deals with the associated morphological findings. Effects of fixation, dehydration and drying are considered. Freeze drying and critical point drying of glutaraldehyde and glutaraldehyde and osmium fixed samples give perfectly acceptable results for scanning electron microscopy. The best volume retention with freeze dried material is matched by the best morphological appearance of the specimen surface, except when ice crystal damage occurs due to a failure to freeze the tissue rapidly enough. For CPD tissues, perforation of the plasmalemma may occur in glutaraldehyde-only fixed tissue, this being prevented by post-osmication if the glutaraldehyde fixation is not unduly prolonged. This perforation may be due to the extraction of some plasmalemma component during dehydration or further solvent substitution on critical point drying. Solvent evaporation drying usually causes recognizable distortion due to shrinkage: this is minimal in the case of solvent evaporation drying in a nearly saturated atmosphere of the same solvent if this is a very volatile solvent. The examples of Freon 113 and diethyl ether are given here. Swelling during early stages of ethanol dehydration can be prevented by using 70% or 100% ethanol as the first step, with marginal reduction in the post CPD volume and no apparent differences in the SEM. The severe swelling causing sample disruption which can occur with GA + OsO4 fixed tissue can also be prevented by treating the sample with divalent cations, such as Ca++ or Cu++, at any stage.

Use of tannic acid as a fixative-mordant to improve the ultrastructural appearance of Candida albicans blastospores.

Abstract: Blastospores of Candida albicans, grown in YM broth (Difco, pH 6.0), were treated with tannic acid as part of a fixation schedule with glutaraldehyde and osmium tetroxide. The results obtained were compared to routine fixation procedures employing glutaraldehyde-osmium tetroxide and potassium permanganate, as well as several schedules using various combinations of the aforementioned fixatives. Glutaraldehyde-osmium tetroxide-tannic acid fixed cells had several morphological structures accentuated, including sharply delineated cytoplasmic and organelle membranes, a detailed cytoplasmic matrix, and an intensified layered cell wall. Tannic acid has been found to react as a mordant between osmium tetroxide and lead citrate, forming a "stabilizing matrix" within the cells. Blastospores appeared to have an improved comprehensive ultrastructural appearance, a result of the tannic acid-matrix preserving cellular components for electron microscopy.

Zum Reaktionsmechanismus von Glutaraldehyd mit Proteinen

Abstract: The interaction of glutaraldehyde with model aliphatic amines was studied in order to understand the crosslinking reaction of glutaraldehyde with proteins. The reaction in organic solvents gave N-alkyl-1,4-dihydropyridines and N,N′-dialkyl-1,5-diiminopentanes. The isolated products are new or were previously described by us for the first time1. Hydration of the reaction products led to stable N-alkylpiperidines and N,N′-dialkyl-1,5-diaminopentanes. In aqueous solution the reaction depends on thepH: at apH above 7, N-alkyl-1,4-dihydropyridines and at apH below 7, polymers were obtained. For the crosslinking reaction of proteins with glutaraldehyde the following mechanism is proposed: Monomeric glutaraldehyde reacts with the protein to give intermediate N-alkyl-2,6-dihydroxypiperidines. Intramolecular dehydration leads to the corresponding N-alkyl-1,4-dihydropyridines. Condensation of the cyclic monohydrate of glutaraldehyde and N-alkyl-2,6-dihydroxypiperidines gives linear polymeric crosslinks containing α-oxo-N-alkylpiperidine units.

Glutaraldehyde/polyethylenimine immobilization of whole microbial cells

Abstract: Whole microbial cells containing enzymes not sensitive to glutaraldehyde such as sucrose mutase or glucose isomerase are immobilized by forming a reaction product of the cells in an aqueous medium with glutaraldehyde, reacting the reaction product with polyethylenimine to flocculate the reaction product, and recovering the flocculated reaction product from the aqueous medium. Reacting the cells with glutaraldehyde prior to reacting with polyethylenimine results in flocculated cells that can be more easily separated from the aqueous medium.

Glutaraldehyde-treated carrier erythrocytes for organ targeting of methotrexate in dogs.

Abstract: Chemotherapeutic doses of methotrexate (MTX) were encapsulated in as little as 1 ml of canine carrier erythrocytes. Carrier erythrocytes prepared by a dialysis technique contained more than 3.0 mg of MTX/ml of cells. In vivo pharmacokinetic studies indicated that most of the drug leaked from the circulating MTX-loaded erythrocytes. In dogs, glutaraldehyde treatment of MTX-loaded erythrocytes prevented drug leakage and targeted more than 50% of the drug to the liver.

A New Synergized Glutaraldehyde-Phenate Sterilizing Solution and Concentrated Disinfectant

Abstract: Research leading to the development of Sporicidin, a new (synergized glutaraldehyde-phenate) sterilant and concentrated disinfectant, is reported. Microbial evaluation and shelf life testing of the new formulation was done using AOAC tests--Use-Dilution, Sporicidal, Tuberculocidal--and the E.P.A. Virucidal Test. Full-strength Sporicidin sterilizes (is sporicidal) in 6 3/4 hours at both 20C and 25C, and disinfects in 1-2 minutes. When diluted with 15 parts of tap water, Sporicidin is tuberculocidal, virucidal, and germicidal in 10 minutes, qualifying as a hospital disinfectant that will not "yellow" the hands or skin. The shelf life of activated Sporidicin, full strength or diluted 1 in 16, is 30 days. According to the data presented, which were the basis of E.P.A. registrations, Sporicidin is: (a) the fastest glutaraldehyde sterilant at room temperature, and (b) the only glutaraldehyde product that can be diluted below 2% and still qualify as a hospital disinfectant.

Method of stabilizing platelets for determining multiple platelet parameters in reference control and calibrator compositions; and diluents thereof

Abstract: Stabilized reference control and calibrator compositions for determining multiple platelet parameters in stand alone platelet controls and whole blood reference controls are prepared from platelets stabilized with a fixative-stabilizing composition containing glutaraldehyde and a non-ionic surfactant which is a mixture of ethoxylates of certain isomeric linear alcohols

Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents.

Abstract: Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.

Extended-life tissue fixative composition and method of using the same

Abstract: Extended-life aqueous tissue fixative compositions or solutions are provided for use in histopathology work and the like. The compositions include glutaraldehyde at controlled pH, a boric acidtetraborate buffer, buffer stabilizer and complexing agent. The compositions, which also may include a compatible surfactant, and their method of application enable efficient fixing such that the treated tissues have low artifactual color and good texture and may readily be sectioned and accurately stained.

Coated Tube Enzyme Immunoassay: Factors Affecting Sensitivity and Effects of Reversible Protein Binding to Polystyrene

Abstract: Coated tube enzyme immunoassay using alkaline phosphatase conjugated to rabbit (anti-human IgG) antiserum was studied to determine conditions of maximum sensitivity. The competitive binding assay utilized showed a large increase in sensitivity with immobilized antigen levels below the levels giving rise to the maximum in the coating-antigen dilution series. The effects of reversible antigen binding to the solid phase were investigated by comparison of untreated polystyrene tubes, polystyrene tubes treated with glutaraldehyde and glass tubes activated with an aminosilane. The use of glutaraldehyde treated tubes reduced, and the use of activated glass tubes prevented the time dependent release of immobilized antigen seen with the untreated polystyrene tubes. By comparison of these solid phases, it is shown that reversible antigen immobilized in a competitive binding assay gives rise to poorer conjugate binding (three-fold), and poorer sensitivity (sixfold). A noncompetitive response was found to oc...

The use of glutaraldehyde and tannic acid to preserve reconstituted collagen for electron microscopy.

Abstract: The effects of glutaraldehyde and tannic acid on the axial periodicity of collagen have been measured. Both fixatives produce axial shrinkage of the collagen but whereas glutaraldehyde produces 7% shrinkage, tannic acid produces only 2% shrinkage. The technique of carbon/platinum shadowing was used to estimate the extent to which the collagen fibrils flatten down when they are dried onto grids for electron microscopy without prior embedding and sectioning. The influence of fixation was studied and it was found that minimum distortion occurred when both tannic acid and glutaraldehyde were used to preserve the protein structure.

Autoradiography of phosphatidyl choline.

Abstract: Saturated choline phosphatides are extracted during conventional tissue processing for electron microscopy. To facilitate autoradiographic subcellular localization of arrhythmogenic myocardial phospholipids, we evaluated tissue processing procedures for preservation of saturated phosphatidyl choline (PC). Suspensions, of a murine plasmacytoma were incubated with negative, unilamellar liposomes containing 14C-choline-labeled PC or 14C-1-palmitate dipalmitoyl PC. Extraction of radioactivity was monitored at each processing step by liquid scintillation spectrometry. Conventional fixation with glutaraldehyde and osmium tetroxide followed by acetone dehydration and Spurr's plastic embedding led to extraction of nearly all radioactivity. However, treatment of cells with 1.5% tannic acid after glutaraldehyde but before osmium tetroxide fixation preserved 93.1 +/- .6% of 14C-choline-labeled PC. Virtually identical results were obtained with dipalmitoyl PC. Autoradiography demonstrated no significant translocation of labeled PC from plasmacytoma cells to unlabeled avian erythrocytes, mixed in equal proportions after fixation but before dehydration and embedding.

Effects of different fixative solutions on labeling of concanavalin-A receptor sites in human T-lymphocytes.

Abstract: We have studied the effect of several fixative solutions on the number of Concanavalin-A·(Con-A) receptor sites of human peripheral blood T-lymphocytes. Cells treated with different fixative solutions (glutaraldehyde (G); formaldehyde (F);G+F; osmium tetroxide (Os); Os+G; Os+F; and Os+G+F) were labeled with a Con-A gold labeled horseradish peroxidase (HRP) complex and the number of gold particles on the lymphocytic surface was evaluated. Comparison of cells treated with the different fixatives used showed significant differences in the density of labeling. After G fixation the number of gold particles was lower than after fixation with Os or F. Moreover, G used in combination with F or Os reduced the labeling obtained when the two latter fixatives were used alone.

Fine structure of intramembranous particle aggregates in ADH-treated frog urinary bladder and skin: influence of glutaraldehyde and N-ethyl maleimide.

Abstract: The fine structure of ADH-induced intramembrane particle aggregates has been studied in different tissues and under different experimental conditions. Particle aggregates similar to those previously observed in the amphibian urinary bladder and in the mammalian collecting duct were also found in the frog skin, another ADH target tissue. In the frog urinary bladder, typical aggregates were observed in the absence of glutaraldehyde fixation. Two experimental approaches were used a) the absence of both fixative and cryoprotectant treatments and b) the absence of only glutaraldehyde treatment. In the latter case the reversal of hydrosmotic action was prevented by exposing the preparations to N-ethyl maleimide. In specimens of frog urinary bladder conventionally fixed with glutaraldehyde, two fracture levels could be observed in the aggregates, suggesting that the aggregated particles span an appreciable part of the membrane thickness.

Effects of glutaraldehyde or osmium tetroxide fixation on the osmotic properties of lung cells.

Abstract: The osmotic properties of lung cells have been tested before and after perfusion fixation of isolated, perfused lungs with either glutaraldehyde or osmium tetroxide. The testing procedure was to add hypertonic sucrose to the perfusate for several minutes and monitor the lung weight response (an 'osmotic transient'). Each lung was perfused with one or the other fixative solutions for 10 min, then the perfusate was changed back to Ringer-lactate before the post-fixation test was conducted. The results indicate that osmium tetroxide makes the cell membranes as permeable to sucrose as to water, and that sucrose thus causes no osmotic volume change. Glutaraldehyde, on the other hand, apparently preserves the impermeability of the cell membranes to sucrose, but the osmotic volume response is attenuated, indicating that significant changes in the cells have occurred.

Immobilization of thermophilic microbial cells in crude egg white

Abstract: Cells of thermophilic archaebacterium Caldariella acidophila have been trapped in chicken egg white by co-crosslinking with glutaraldehyde The activity of cytosolic β-galactosidase in trapped cells has been followed by varying temperature, pH, organic solvents A biocatalyst with magnetic properties has been prepared by including magnetite in the resin