Effect Of Glutaraldehyde Fixation Research Articles

Glutaraldehyde fixation of venous valve tissue: A benchmark for alternative fixation methods.

Bioprosthetic venous valves have yet to achieve long-term patency due to issues with calcification following implantation that is influenced by current xenograft fixation methods, most notably glutaraldehyde. The goal of this study was to investigate the effects of glutaraldehyde fixation on the functional properties of venous tissue to establish a benchmark for the evaluation of alternative fixation methods. The degree of crosslinking was evaluated by determining shrink temperature and the stability of tissue with pronase and collagenase digestion. Glutaraldehyde fixation of venous tissue was confirmed by a significant difference in the shrink temperature between fresh and glutaraldehyde treated samples. Significant differences in the amount of tissue remaining following digestion were observed for venous versus cardiac tissue. This study demonstrates the importance of tissue-specific evaluation in the development of alternative xenograft fixation methods to improve outcomes with bioprosthetic venous valves.

Effect of glutaraldehyde fixation on the frictional response of immature bovine articular cartilage explants

This study examined functional properties and biocompatibility of glutaraldehyde-fixed bovine articular cartilage over several weeks of incubation at body temperature to investigate its potential use as a resurfacing material in joint arthroplasty. In the first experiment, treated cartilage disks were fixed using 0.02, 0.20 and 0.60% glutaraldehyde for 24h then incubated, along with an untreated control group, in saline for up to 28d at 37°C. Both the equilibrium compressive and tensile moduli increased nearly twofold in treated samples compared to day 0 control, and remained at that level from day 1 to 28; the equilibrium friction coefficient against glass rose nearly twofold immediately after fixation (day 1) but returned to control values after day 7. Live explants co-cultured with fixed explants showed no quantitative difference in cell viability over 28d. In general, no significant differences were observed between 0.20 and 0.60% groups, so 0.20% was deemed sufficient for complete fixation. In the second experiment, cartilage-on-cartilage frictional measurements were performed under a migrating contact configuration. In the treated group, one explant was fixed using 0.20% glutaraldehyde while the apposing explant was left untreated; in the control group both explants were left untreated. From day 1 to 28, the treated group exhibited either no significant difference or slightly lower friction coefficient than the untreated group. These results suggest that a properly titrated glutaraldehyde treatment can reproduce the desired functional properties of native articular cartilage and maintain these properties for at least 28d at body temperature.

Effect of glutaraldehyde fixation on bacterial cells observed by atomic force microscopy

Atomic force microscopy (AFM) is a promising microscopy technique that can provide high-resolution images of bacterial cells without fixation. Three species of bacteria, Xanthomonas campestris, Pseudomonas syringae, and Bacillus subtilis, were used in this study. AFM images were obtained from unfixed and glutaraldehyde-fixed cells, and cell height was measured. The mean height of bacterial cells prepared by fixation was higher than that of those prepared by nonfixation. However, the height changes were different between Gram-negative and Gram-positive bacteria: the mean height of two fixed Gram-negative bacteria, X. campestris and P. syringae, increased by 112.31 and 84.08%, respectively, whereas Gram-positive bacterium, B. subtilis, increased only by 38.79%. The results above indicated that glutaraldehyde fixation could affect the measured height of cells imaged by AFM; further more, the effect of glutaraldehyde fixation on the measured height of Gram-negative bacterial cells imaged by AFM seemed much more than on that of Gram-positive bacterial cells.

Imaging of <i>Acidithiobacillus Ferrooxidans</i> by AFM and Analysis of AFM Curves

The aim of the present investigation is to obtain tapping mode images of Acidithiobacillus ferrooxidans and interfacial forces between Acidithiobacillus ferrooxidans cells and silicon nitride tip of AFM. The effect of exposure of cells to toxic metal ions like arsenic on the morphology of the cells was investigated by the section analysis of the AFM images obtained. Effect of glutaraldehyde fixation on force curves is also analyzed.

An X‐ray microscopy perspective on the effect of glutaraldehyde fixation on cells

X-ray microscopy (XRM) is the only microscopy technique that can provide high-resolution (30 nm) imaging of biological specimens without the need to fix, stain or section them. We aim to determine the effect, if any, of glutaraldehyde fixation on algae cells from the XRM perspective and thus provide beneficial information for both X-ray and electron microscopists on artefacts induced by glutaraldehyde fixation. Three species of microalgae, Microcystis aeruginosa, Anabaena spiroides and Chlorella vulgaris, were used in this study. XRM images were obtained from unfixed and glutaraldehyde-fixed cells and cell diameter and percentage X-ray absorbency were measured. The mean diameter of cells from fixed preparations was smaller than from unfixed preparations; the mean diameter of M. aeruginosa cells was significantly reduced from 3.92 microm in unfixed cells to 3.43 microm in fixed cells (P < 0.05); in C. vulgaris the diameter of cells was also significantly reduced from 3.50 microm in unfixed to 2.98 microm in fixed samples (P < 0.05); whereas there was no significant reduction in the diameter of A. spiroides cells (4.04-3.90 microm). The protein crosslinking mechanism of glutaraldehyde probably generated free water molecules, which play an important role in radiation damage induced by X-rays. This was seen as mass loss and cell shrinkage, which in the present study occurred more frequently in fixed cells than in unfixed cells. In addition, we demonstrated that the uptake of glutaraldehyde by cells makes all protein constituents in the cell organize into a closely packed configuration, thus causing a rise in the percentage of X-ray absorbency. In fixed cells, this rise was approximately by a factor of two compared with unfixed samples in which protein constituents inside the cell are arranged in their native form.

Effect of glutaraldehyde-fixation on the immunogenicity, particle stability and antigenic reactivity of alfalfa mosaic virus, and the specificity of elicited antibodies

Glutaraldehyde-fixation was shown to stabilize the structural integrity of alfalfa mosaic virus (AMV) particles as well as to increase their immunogenicity and antigenic reactivity. The antigenic reactivity of the particles was substantially increased irrespective of whether the antibodies were from animals immunized with native or fixed AMV, or preparations of coat protein subunits isolated from the virus. No significant changes in the antigenic specificity of AMV particles were detected following glutaraldehyde-fixation. Thus it is possible to raise antisera to AMV with higher titres by using fixed virus as immunogen.

Microtubules and tubulin sheet polymers elongate from isolated axonemes in vitro as observed by negative-stain electron microscopy

Microtubules are an important cytoskeletal component involved in cell motility and morphogenesis. They are unique polymers because they are highly dynamic in vivo and in vitro, displaying spontaneous transitions between phases of elongation and rapid shortening. This property has been termed microtubule dynamic instability. Here we describe the application of negative-stain electron microscopy to examine the morphology of microtubules. The purpose was to provide insight into the structural basis of dynamic instability. Highly purified porcine brain tubulin was seeded from isolated axoneme seeds and the morphologies of the tubulin polymers were examined. As previously reported, tubulin polymer sheets in addition to intact MTs were observed during elongation. Cross-sections of identical preparations displayed intact circles (MTs) and c-shaped polymers. The fraction of sheets (28%) observed by negative staining was identical to the fraction of c-shaped polymers in cross-sections. These results suggest that tubulin polymer elongation is not strictly helical due to the lack of helical symmetry of tubulin sheets. Finally, we document the novel effect of glutaraldehyde fixation on MTs. Fixation of MTs in 1% glutaraldehyde for longer than 2 min severely disrupted MT protofilament structure and induced MT curvature at a gross level. Even at 1 min, thin, thread-like structures were observed that were only present in glutaraldehyde-fixed samples. It is therefore an advantage to minimize the extent of glutaraldehyde fixation.

Effect of glutaraldehyde fixation on cell surface binding capacity of Candida albicans

The ability of viable and glutaraldehyde-fixed, stationary-phase yeast cells of Candida albicans to bind concanavalin A and monospecific antiserum for antigenic factor 1 was examined. Both fluorescence flow cytometric analysis and transmission electron microscopy indicated that glutaraldehyde-fixed cells bound less of the two reagents than did unfixed viable cells.

Effects of glutaraldehyde fixation on the body size of red sea bream ( Pagrus major) larvae

The use a buffered 2.5% glutaraldehyde solution as a fixative for red sea bream larvae was investigated. Morphometric data were obtained from live larvae 3, 12, 18, and 25 days old. These were then fixed for a period of 6 months and were remeasured. The only significant changes between live and preserved specimens were in body and muscle depth in 3-day-old specimens. Compared to conventional formalin-fixed specimens, the glutaraldehyde-fixed specimens had negligible shrinkage and a more natural appearance.

Thiaminepyrophosphatase activity in the plasma membrane of microglia.

An intense thiaminepyrophosphatase (TPPase) activity was demonstrated in glial cells and blood vessels in the central nervous system (CNS), when incubation was carried out with thiaminepyrophosphte (TPP, cocarboxylase), using the method of Novikoff and Goldfischer (1961). Glial cells with TPPSase activity were identified as microglia because they were morphologically similar to microglial cells in the sections stained with silver impregnation. TPPase activity was localized in the microglial perikaryon and in the processes, as viewed under a light microscope. Electron microscopically, enzyme activity was localized in the plasma membrane of microglia. We consider this activity to be a true TPPase activity hydrolyzing TPP, and we then went on to examine the substrate specificity, optimum pH, effect of chemical inhibitors and activators, and the effect of glutaraldehyde fixation. Our data are reported herein.

The myocardial plasma membrane during development: influence of glutaraldehyde fixation on the density and size of intramembranous particles.

The ultrastructure of the plasma membrane of mouse myocardial cells has been studied during development (from 10 days post-coïtum to the adult stage) using the freeze-cleave method, and the effect of glutaraldehyde-fixation on this structure has also been evaluated. Blebs, free of intramembranous particles (IMP), were found to be dependent upon the developmental stage and upon fixation with glutaraldehyde. On P and E fracture faces, the presence of IMP-free areas was age-dependent and fixation-independent. No difference was demonstrated in the IMP density during heart development. Fixation with glutaraldehyde retains in the plasma membrane approximately 40% of IMP, probably by preventing an alteration induced by glycerol. Whatever the developmental stage, E face-associated particles are significantly larger than P face-associated particles. The diameter of both types of particle increases slightly but irregularly as myocardial cells mature. Fixation reduces the particle size on both fracture faces in such a way that no significant difference between them can be demonstrated.

Effects of glutaraldehyde fixation on the structure of tight junctions. A quantitative freeze-fracture analysis

The effects of aldehyde fixation on the structure of tight junctions in rat liver and small intestine were studied by quantitations of freeze-fractured material. The length of fibrils in percentage of the total length of the junctional strands (ridges) on P-faces and the number of particles per micrometer of E-face groove were calculated in a series of experiments. After fixation in 2.5% glutaraldehyde (GA) for 1 or 3 hr, the tight junctions of liver and small intestine were comprised of strands with about 60–80% fibrils on the P-faces, whereas the E-face grooves exhibited only a very few particles (2–5/ μm). When the tissue was fixed in 0.1% GA for 24 hr similar results were obtained. However, with decreasing time of fixation (3 hr, 1 hr, 15 min, and 0 min) in 0.1% GA before glycerol cryoprotection, a significant decrease in percentage fibrils and a corresponding increase in number of particles per micrometer of E-face groove were seen. In the 15- and 0-min material, values of only about 1–3% fibrils and as much as about 60 particles per micrometer were obtained. In fresh-frozen material (nonfixed, noncryoprotected) fibrils were completely absent and all tight junction particles (approximately 65/μm) were located in chains on the E-faces. After fixation in 1.5% formaldehyde for 1 and 3 hr, only about 6 and 9% fibrils in the liver, and about 4 and 15% fibrils in the small intestine, were obtained. With respect to E-face grooves, about 31 and 25, and 44 and 42 particles per micrometer of liver and small intestine, respectively, were obtained. These results indicate that the tight junction fibrils may be polymerization products caused by the formidable crosslinking effect of GA. It is suggested that tight junctions in vivo are comprised of discrete integral proteins with stronger binding to the external side of the membrane or to opposing junctional proteins in the adjacent membrane than to the inner side of the membrane. However, GA fixation causes (1) lateral linking of the junctional proteins resulting in the formation of fibrils, and (2) binding of the fibrils to the protein-rich inner or cytoplasmic side of the membrane. Some functional consequences of this concept are considered.

Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.

Analysis of membrane structure in the transitional epithelium of rat urinary bladder: 1. The luminal membrane

The structure of the luminal membrane of rat urothelium as observed with the frozen-surface and freeze-fracture replica techniques is described. Plaques, which are composed of hexagonally arranged subunits, constitute 73% of the membrane area. Data on the size and organisation of the subunits and other membrane components are presented. The structure of the subunits as seen in optically filtered and unprocessed images of the E-surface and the E- and P-fracture faces of the membrane is discussed with reference to current concepts of the events associated with the fracturing and replication of frozen membranes. Differences between the E-face and E-surface subunit structure are explicable in terms of deformation induced by fracturing and other technical considerations. The effect of glutaraldehyde fixation on the fracturing behavior of subunits and the occurrence of subunits in a dispersed arrangement are also discussed. The interplaque areas of the luminal membrane exhibit small, inconspicuous intramembrane particles on the P-face and E-face, and a rugose appearance at the E-surface. These structures may correspond to protein components other than those of the subunits which have been characterized in biochemical studies.

Glutaraldehyde fixation and the mechanism of erythrocyte agglutination by concanavalin a and soybean agglutinin

To evaluate the importance of lectin receptor mobility and clustering for enhanced cell agglutinability, the effect of glutaraldehyde fixation on the agglutinability of human erythrocytes by concanavalin A and soybean agglutinin was investigated. Agglutinability was evaluated in unperturbed Microtiter ® plates. Fixation increased slightly the agglutinability of the erythrocytes by both lectins. Fixation did not alter trypsin-enhanced agglutinability. Furthermore, when fixed erythrocytes were trypsinized, their agglutinability increased to the level of unfixed, trypsinized erythrocytes. The kinetics of agglutination of fixed and unfixed erythrocytes were monitored in an electronic particle counter. The shear forces associated with the kinetic experiments diminished fixed-cell to fixed-cell agglutination, i.e., both lectins gave slower kinetics of agglutination with fixed erythrocytes than with unfixed erythrocytes. In contrast, the kinetics of concanavalin A-mediated agglutination of trypsinized-fixed erythrocytes mixed with equal numbers of trypsinized-unfixed erythrocytes were indistinguishable from the rapid kinetics of agglutination of trypsinizedunfixed erythrocytes alone. Light microscopy revealed aggregates composed of fixed and unfixed erythrocytes. We conclude that glutaraldehyde fixation does not diminish the agglutinability of human erythrocytes under low-shear conditions. Our results indicate that the enhanced agglutination of trypsinized erythrocytes is not dependent on clustering of lectin receptors. The disruption of agglutination of fixed erythrocytes by shear forces that do not disrupt agglutination of fixed erythrocytes with unfixed erythrocytes suggests that the rigidity of the fixed erythrocyte may prevent stable aggregate formation by fixed erythrocytes alone.

Sensitivity of glucose 6-phosphatase activity to glutaraldehyde

The effect of glutaraldehyde fixation on glucose 6-phosphatase activity in mouse liver was investigated. After transparenchymal perfusion with 2% glutaraldehyde for 1.5 minutes, the activity of the recovered enzyme was higher than those reported for acid phosphatase and aryl sulfatase activities after fixation under similar condition, and an abundant deposition of reaction product was observed in hepatocytes. Subsequent immersion in the same fixative solution for 30 minutes after 4 degrees C resulted in only a slight decrease in the activity. However, the activity was almost completely destroyed after 3 hours of immersion fixation at 4 degrees C following the perfusion. Therefore, the enzyme can be said to be aldehyde-sensitive when a long fixation time is used, but not aldehyde-sensitive during a short fixation time.

Effect of glutaraldehyde fixation on erythrocyte agglutinability.

SummaryThe fixation of human erythrocytes with glutaraldehyde causes a striking reduction in the agglutinability by ABO–specific antisera. However, glutaraldehyde does not appear to destroy the specific antigenic receptors, since the fixed cells retain the capacity to adsorb specific antibodies from solution.

The effect of glutaraldehyde fixation on the primary photochemical processes in bacterial photosynthesis

In Chromatium D the half-time for laser-induced (20–30-nsec flash) cytochrome C553 oxidation in redox poised chromatophores (1 μsec) and cytochrome C555 oxidation in whole cells (2.5μsec) is not affected by glutaraldehyde fixation. The reduction half-times for both cytochromes, however, increase as different functions of the glutaraldehyde concentration during the whole cell fixation process. At a cell-fixing concentration of 0.8%, cytochrome C555 but not C553 is observed after a laser flash. Steady light-induced spectra using similar preparations suggest the possibility of four components observable in the 500–620-nm range. These are cytochrome C555, P600, a species peaking at 560 nm and a component displaying a light-induced blue shift in the 500–540-nm region which may be a carotenoid response. The wavelength expected for the α-peak (reduced- minus-oxidized) of cytochrome cc′ is 560 nm, but the lack of a corresponding Soret peak makes identification uncertain and raises the possibility that we are observing a totally new component. Comparison of the amount of cytochrome oxidized by steady illumination and by a laser flash shows that on the average there are three cytochrome C555 molecules per reaction center in both whole cells and chromatophores. If the glutaraldehyde acts directly on the reaction center cytochromes then it is clear that cytochrome reduction requires large amplitude motion, but that oxidation does not. However, glutaraldehyde fixation may simply block the path of reducing electrons before they reach reaction center bound cytochromes.

Effect of glutaraldehyde fixation on the localization of various oxidative and hydrolytic enzymes in the brain of rhesus monkey, Macaca mulatta.

Histochemical investigations have been made on the localization of certain oxidative and hydrolytic enzymes in the different areas of rhesus monkey brain using unfixed, freshfrozen tissue and 3% glutaraldehyde-fixed material. After glutaraldehyde fixation, the oxidative enzymes lose most of their activity normally demonstrable in the fresh-frozen section. The hydrolytic enzymes are somewhat resistant to fixation but also lose about half of the enzyme activity observed after no fixing procedure. The glycogen is better preserved in the glutaraldehyde-fixed material compared to fresh-frozen or even formaldehyde-fixed tissue. The significance of these observations is discussed in relation to glutaraldehyde as a fixative of choice in electron histochemistry.

The effect of glutaraldehyde fixation on the elucidation of the morphogenesis of annulate lamellae in oocytes ofRana pipiens

Primary fixation of the frog oocyte with glutaraldehyde, compared to osmium tetroxide, alters the appearance of components involved in the morphogenesis of annulate lamellae. With glutaraldehyde, the outer layer of the nuclear envelope is connected with membranous laminae of variable length. Rounded blebs of the outer layer of the nuclear envelope are infrequently observed. Further, rather than clusters or rows of cytoplasmic vesicles as are observed in osmium tetroxide-fixed cells; numerous and long, smooth-surfaced lamellae are present in the ooplasm after glutaraldehyde fixation. The long membranous laminae then become concentrated in several ooplasmic packets. This is followed by the progressive alignment or orientation of the laminae within the packet. Eventually, those aligned and formerly smooth-surfaced lamellae are converted into annulate lamellae.