Direct measurement of the osmotic effects of buffers and fixatives in Nitella flexilis

The pressure probe has been used to investigate the amount and time course of changes in turgor induced by agents used in fixation of biological material for electron microscopy. All buffers tested decreased the turgor pressure of internodal cells of the green alga Nitella flexilis in proportion to their osmotic concentration. With the exception of s-collidine, the buffers were impermeant, as shown by the stability of the reduced turgor. The fixatives formaldehyde and glutaraldehyde caused a rapid reduction in turgor followed by recovery of turgor as the aldehyde penetrated the cell. Plasmolysis of cells can be avoided by a procedure involving stepwise increase of fixative concentration.     

The effect of six fixatives on the areas of rabbit neurons and rabbit and rat cerebral slices.

Cell bodies of cerebral neurons from rabbits were isolated by hand, transferred to a microscope slide in a ‘199’ medium, and the projected areas of their cytoplasm and nuclei were measured. In sixty-four cells there was a strong correlation between the projected areas of the cytoplasm and the nuclei (r=0.66, P < 0001), and the ratio of the projected areas was 11.6. The medium was then replaced by the following fixatives: formalin (10% v/v), Bouin's, Carnoy's, Susas, glutaraldehyde (5% v/v), and osmium tetroxide (1% w/v). Cerebral slices were obtained from the grey and white matter of rabbit and rat, and were also measured before and after treatment with similar fixatives. Relative to the unfixed areas, glutaraldehyde and osmium tetroxide had no significant effect on the projected areas of isolated cells, Carnoy's fixative shrunk the areas of the cytoplasm by means of 16.26%, Bouin's by a mean of 49%, and Susas by a mean of 65%. The shrinkage of the cytoplasm and the nuclei was not significantly different from that of the nuclei for each of these three fixatives individually, but with formalin the mean shrinkage of the cytoplasm was 46% while the nuclei did not shrink significantly. Using the same fixatives the effect on the areas of the cerebral slides from rabbit and rat were as follows: glutaraldehyde and osmium tetroxide caused no change in area; Carnoy's, formalin and Bouin's fixative diminished the areas by a mean of 10–20%, and Susa's by a mean of 35%. It was concluded that a particular fixative often caused a different degree of shrinkage to the cytoplasm, nuclei and cerebral slice. In general, the lower the osmotic pressure of the fixative, the less shrinkage it induced.     

The avian erythrocyte: a study of fixation for electron microscopy.

The quality of ultrastructural preservation of the avian erythrocyte achieved using various fixation techniques is evaluated. Different combinations of initial fixatives, buffers and post-fixation procedures were tested as well as variations in fixative osmolarity, pH and temperature. Of the commonly used initial fixatives (glutaraldehyde, acrolein and formaldehyde), 2% glutaraldehyde, alone in a slightly hypertonic buffer containing divalent ions, produced optimum erythrocyte preservation. The osmolarity was balanced using a non-electrolyte such as a sucrose. The addition of 12% hexylene glycol to the buffer solutions also improves erythrocyte preservation, as evidenced by the increased stability of the marginal microtubules, microfilaments and proteinaceous material. The use of Spurr low-viscosity epoxy resin enables the cells to be collected using low gravitational centrifugation.     

Atomic force microscopy of BHK-21 cells: an investigation of cell fixation techniques

The atomic force microscope (AFM) has been used to image a wide variety of biological samples, including cultured cells, in air. Whilst cultured cells have been prepared for AFM analysis using a variety of matrices and fixatives, a definitive study of sample preparation and its effects on cell morphology has not, as far as the authors are aware, previously been reported. Although a considerable number of cell fixatives exist, no single fixative is ideal for all investigations. Prior to the performance of specialised techniques, such as atomic force microscopy of cultured cells in air, the cell fixation method must be investigated and optimised. The fixative abilities of 2% paraformaldehyde-lysine-periodate, 0.25% glutaraldehyde, paraformaldehyde-glutaraldehyde, 4% phosphate-buffered formal saline, 1% formaldehyde, methanol:acetone, formal saline, 4% paraformaldehyde and ethanol:acetic acid were assessed in this study. A qualitative assessment system was used to evaluate the efficacy of the above fixatives using conventional fixation criteria (i.e. the presence of fibroblastic morphology consistent with optical microscopy and the absence of fixation artifacts). The optimal fixative was identified as 4% paraformaldehyde, which was capable of providing optically consistent images of BHK-21 (fibroblastic) cells, whose heights remained within the measurement capability of the AFM instrument used in this study.     

Localization and identification of crustacean hyperglycemic hormone producing neurosecretory cells in the eyestalk of blue swimmer crab,Portunus pelagicus

This study intensely focuses on to the localization and identification of crustacean hyperglycemic hormone (CHH) producing neurosecretory cells in the eyestalk of the blue swimmer crab Portunus pelagicus. Anti‐Carcinus maenas‐CHH was used to identify the location of CHH neurosecretory cells by immunohistochemistry. Ten pairs of eyestalks were collected from intact adult intermoult female crab and fixed in Bouin's fixative. Eyestalks were serially sectioned and stained with chrome‐hematoxylin‐phloxine stain. Histological studies show the presence of different types of neurosecretory cells namely A (multipolar), B (tripolar), C (bipolar), D (unipolar), E (oval), and F (spherical) in the medulla interna, externa, and terminalis regions based on their size, shape, and tinctorial properties. The neurohemal organ, sinus gland (SG) was observed laterally between medulla interna and terminalis regions. Immunohistochemical studies showed the presence of distinct CHH‐like immunoreactivity in the optic ganglia. Divergent group of neurosecretory cells with varying degree of immunoreactivity with Anti‐Carcinus maenas‐CHH (low, moderate, and intense reactivity) were identified in medulla terminalis, medulla interna, medulla externa, and sinus gland. The present study maps the various types of neurosecretory cells in the optic ganglia and also shows the presence of CHH‐like immunoreactivity in various regions of optic ganglia in P. pelagicus. The presence of these unique neurosecretory cell types with larger cell diameter in medulla terminalis, a region that bears the neurosecretory cell bodies, suggest high secretory activity.     

Effects of glutaraldehyde fixative osmolarities on smooth muscle cell volume,and osmotic reactivity of the cells after fixation

The contribution of glutaraldehyde (GA) to the effective osmolarity of GA fixatives, the osmotic reactivity of the cells after fixation in GA, and also the duration of fixation in GA on cell volume, were investigated using cultured smooth muscle cells (SMC) and spiral aortic strips. Four fixation procedures were studied. We found that GA contributes to the total effective osmolarity of the fixatives, and that the type of buffers used for the fixatives can also affect the cell volume differently during GA fixation. After GA fixation, the cells were still osmotically reactive, regardless of the buffer types for making up the GA fixatives, so that the osmolarity of the wash buffer after GA fixation is important. However, OsO₄ eliminates osmotic responses, thus the osmolarity of OsO₄ fixative and wash buffer have negligible influence on the cell volume. Longer fixation time up to 4 h had no effect on the cell volume.     

Freeze-substitution techniques for preparing nematodes for scanning electron microscopy

The effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated. Hammerschmidtiella diesingi adults and Trichostrongylus colubriformis infective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible. Panagrolaimus davidi was not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO₄/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.     

The innovative safe fixative for histology,histopathology, and immunohistochemistry techniques: “Pilot study using shellac alcoholic solution fixative”

The concerns over health and workplace hazards of formalin fixative, joined to its cross‐linking of molecular groups that results in suboptimal immunohistochemistry, led us to search for an innovative safe fixative. Shellac is a natural material which is used as a preservative in foods and pharmaceutical industries. This study was undertaken to evaluate the fixation adequacy and staining quality of histopathological specimens fixed in the “shellac alcoholic solution” (SAS), and also to determine the validity of immunohistochemical staining of SAS‐fixed material in comparison to those fixed in formalin. Fresh samples from 26 cases from various human tissues were collected at the frozen section room of King Abdulaziz University Hospital, and fixed in SAS fixative or in neutral buffered formaldehyde (NBF) for 12, 18, 24, and 48 h, and processed for paraffin sectioning. Deparaffinized sections were stained with hematoxylin and eosin (H&E) and immunostained for different antigens. The tissues fixed in SAS for >18 h showed best staining quality of H&E comparable to NBF‐fixed tissues. Comparison of the immunohistochemical staining of different tissues yielded nearly equivalent readings with good positive nuclear staining quality in both fixatives. These findings support the fixation and preservation adequacy of SAS. Furthermore, it was concluded that the good staining quality obtained with SAS‐fixed tissues, which was more or less comparable with the quality obtained with the formalin fixed tissues, supports the validity of this new solution as a good innovative fixative. Microsc. Res. Tech. 77:385–393, 2014. © 2014 Wiley Periodicals, Inc.     

Influence of fixative osmolality on the morphometric determination of extracellular space in normal and reperfused ischaemic myocardium

Morphometric determination of extracellular space in control and post-ischaemic reperfused rabbit myocardium was evaluated using two fixatives differing in their composition and total osmolality. Measurement of control extracellular space in an isotonic fixative (294 mOsm/kg water) was 20.8% and in a hypertonic fixative (1816 mOsm/kg water) was 22.2%. These values were not statistically different. Ischaemic durations of 15, 30, 60 and 90 min, followed by an equivalent period of reperfusion, created significant increases in extracellular space. The size of the extracellular space determined by both fixatives was found to be the same. Total fixative osmolality does not appear to influence morphometric evaluation of the extracellular space in control tissue or in tissue damaged by ischaemia and reperfusion.     

Monitoring of the course of fixation of plant cells

The fixation of cells of petiolar hairs of tomato was monitored by phase contrast and Nomarski interference contrast microscopy. Because the cuticle covering these hairs is relatively impermeable some fixatives enter only the base of the hair so that a ‘fixation front’ can be followed through a single cell. The basal end of such a cell may be immobilized by the fixative while the apical end is still streaming. A variety of fixatives were tested at room temperature and at 273–278 K. All procedures tested failed to stabilize a pleomorphic canalicular system that is a dominant feature of these cells so that no clue to the presence of this system can be seen in sectioned material. It is therefore very desirable that images of fixed cells be compared to the structure of similar cells which are still alive.     

Cytochrome oxidase activity and confocal laser scanning microscopic analysis of the hamster submandibular gland using microwave irradiated fixation

Submandibular glands of the hamster were irradiated in 2% paraformaldehyde (pFA)-0.5% pure glutaraldehyde (PGA) with a microwave (MW) processor at temperatures of 10 degrees and 37 degrees C. Electron microscopy showed that cytochrome oxidase activity was taking place in the mitochondrial intermembrane-intracristal space of the granular duct cell when the temperature of the MW-irradiated fixatives was at 10 degrees C. However, a decrease of this activity was observed when we took care to keep the temperature of the MW-irradiated fixatives at 37 degrees C. The distinct reduction of cytochrome oxidase activity allowed by MW irradiation seems to be due the thermal affects of fixatives. Of course, the possibility cannot be excluded that MW irradiation caused other undetectable membrane damage. Then, we used confocal laser scanning microscopy for the preservation check of the mitochondrial membrane for cytochemistry with MW-irradiated fixation. The fluorescence of rhodamine 123 was observed in the inner spaces of the mitochondria at temperatures of 10 degrees and 37 degrees C. When the same tissues were fixed with 2% pFA using an MW processor as the sole fixative at 10 degrees C, no mitochondrial fluorescence was observed. Cytochrome oxidase activity, by contrast, could be seen in the mitochondrial intermembrane-intracristal spaces in the same condition. Formaldehyde is not the best aldehyde for the purpose of ultrastructural preservation. On the other hand, light and electron microscopy showed that the endogenous peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum, secretory granules, and Golgi apparatus of the hamster submandibular gland using 2% pFA-0.5% PGA fixative with and without MW irradiations at temperatures of 10 degrees and 37 degrees C. Some of the same cells were fixed with only 2% pFA under MW irradiation at 10 degrees C; however, marked diffuseness of the peroxidase activity was observed. Therefore, these results indicated that cytochrome oxidase activity was sensitive to heat with MW-irradiated fixation. Peroxidase activity was very resistant to heat with MW-irradiated fixation but not with pFA solo fixation, therefore, PGA had to be used.     

Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue

Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ , are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 °C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.     

Aldehyde fixatives: Quantification of acid-producing reactions

Under conditions commonly used for the preservation of tissue for electron microscopy, substantial amounts of hydrogen ions were produced when either formaldehyde or glutaraldehyde was added to solutions of amines or proteins, or to tissue homogenates. In glycine-aldehyde reactions, hydrogen ions were generated in linear proportion to the glycine concentration over the range of 0.001 M to 0.100 M glycine. In the presence of 1% formaldehyde or 1% glutaraldehyde, 0.3–0.9 equivalents of acid were produced per mole of primary amine. This variation is a function of the specific primary amine used. Acid production increased with increasing formaldehyde concentration but did not increase appreciably with increasing glutaraldehyde concentration. The concentration of amines in tissue is high. These amines are probably responsible for much of the acid generated when aldehydes react with tissue homogenates. In amine-aldehyde reactions, the positive charge originally associated with the parent amine is simultaneously neutralized with the formation of hydrogen ions. Thus, in tissues exposed to aldehydes, there is a decrease in the overall positive charge which is related to the amount of acid generated. The inherent buffering capacity of liver and muscle was not sufficient to prevent large pH decreases when aldehydes were added to homogenates of these tissues. These data suggest that significant but transient pH decreases may occur within cells exposed to aldehydes. To minimize the aldehyde-induced pH decreases, a buffer should be chosen so that its pK_a is 0.2–0.3 pH units less than the pH desired for a particular experiment. In addition, buffer concentrations of at least 0.10 M are suggested for fixatives in order to provide adequate buffering capacity during tissue preservation.     

Fixation induces differential polarized translocations of organelles in hyphae of Saprolegnia ferax

Saprolegnia hyphal tips were examined during fixation, and living or freeze-substituted tips were quantitatively compared with those fixed in commonly employed formulations of paraformaldehyde and glutaraldehyde. Treating hyphae with fixatives induced extensive longitudinal translocations of the cytoplasm and organelles, usually beginning with contractions toward the tip. These translocations were minimal in the extreme apex (~10 μm) and more extensive subapically. Hypertonic media or hypotonic buffers seldom or never induced translocations, respectively; in contrast, hypotonic buffers containing detergents or the Ca²⁺ -ionophore, A23187, frequently induced contractions. All fixations caused net nucleus movement away from the tip, with the amount of displacement depending on the pre-fixation distance from the tip. Similarly, all fixations moved the most-apical of saltatory vesicles away from the tip, but the total number in the apex increased or decreased depending on the fixative used. The patterns of these results suggest that nucleus and vesicle distribution controls may be related (with respect to most-apical organelles) but also at least partially independent (with respect to organelle populations in hyphal tips). Hyphal diameter was reduced by some, but not all fixations; this variability did not correlate with displacements of either organelle, nor with fixative osmotic pressure. Evidently fixative-induced changes are more complex and systematic in highly polarized tip-growing cells than previously reported in other, less polarized, cell types. These results also suggest that hyphae contain multiple and complex organelle distribution and hyphal diameter control systems which can be readily altered, often subtly, by fixation protocols commonly and uncritically employed in immunocytochemical and ultrastructural analyses, and that fixation can cause serious cellular reorganization.     

Mercuric chloride in alcohol and chloroform used as a rapidly acting fixative for contracting muscle fibres

A fixative which preserves the correct alignment of the filaments in actively contracting muscle fibres must prevent sliding during fixation. Glutaraldehyde is unsuitable because the fibres are capable of relaxation and contraction for several seconds. A modification of Carnoy's fixative rapidly fixes tetanized fibres without relaxation or further activity. The fixative contained ethanol and chloroform saturated with mercuric chloride. Fibres were thereby fixed and dehydrated at the same time. They were then stained with uranyl acetate, embedded, and stained with lead in the usual way. Isolated fibres from R. temporaria were tetanized with low voltage pulses in air, and normal tetanic tensions were recorded prior to fixation. At least 50% of the tension remained after fixation. Unstimulated fibres were activated by the fixative, but fibres depolarized with K+ developed no tension. Embedded fibres were examined intact in polarized light and closely resembled fresh fibres. Electron micrographs showed that, although the membranes were disrupted, the myofibrils were well preserved to 15 μm below the surface and the band pattern was maintained throughout the fibre. There was some local variability in the width of the overlap zone, especially where myofibrils branched. The fine structure of the filaments was similar to that seen after conventional fixation. The cross-bridges were visible, but the alcoholic dehydration probably interfered with their orientation. Measurements of filament length, and of troponin and cross-bridge periods by optical diffraction of calibrated electron micrographs, indicated differential shrinkage between the I-band, overlap zone and H-zone similar to that seen with other fixatives. Criteria for satisfactory fixation of contracting muscle are discussed. The fixative is considered to be suitable for examining the longitudinal alignment of the filaments, and also for periodicity measurements if the small shrinkage artefact is allowed for. It is unsuitable for studying the reticulum, or for measuring the volume ratio of the myofibrils.     

Electron-microscope preparation techniques applied to the light microscopy of the cambium and its derivatives in Pinus radiataD. Don.

Epon 812 was used successfully as an embedding medium for the preparation of the secondary meristem and its derivatives in Pinus radiata for light microscopy. Specimens were fixed in glutaraldehyde followed by osmium tetroxide solution, using a schedule developed for the electron microscopy of these tissues. Sections from 2 to 5 μm thick were cut using an LKB Ultratome III and glass knives. Sections were evaluated without prior removal of the embedding medium using bright-field, phase-contrast and interference-contrast microscopy. Results for cambium were generally superior to those obtained by using the conventional fixatives for light microscopy and paraffin embedding. The advantages and disadvantages of this technique are discussed and it is concluded that the use of Epon for embedding this type of material is easier and gives better results than older established methods. It is suggested that methods of electron microscopy be considered when the preparation of a material for light microscopy has proved difficult or impossible by other means, or when particularly thin sections are required.     

Changes of particle frequency in freeze-etched erythrocyte membranes after fixation

The frequency of particles on the membrane fracture faces of freeze-etched human erythrocytes was measured, and the effect of fixation procedures on the particle frequencies was studied. Fresh blood, buffer washed cells and cells fixed in one of the following ways were examined: glutaraldehyde, glutaraldehyde followed by osmium tetroxide, osmium tetroxide alone. Quantitative analyses showed that some treatments produced a significant reduction in the number of particles on the fracture faces as compared with the fresh cells. After both osmium tetroxide fixations, the loss of particles was greater from the outer fracture face (OFF) than the inner fracture face (IFF), whilst after the other treatments approximately the same number of particles were lost from both fracture faces. The results are discussed with respect to some current concepts of the molecular architecture of the erythrocyte membrane and the action of fixatives. The reduction of particle frequencies is thought to be due to both leaching of membrane proteins, and deviations of the usual fracture plane within the membrane. Glutaraldehyde alone was shown to have less effect on particle frequency than the other fixatives and it is therefore a suitable fixative for the preparation of freeze-etch specimens.     

Oxygen content of the fixative is important in the interpretation of the ultrastructure of ischaemic myocardium

Isolated rat hearts were subjected to 15, 45, or 60 minutes of global ischaemia and then fixed by perfusion at 37°C with glutaraldehyde containing various amounts of oxygen. This either had been bubbled with 100% oxygen (PO₂ 620 mm Hg) or with 100% nitrogen (PO₂ 40 mm Hg) immediately before use, or it had been routinely prepared and stored exposed to atmospheric oxygen (PO₂ 245 mm Hg). The ultrastructure of myocytes and endothelial cells subjected to 15 minutes of ischaemia was not affected by the treatment of the fixative. However, when the tissue subjected to longer periods of ischaemia was fixed with routinely prepared or oxygen-bubbled glutaraldehyde, ultrastructural changes characteristic of reoxygenation damage were uniformly evident in both the microvasculature and myocytes. These qualitatively distinct changes included mitochondrial swelling, cell swelling, endothelial bleb formation, and narrowing of capillary lumina. These abnormalities were not observed in tissue fixed with nitrogen-bubbled glutaraldehyde. These findings indicate that deliberate steps should be taken to reduce or eliminate dissolved oxygen from the fixatives used to study ischaemic tissues. Otherwise artefactual reoxygenation damage in vitro may occur and make valid ultrastructural interpretation difficult or impossible.     

Quantitative assessment of chemical fixation,including the use of malachite green,for electron microscopy of vacuolate mesophyll cells from leaves of a grass

Various chemical fixation regimes were studied using leaves of the grass Lolium multiflorum Lam. as a test system. To evaluate the quality of fixation for electron microscopy two parameters were chosen; the extent of cell damage to mesophyll cells and damage and change in size of chloroplasts. Reducing the concentration of glutaraldehyde from 3% to 1% resulted in extensive cell damage. The addition of paraformaldehyde to 1% glutaraldehyde did not totally eliminate cell damage and resulted in dilation of thylakoids within the chloroplasts. In tissue fixed and washed in solutions matched to the total osmolality of the primary fixative solution there was little cell damage but the chloroplasts were larger, diameters 4.5times2.9 μm, than when only the buffer vehicle osmolality was maintained during subsequent washings, diameters 3.5times1.6 μm. Malachite green added to the primary fixative solution abolished cell damage regardless of buffer concentration and resulted in the appearance of osmiophilic droplets on the surface of and within the chloroplasts which were not present in the absence of malachite green. It is suggested that malachite green stabilizes cell membranes during primary fixation.     

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

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.