Conventional Chemical Fixation Research Articles

Synchrotron X-rays reveal the modes of Fe binding and trace metal storage in the brown algae Laminaria digitata and Ectocarpus siliculosus.

Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (μXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation-freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component.

A conventional fixation volume electron microscopy protocol for plants.

Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies, environmental effects and microbial interactions with large plant structures, to name a few. Due to large air voids central vacuole, cell wall and waxy cuticle, many plant tissues pose challenges when trying to achieve high quality morphology, metal staining and adequate conductivity for high-resolution volume EM studies. Here, we applied a robust conventional chemical fixation strategy to address the special challenges of plant samples and suitable for, but not limited to, serial block-face and focused ion beam scanning electron microscopy. The chemistry of this protocol was modified from an approach developed for improved and uniform staining of large brain volumes. Briefly, primary fixation was in paraformaldehyde and glutaraldehyde with malachite green followed by secondary fixation with osmium tetroxide, potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide and finally uranyl acetate and lead aspartate staining. Samples were then dehydrated in acetone with a propylene oxide transition and embedded in a hard formulation Quetol 651 resin. The samples were trimmed and mounted with silver epoxy, metal coated and imaged via serial block-face scanning electron microscopy and focal charge compensation for charge suppression. High-contrast plant tobacco and duckweed leaf cellular structures were readily visible including mitochondria, Golgi, endoplasmic reticulum and nuclear envelope membranes, as well as prominent chloroplast thylakoid membranes and individual lamella in grana stacks. This sample preparation protocol serves as a reliable starting point for routine plant volume electron microscopy.

Overcoming the challenges of preserving lipid-rich Cannabis sativa L. glandular trichomes for transmission electron microscopy.

Cannabis glandular trichomes produce and store an abundance of lipidic specialised metabolites (e.g. cannabinoids and terpenes) that are consumed by humans for medicinal and recreational purposes. Due to a lack of genetic resources and inherent autofluorescence of cannabis glandular trichomes, our knowledge of cannabinoid trafficking and secretion is limited to transmission electron microscopy (TEM). Advances in cryofixation methods has resulted in ultrastructural observations closer to the 'natural state' of the living cell, and recent reports of cryofixed cannabis trichome ultrastructure challenge the long-standing model of cannabinoid trafficking proposed by ultrastructural reports using chemically fixed samples. Here, we compare the ultrastructural morphology of cannabis glandular trichomes preserved using conventional chemical fixation and ultrarapid cryofixation. We show that chemical fixation results in amorphous metabolite inclusions surrounding the organelles of glandular trichomes that were not present in cryofixed samples. Vacuolar morphology in cryofixed samples exhibited homogenous electron density, while chemically fixed samples contained a flocculent electron dense periphery and electron lucent lumen. In contrast to the apparent advantages of cryopreservation, fine details of cell wall fibre orientation could be observed in chemically fixed glandular trichomes that were not seen in cryofixed samples. Our data suggest that chemical fixation results in intracellular artefacts that impact the interpretation of lipid production and trafficking, while enabling greater detail of extracellular polysaccharide organisation.

Ultrastructural examination of mouse kidney glomerular capillary loop by sandwich freezing and freeze-substitution.

Sandwich freezing is a method of rapid freezing by sandwiching specimens between two metal disks and has been used for observing exquisite the close-to-native ultrastructure of living yeast and bacteria. Recently, this method has been found to be useful for preserving cell images of glutaraldehyde-fixed animal and human tissues. In the present study, this method was applied to observe the fine structure of mouse glomerular capillary loops. Morphometry was then performed, and the results were compared with the data obtained by an in vivo cryotechnique, which may provide the closest ultrastructure to the native state of living tissue. The results show that the ultrastructure of glomerular capillary loops obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation was close to that of the ultrastructure obtained by in vivo cryotechnique not only in the quality of cell image but also in quantitative morphometry. They indicate that the ultrastructure obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation may more closely reflect the living state of cells and tissues than conventional chemical fixation and dehydration at room temperature and conventional rapid freezing-freeze-substitution of excised tissues without glutaraldehyde fixation. Sandwich freezing-freeze-substitution techniques should be used routinely as a standard method for observing the close-to-native ultrastructure of biological specimens.

A cryo-fixation protocol to study the structure of the synaptonemal complex.

Genetic variability in sexually reproducing organisms results from an exchange of genetic material between homologous chromosomes. The genetic exchange mechanism is dependent on the synaptonemal complex (SC), a protein structure localized between the homologous chromosomes. The current structural models of the mammalian SC are based on electron microscopy, superresolution, and expansion microscopy studies using chemical fixatives and sample dehydration of gonads, which are methodologies known to produce structural artifacts. To further analyze the structure of the SC, without chemical fixation, we have adapted a cryo-fixation method for electron microscopy where pachytene cells are isolated from mouse testis by FACS, followed by cryo-fixation, cryo-substitution, and electron tomography. In parallel, we performed conventional chemical fixation and electron tomography on mouse seminiferous tubules to compare the SC structure obtained with the two fixation methods. We found several differences in the structure and organization of the SC in cryo-fixed samples when compared to chemically preserved samples. We found the central region of the SC to be wider and the transverse filaments to be more densely packed in the central region of the SC.

Improved chemical fixation of lipid-secreting plant cells for transmission electron microscopy.

Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone pigments in Boraginaceae. The new fixation method combined glutaraldehyde containing malachite green, imidazole–osmium and p-phenylenediamine treatments, and cells were then observed with a transmission electron microscope. The method prevented the extraction of lipids, including shikonin derivatives, and improved the visualization of subcellular structures, especially the membrane system, when compared with that of conventional fixation. The improved quality of the transmission electron micrographs is because malachite green ionically binds to the plasma membrane, organelles and lipids and acts as a mordant for electron staining with osmium tetroxide. Imidazole promotes the reaction of osmium tetroxide, leading to enhanced electron staining. p-Phenylenediamine reduces osmium tetroxide bound to cellular materials and increases the electron density. This protocol requires only three additional reagents over conventional chemical fixation using glutaraldehyde and osmium tetroxide.

New insights on Laminaria digitata ultrastructure through combined conventional chemical fixation and cryofixation

Abstract The objective of the present study is to examine the fine structure of vegetative cells of Laminaria digitata using both chemical fixation and cryofixation. Laminaria digitata was chosen due to its importance as a model organism in a wide range of biological studies, as a keystone species on rocky shores of the North Atlantic, its use of iodide as a unique inorganic antioxidant, and its significance as a raw material for the production of alginate. Details of the fine structural features of vegetative cells are described, with particular emphasis on the differences between the two methods used, i.e. conventional chemical fixation and freeze-fixation. The general structure of the cells was similar to that already described, with minor differences between the different cell types. An intense activity of the Golgi system was found associated with the thick external cell wall, with large dictyosomes from which numerous vesicles and cisternae are released. An interesting type of cisternae was found in the cryofixed material, which was not visible with the chemical fixation. These are elongated structures, in sections appearing tubule-like, close to the external cell wall or to young internal walls. An increased number of these structures was observed near the plasmodesmata of the pit fields. They are similar to the “flat cisternae” found associated with the forming cytokinetic diaphragm of brown algae. Their possible role is discussed. The new findings of this work underline the importance of such combined studies which reveal new data not known until now using the old conventional methods. The main conclusion of the present study is that cryofixation is the method of choice for studying Laminaria cytology by transmission electron microscopy.

Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx.

Simple SummaryThe glycocalyx (GCX) is a hydrated, gel-like layer of biological macromolecules attached to the cell membrane. The GCX acts as a barrier and regulates the entry of external substances into the cell. The function of the GCX is highly dependent on its structure and composition. Pathogenic factors can affect the protective structure of the GCX. We know very little about the three-dimensional organization of the GXC. The tiny and delicate structures of the GCX are difficult to study by microscopic techniques. In this study, we evaluated a method to preserve and label sensitive GCX components with antibodies for high-resolution microscopy analysis. High-resolution microscopy is a powerful tool because it allows visualization of ultra-small components and biological interactions. Our method can be used as a tool to better understand the role of the GCX during the development and progression of diseases, such as viral infections, tumor invasion, and the development of atherosclerosis.The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.

Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Self-pressurised rapid freezing, a time-efficient and affordable cryo-fixation method for ultrastructural observations on unhatched cyst nematodes

Summary Ultrastructural analysis of the early development of nematodes is hampered by the impermeability of the eggshell to most commonly used fixatives. High-pressure freezing (HPF), a physical cryo-fixation method, facilitates a fast rate of fixation, and by using this method the issue of the uneven delivery of fixative is circumvented. Although HPF results in a superior preservation of the fine structure, the equipment costs impede a wider application of this method. Self-pressurised rapid freezing (SPRF) is an alternative low-cost cryo-fixation method, and its usefulness was evaluated in an ultrastructural study of the eggshell and the cuticle of unhatched second-stage juveniles (J2) of Globodera rostochiensis and Heterodera schachtii. A comparison with conventional (chemical) fixation demonstrates that SPRF fixation results in a remarkably well-preserved ultrastructure of the entire egg including both the eggshell and the cellular details of the unhatched J2. Therefore, SPRF fixation is proposed as an affordable, relatively easy-to-use and time-efficient technique to study the ultrastructure of unhatched J2 and eggs of nematodes.

Transmission Electron Microscopy (TEM) to Study Histology of Pollen and Pollen Tubes.

Here, we describe methods of transmission electron microscopy (TEM) based on conventional chemical fixation and high-pressure freezing (HPF) and freeze-substitution (FS) to examine the ultrastructure of Arabidopsis pollen grains and pollen tubes. Compared to other plant samples, such as root or leaf, pollen grains have thick pollen coat and cell wall which limit the permeation of fixative. Thus, it is difficult to obtain high-quality ultrastructural images of pollen. Moreover, pollen tube is very soft and the traditional procedure is too harsh to get an intact pollen tube sample. Up to now, there is no available mature protocol for TEM sample preparation of Arabidopsis pollen tube. Here, we describe the details and step-by-step procedures of chemical fixation, HPF, FS, and resin-embedding protocols for Arabidopsis pollen and pollen tube. In addition, we also provide a method on how to get longitudinal sections of pollen tubes.

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy.

SummaryTrace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.

Fine structure of the Arabidopsis stem cuticle: effects of fixation and changes over development.

The Arabidopsis cuticle, as observed by electron microscopy, consists primarily of the cutin/cutan matrix. The cuticle possesses a complex substructure, which is correlated with the presence of intracuticular waxes. The plant cuticle is composed of an insoluble polyester, cutin, and organic solvent soluble cuticular waxes, which are embedded within and coat the surface of the cutin matrix. How these components are arranged in the cuticle is not well understood. The Arabidopsis cuticle is commonly understood as 'amorphous,' lacking in ultrastructural features, and is often observed as a thin (~80-100nm) electron-dense layer on the surface of the cell wall. To examine this cuticle in more detail, we examined cuticles from both rapidly elongating and mature sections of the stem and compared the preservation of the cuticles using conventional chemical fixation methods and high-pressure freezing/freeze-substitution (HPF/FS). We found that HPF/FS preparation revealed a complex cuticle substructure, which was more evident in older stems. We also found that the cuticle increases in thickness with development, indicating an accretion of polymeric material, likely in the form of the non-hydrolyzable polymer, cutan. When wax was extracted by chloroform immersion prior to sample preparation, the contribution of waxes to cuticle morphology was revealed. Overall, the electron-dense cuticle layer was still visible but there was loss of the cuticle substructure. Furthermore, the cuticle of cer6, a wax-deficient mutant, also lacked this substructure, suggesting that these fine striations were dependent on the presence of cuticular waxes. Our findings show that HPF/FS preparation can better preserve plant cuticles, but also provide new insights into the fine structure of the Arabidopsis cuticle.

Morphofunctional Merits of an In Vivo Cryotechnique for Living Animal Organs: Challenges of Clinical Applications from Basic Medical Research.

Recent advances in molecular and genetic techniques have led to establishment of new biomedical fields; however, morphological techniques are still required for a more precise understanding of functioning cells and tissues. Conventional preparation procedures involve a series of chemical fixation, alcohol dehydration, paraffin or epoxy resin embedding, sectioning, and staining steps. In these steps, technical artifacts modify original morphologies of the cells being examined. Furthermore, difficulties are associated with capturing dynamic images in vivo using conventional chemical fixation. Therefore, a quick-freezing (QF) method was introduced for biological specimens in the 20th century. However, specimens have to be resected from living animal organs with blood supply, and their dynamical morphologies have not been investigated in detail using the QF method. In order to overcome these issues, the tissue resection step of organs had to be avoided and samples needed to be frozen under blood circulation. Our in vivo cryotechnique (IVCT) was an original technique to cryofix samples without resecting their tissues. The most significant merit of IVCT is that blood circulation into organs is preserved at the exact moment of freezing, which has been useful for arresting transient physiological processes of cells and tissues and maintaining their components in situ.

A Method for Preparing Difficult Plant Tissues for Light and Electron Microscopy.

Although the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation of Banksia prionotes (Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs.

Inner ear tissue preservation by rapid freezing: Improving fixation by high-pressure freezing and hybrid methods

In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues.

Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature

Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the biosafety area. Freeze-substitution with osmium-acetone (OA) at ultralow temperature (−85°C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for freeze-substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel freeze-substitution preparation method that can be performed using a portable device in a biosafety cabinet at biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid freeze-substitution (RFS) after fixation with glutaraldehyde (GA>RFS) are of comparable quality to those obtained using standard RFS. GA>RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA>RFS was not significantly different (6987.8±2181.0 and 6888.9±1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7±2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS.

Ultrastructure of the rickettsial endosymbiont “MIDORIKO” in the green alga Carteria cerasiformis as revealed by high-pressure freezing and freeze-substitution fixation

Bacterial endosymbionts belonging to the family Rickettsiaceae were recently identified in the unicellular green alga Carteria cerasiformis, providing the first molecular evidence of rickettsial endosymbionts within photosynthetic eukaryotes. However, previous morphological studies using transmission electron microscopy (TEM) with conventional chemical fixation did not demonstrate whether the endosymbionts of C. cerasiformis have the diagnostic characteristics of the family Rickettsiaceae. In this study, we observed the rickettsial endosymbionts "MIDORIKO" within C. cerasiformis cells by TEM with high-pressure freezing and freeze-substitution fixation. The rickettsial endosymbionts resided directly in the C. cerasiformis cytoplasm without engulfing or encompassing membranes or vacuoles. The endosymbionts had a Gram-negative cell envelope composed of outer and inner bilayer membranes. The thicknesses of the outer and inner leaflets of the bacterial cell wall were almost identical. These morphological characteristics are consistent with those of the genus Rickettsia, but the cell wall structure differed from that of the genus Orientia within the family Rickettsiaceae.

A convenient sample preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage

Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM) provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving pre-fixation with 75mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained from stems stood into water. The lysine-based protocol was compared with four other processing protocols of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with CPD. The non-fixed control, FD and the glutaraldehyde fixation with HMDS drying gave poor preservation of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was superior to these three methods in terms of better preserving the EPS. However, this fourth method gave condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for examination of bacterial stem-end blockage in cut ornamentals.

Improvement of Ultrastructural Preservation of Eimeria Oocysts by Microwave-assisted Chemical Fixation or by High Pressure Freezing and Freeze Substitution

Fixation and preparation for electron microscopy of coccidian oocysts is a general problem. Especially in sporulated oocysts, proper fixation and resin infiltration are hindered by the robust oocyst wall. Conventional chemical fixation therefore leads to artefacts that obscure cellular details in the oocysts. In this study, sporulated oocysts of Eimeria nieschulzi were subjected to different fixation and embedding protocols: conventional chemical fixation and embedding in Spurr's resin, microwave-assisted fixation and processing followed by embedding in epon, and high pressure freezing followed by freeze substitution and epon embedding. The samples were finally studied by transmission electron microscopy. Many ultrastructural features of the oocyst wall and the sporozoites were already substantially improved after microwaved-assisted fixation and processing. However, the fine structural preservation still suffered from shrinkage and artificial extraction, which occured during dehydration and infiltration. High pressure freezing (HPF) and freeze substitution (FS) revealed much better preservation. Oocyst walls retained their ovoid shape, and the ultrastructure of sporozoites was well preserved with no signs of shrinkage or extraction. HPF and FS are therefore a suitable method for the ultrastructural analysis of coccidian oocysts.