APPLICATIONS OF FREEZE-SUBSTITUTION TO ELECTRON MICROSCOPE STUDIES OF INVERTEBRATE OOCYTES

A modified freeze-substitution process is described which gives a low percentage (less than 5 per cent) of preparations of invertebrate eggs which appear to be ice crystal-free at the resolution of the electron microscope. The mitochondria show no membranes in these preparations but can be recognized by internal spaces with the size and the distribution of the cristae. The Golgi bodies resemble those seen with diffusion fixatives, but the limiting membranes are here double; that is, they appear to be a triple-layered sandwich with two outer dark approximately 25A layers and an inner light layer of the same thickness. The endoplasmic reticulum is clearly present and resembles that seen with diffusion fixatives. Here again, the limiting membranes are double with the same dimensions as those in the Golgi bodies. The membranes of the Golgi bodies and ER are seen after permanganate but not lead hydroxide staining. The hyaloplasm (or "cell sap") is crowded with 150 to 200A particles and these are also seen lining the ER membranes. In general, the structures as seen with the present technique show considerable similarity to those seen with diffusion fixatives.     

Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas

Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, kappa and lambda light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3-8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when kappa and lambda chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse alpha-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.     

Diatom elemental and morphological changes in response to iron limitation: a brief review with potential paleoceanographic applications

Diatoms are a major group of phytoplankton that account for approximately 40% of the ocean carbon fixation and the vast majority of biogenic silica production through the construction of their cell walls (termed frustules). These frustules accumulate and are partially preserved in the ocean sediments. Diatom growth and nutrient utilization in high‐nitrate, low‐chlorophyll regions of the world’s oceans are mostly regulated by iron availability. Diatoms acclimate to iron limitation by decreasing cell size. The associated increase in surface area‐to‐volume ratio and decrease in diffusive boundary layer thickness may improve nutrient uptake kinetics. In parallel, cellular silicon (Si) contents are elevated in iron‐limited diatoms relative to nitrogen (N) and carbon (C). Variations in degree of silicification and nutritional requirements of iron‐limited diatoms have been hypothesized to account for higher cellular Si and/or lower cellular N and C, respectively. However, in some diatoms, frustule silicification does not significantly change when cells are iron‐limited. Instead, changes in the Si‐containing valve surface area relative to volume within these diatoms is hypothesized to be responsible for the variations in the cellular Si : N and Si : C ratios. In particular, some examined iron‐limited pennate diatoms have reduced widths relative to their lengths (i.e. lower length‐normalized widths, LNW) compared to iron‐replete cells. In the pennate diatom Fragilariopsis kerguelensis, the mean LNWs of valves preserved in sediments throughout the Southern Ocean (a well‐characterized iron‐limited region) is positively correlated with satellite‐derived, climatological net primary productivity in the overlying waters. Because of the specific morphological changes in pennate diatom frustules in response to iron availability, the valve morphometerics (e.g. LNWs) can potentially be used as a diagnostic tool for iron‐limited diatom growth and relative changes in the Si : N (and Si : C) ratios in extant diatom assemblages as well as those preserved in the sediments.     

Choice of fixatives for the immunohistochemical study of antigens using immunoadsorption

The effect of fixatives on the rat bone marrow antigens was studied by a method combining immunoadsorption and immunodiffusion. This method allows to estimate rapidly and reliably the effect of fixation on immunochemical properties of the antigen under study, using polyvalence antisera. Acetone and ethanol proved to be the best fixatives for the rat bone marrow antigens since they preserved their immunochemical properties. Aldehydes (formaldehyde and glutaraldehyde) "inactivated" the bone marrow antigens.     

Particle morphology characterization and manipulation in biomass slurries and the effect on rheological properties and enzymatic conversion

An improved understanding of how particle size distribution relates to enzymatic hydrolysis performance and rheological properties could enable enhanced biochemical conversion of lignocellulosic feedstocks. Particle size distribution can change as a result of either physical or chemical manipulation of a biomass sample. In this study, we employed image processing techniques to measure slurry particle size distribution and validated the results by showing that they are comparable to those from laser diffraction and sieving. Particle size and chemical changes of biomass slurries were manipulated independently and the resulting yield stress and enzymatic digestibility of slurries with different size distributions were measured. Interestingly, reducing particle size by mechanical means from about 1 mm to 100 μm did not reduce the yield stress of the slurries over a broad range of concentrations or increase the digestibility of the biomass over the range of size reduction studied here. This is in stark contrast to the increase in digestibility and decrease in yield stress when particle size is reduced by dilute-acid pretreatment over similar size ranges.     

Rapid-freezing cytochemistry: preservation of tubular lysosomes and enzyme activity

We show that tubular structures present in phorbol ester-stimulated macrophages are sensitive to commonly used chemical fixatives (i.e., they usually become fragmented during fixation). These structures are well preserved in macrophages that are physically fixed by rapid-freezing and subsequent freeze-substitution in osmium-acetone. We have developed methods that combine rapid-freezing, freeze-substitution, and enzyme cytochemistry for preservation of these tubular structures and for detection of endocytosed material (i.e., horseradish peroxidase). This method of rapid-freeze cytochemistry may be useful in other situations where chemical fixation does not adequately preserve cell structures, particularly of membrane compartments.     

Laboratory sources of error for algal community attributes during sample preparation and counting

Applied algal studies typically require enumeration of preserved cells. As applications of algal assessments proliferate, understanding sources of variability inherent in the methods by which abundance and species composition data are obtained becomes even more important for precision of measurements. We performed replicate counts of diatoms on permanently fixed coverglasses and all algae in Palmer–Maloney chambers to assess precision and accuracy of measurements derived from common counting methods. We counted diatoms and all algae with transects and random fields. Variability estimates (precision) of diatom density, species diversity, and species composition on permanent coverglasses were low between replicate subsamples and between replicate transects. However, average density estimates of diatoms settled on coverglasses determined with transect methods were 42–52% greater than density estimates made with random fields. This bias was due to a predictable, nonrandom distribution of diatoms on the coverglass with few diatoms near edges. Despite bias in density when counting diatoms along coverglass transects, no bias was observed in estimates of species composition. Estimates of density and taxa richness of all-algae in Palmer–Maloney chambers also had low variability among multiple transects and high similarity in species composition between transects. In addition, counting method in Palmer–Maloney chambers did not affect estimates of algal cell density, taxa richness, and species composition, which suggested that counting units were distributed randomly in the chambers. Thus, most sources of variability in sample preparation and analysis are small; however, transect counts should not be used to estimate cell density, and sufficient numbers of random fields must be counted to account for edge effects on cell distribution with material settled on permanently fixed coverglasses.     

Paleodepth determination from Antarctic benthic diatom assemblages

Analysis of modern surface sediments from fjords in the Vestfold Hills (Antarctica) indicates that 58% of the variation in benthic diatom assemblages can be attributed to changes in environmental parameters with water depth. Attenuation of light through the water column is suggested to account for 45% of the variation, and the decrease in substrate grain size with depth possibly accounts for a further 13%. Cluster analysis and principal component analysis were used to objectively circumscribe five floral zones between the surface and 35 m depth. The depth distribution of the benthic diatoms was then used to interpret the paleodepth of relict fjord (Holocene) sediments exposed around Deep Lake in the Death Valley (Vestfold Hills). Paleodepths measured from the sea-ice bench around Deep Lake combined with data from grain size analysis indicate that the relict fjord sediments have no analogue amongst the modern fjord sediments sampled in the Vestfold Hills. Without a comparable modern habitat on which to model the diatom depth zones, however, this study was unable to accurately determine the paleowater depth at Deep Lake using diatoms. Paleodepth determination will be possible using grain size analysis and diatom data when the substrate and light requirements of benthic diatoms are understood.     

The Dry Preservation of Fixed Plant Material

A method for the dry-preservation of fixed plant material, root tips and buds, is described. The method seems to be advantageous on long expeditions and when material has to be sent away.

The material is transferred from the fixative to 70% alcohol (3 changes, 1/2 hour in the last). It is dried on blotting-paper. The dried material may be preserved a long time. Material kept dry for 4 1/2 years has proved to be quite satisfactory. Drying has been tried after fixation with CRAF-solutions (Webber and Randolph modifications) and fixatives containing osmic acid (Fleming-Benda and 2BD).

The dry material is swollen by keeping for 2 days in 10% alcohol. It is embedded in paraffin according to the usual method. A satisfactory staining has been obtained after these fixatives using iodine-gentian-violet and Feulgen stainings. In addition to chromosome counts dry material may be used for chromosome morphology studies.

Dried material fixed in aceto-alcohol (1:3) has not turned out to be specially suitable for squash preparations owing to the fragility of the chromosomes. If strong pressure is not applied, satisfactory results may, however, be obtained.     

The use of diatoms to assess past and present water quality

Abstract Diatoms possess a number of attributes which contribute to their suitability as biological indicators. They are highly sensitive to water chemistry changes, abundant in aquatic environments, largely cosmopolitan in distribution, less habitat dependent than macroinverte-brates and have a well-studied taxonomy and ecology. Furthermore, the preservation of diatom valves in lake sediments means that they can provide otherwise unavailable baseline data which can be used to assess and contextualize human impacts on aquatic ecosystems. The value of diatoms as bioindicators in contemporary and palaeolimnological studies has been well established overseas. Despite this, they have been under-utilized in Australia. This paper outlines some of the applications and potential for the use of diatoms as biological indicators in Australia.     

Influence of bacteria on cell size development and morphology of cultivated diatoms

Vegetative cell division in diatoms often results in a decreased cell size of one of the daughter cells, which during long‐term cultivation may lead to a gradual decrease of the mean cell size of the culture. To restore the initial cell size, sexual reproduction is required, however, in many diatom cultures sexual reproduction does not occur. Such diatom cultures may lose their viability once the average size of the cells falls below a critical size. Cell size reduction therefore seriously restrains the long‐term stability of many diatom cultures. In order to study the bacterial influence on the size diminution process, we observed cell morphology and size distribution of the diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea for more than two years in bacteria‐free conditions (axenic cultures) and in cultures that contain bacteria (xenic cultures). We found considerable morphological aberrations of frustule microstructures in A. minutissimum and C. affiniformis when cultivated under axenic conditions compared to the xenic cultures. These variations comprise significant cell length reduction, simplification and rounding of the frustule contour and deformation of the siliceous cell walls, features that are normally found in older cultures shortly before they die off. In contrast, the xenic cultures were well preserved and showed less cell length diminution. Our results show that bacteria may have a fundamental influence on the stability of long‐term cultures of diatoms.     

PLANT MICROTECHNIQUE: SOME PRINCIPLES AND NEW METHODS

Some easily seen structural features of living plant cells are destroyed or badly distorted by most of the common fixatives and embedding media used in plant histology. In stained sections of plant tissues fixed in FAA (formalin-acetic acid-alcohol mixtures) and embedded in paraffin wax, for example, mitochondria and fine transvacuolar strands of cytoplasm are usually not visible. Many structural features such as these can be preserved, however, with suitable fixatives and embedding media. Specifically we recommend fixation in non-coagulant fixatives (e.g., osmium tetroxide, acrolein, glutaraldehyde, formaldehyde) and the use of plastics as embedding media, and we describe in detail a method of fixation in acrolein and embedding in glycol methacrylate polymer. In a wide range of plant specimens prepared in this way, stained sections 1–3 microns thick showed excellent preservation of tissue and cell structures.     

Effect of fixation to the degradation of nuclear and mitochondrial DNA in different tissues.

Samples of different tissues were preserved in seven fixatives for periods of time extending from 1 to 336 days, to determine which fixatives reduce the time-dependent degradation of DNA and preserve the histological structure. To achieve these results, three PCR systems were used: FGA and TC11 (both for nuclear DNA) and HV1 for mitochondrial DNA (mt-DNA). For long-term storage in combination with amplification of nuclear and mt-DNA, consistent results were obtained in Carnoy's solution and glutaraldehyde. Variable results were observed for buffered formalin; an mt-DNA product could be detected even after 3 months of fixation. In regard to comparison of the different tissues, the quantities recovered from skeletal muscles and kidneys were higher than from other tissues.     

Autecological investigations on marine diatoms. I. Experimental results in biogeographical studies

Summary The upper and/or lower temperature limits for growth of 43 marine diatoms have been determined. According to the extent of the temperature tolerance range for growth and its position within the ecological relevant temperature range, the diatoms were divided in oligo-eurytherm (cold-, temperate-, warm-), meso-eurytherm (cold-, warm-) and eu-eurytherm species.The definition of the terms cosmopolites and circum-tropical could be enlarged, when the experimentai results and the distribution data from literature (19 distribution maps were combined.The results showed a significant relation between lower temperature limit for growth and both volume and cell size. In this way the lower temperature limit influences the size distribution of the phytoplankton in the oceans. Large diatoms are absent in cold water because of their high lower temperature limit for growth.The use of experimental results to reduce the number of sampling stations for determination of geographical distribution of diatoms, is discussed.     

Preservation of extracellular space during fixation of the brain for electron microscopy

Adult mammalian brain contains about 20% extracellular space, but fixatives cause the cellular processes to ingest the extracellular fluid, and the space is not preserved in electron micrographs prepared by any of the conventional methods. This distortion can be prevented by replacing part of the sodium chloride in the extracellular fluid by an impermeant solute such as sucrose. To do this, the blood-brain barrier can be opened by vascular perfusion at 300 mmHg pressure, or the barrier can be bypassed by immersion of thin slices of fresh brain in the impermeant solution. In either case, addition of aldehyde fixatives and conventional processing then leads to the preservation of extracellular space in electron micrographs. Both procedures are as easy to use for routine fixation as conventional methods. In well fixed tissue the cellular processes are different in size, shape and electron density from the inflated profiles seen after the ingestion of extracellular fluid that accompanies conventional fixation. Moreover, extracellular space is found to separate widely some cellular elements, while leaving others contiguous.     

Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy

Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical technique for the simultaneous analysis of lipids, proteins, carbohydrates, and a variety of phosphorylated molecules within intact cells. SR-FTIR microspectroscopy can be used in the imaging mode to generate biospectroscopic maps of the distribution and intensity profiles of subcellular biomolecular domains at diffraction-limited spatial resolution. However, the acquisition of highly spatially resolved IR images of cells is not only a function of instrumental parameters (source brightness, sampling aperture size) but also the cell preparation method employed. Additionally, for the IR data to be biochemically relevant the cells must be preserved in a life-like state without introducing artefacts. In the present study we demonstrate, for the first time, the differences in biomolecular localizations observed in SR-FTIR images of cells fixed by formalin, formalin-critical point drying (CPD), and glutaraldehyde-osmium tetroxide-CPD, using the PC-3 prostate cancer cell line. We compare these SR-FTIR images of fixed cells to unfixed cells. The influence of chemical fixatives on the IR spectrum is discussed in addition to the biological significance of the observed localizations. Our experiments reveal that formalin fixation at low concentration preserves lipid, phosphate, and protein components without significantly influencing the IR spectrum of the cell.     

Summer phytoplankton community structure and growth in a regional upwelling area off hachijo Island,Japan

Phytoplankton community structure both in terms of taxonomic composition and size distribution, and growth were examined in a regional upwelling which occurred in a Kuroshio region in summer. Organic carbon abundance of diatoms was markedly enhanced by the upwelling and they accounted for 87% of the total phytoplankton carbon. Nitzschia pungens Grunow was numerically most prominent accompanied by Chaetoceros and Rhizosolenia species. On the other hand, only a small increase was observed in phytoplankton other than diatoms, which consisted of unicellular cyanobacteria, coccolithophorids, gymnodinoids, and so called monads and flagellates. Division rate of diatoms from the upwelled water was twice that of the other forms. This difference in growth response among phytoplankton groups resulted in a shift of community structure to dominance of diatoms from that of the other forms. The time needed for this shift was estimated to be at least ≈ 3 days.     

Establishment of HEp-2 cell preparation for automated analysis of ANA fluorescence pattern.

BACKGROUND: Identification of antinuclear antibodies (ANAs) has large clinical importance for the assessment of autoimmune diseases. HEp-2 cell preparations on microscopic slides are commonly used as antigenic substrate. Methods used for cell preparation are important for ANA pattern analysis; however, these methods differ widely and are mostly not specified. METHODS: HEp-2 cells were fixed using acetic acid-ethanol, methanol-acetone, acetone, formaldehyde, paraformaldehyde, or glutaraldehyde. Morphological analysis was done after haematoxylin-eosin staining and DAPI-staining of cell nuclei. RESULTS: The results demonstrate a high variability of cell and nuclear morphology depending on the used fixatives. Aldehyde fixatives conserved the cell structures best, acetone fixatives revealed remarkable changes. CONCLUSIONS: After selecting appropriate fixation procedures to preserve nuclear structures further experiments are necessary to find out which fixation procedure preserves the disease-linked antigens the best way and are, therefore, suitable to be used in ANA-testing of AABs.     

Preparation of Permanent Whole Mounts of Marine Centric Diatoms to Permit Observations of Cytoplasmic Inclusions

Marine centric diatoms are highly vacuolated plant cells which often exhibit a marked tendency to plasmolyze during fixation or other manipulatory stages required to make permanent preparations which pemit the observation of cytoplasmic inclusions. Altmann's, Schaudinn's, vom Rath's, 5% acrolein, and Allen's PFA₃ are fixatives which have been found to induce relatively little plasmolysis in the species studied. If, following fixation, the fixative is removed and desalting and dehydration are carried out in a filter assembly which allows gentle suction to make gradual changes in reagent concentration and if the cells are then placed in dilute mounting medium, only a relatively small percentage of them will be plasmolyzed. In such preparations, depending upon the fixatives employed, various cytoplasmic organelles or other inclusions will be visible in the unstained material when examined with phase contrast. Fixation with Altmann's fixative and mounting the material in Zeiss phase-contrast mounting medium (L-15) after gradual desalting and dehydration gave the best results. Standard cytochemical techniques for the detection of saccharides, protein, and lipids may be incorporated in this procedure; or, all of the steps needed for embedding the material in resins, excepting polymerization, are feasible also.