An alginate matrix double-embedding method for paraffin sectioning of minute specimens

A method has been devised for placing minute specimens in viscous aqueous sodium alginate solution, which is polymerized in 0.25 molar calcium chloride. The resulting matrix can then be dehydrated, embedded, and sectioned, maintaining the orientation of the specimens. The sectioned alginate can be dissolved in 0.1 molar EDTA if desired. Tissue sections respond to normal staining procedures.     

A Quick Preparative Method for Electron Microscopy Observations of Delicate Objects Using Alginate Embedding Medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

A quick preparative method for electron microscopy observations of delicate objects using alginate embedding medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

An improved method for preparing microorganism laden alginate bead specimens for accurate Scanning Electron Microscope examination

Summary The distribution of biomass encapsulated within alginate beads can be examined using a Scanning Electron Microscope (SEM). Existing methods for the preparation of suitable specimens are extremely time consuming, involving fixing of samples with glutaraldehyde, dehydrating with successive acetone washings and final drying using a critical point technique. These preparations are necessary to avoid significant specimen shrinkage, however, the resultant specimens are sometimes difficult to analyse using an SEM or light microscope. A reliable quick method has been developed where alginate specimens are directly mounted using a water based adhesive, then dried under controlled conditions. These specimens were found to be robust enough for SEM processing and gave true measurements of the original alginate beads including microorganism orientation.     

Isolation and characterizartion of alginic acid from commercially cultured Nemacystus decipiens (Itomozuku)

An alginate was isolated from commercially cultured Nemacystus decipiens which had been harvested in Yonashiro Town (Okinawa, Japan). The yield of the alginate was 1.6% (w/w of wet alga), and the uronic acid, ash and moisture contents of the alginate were 86.0%, 12.0%, and 2.3% (w/w), respectively. The molecular mass of the alginate was estimated to be about 1.5 x 10(5). The infrared spectrum and optical rotation of the alginate were in agreement with those of the standard alginate. D-Mannuronic acid and L-guluronic acid were identified by 1H- and 13C-NMR spectroscopy, the molar ratio of both sugar residues being estimated to be 0.72:1.00.     

International Society for Biological and Environmental Repositories

Genetically altered mice are an important tool for biomedical research. Several transgenic mice have been created in which activation of the transgene results in production of β-galactosidase that can be detected by histological means. While preservation and subsequent visualization of enzyme activity in soft tissues can be complicated, it is particularly difficult in bone specimens, especially those that have been decalcified. For these studies, we examined the bones of parathyroid hormone-related peptide (PTHrP) knock-in mice in which expression of PTHrP resulted in β-galactosidase production. During the past decade, several studies have demonstrated the importance of PTHrP in bone. Thus, it is important to preserve and detect β-galactosidase enzymatic activity in bone for these studies. We demonstrate here that β-galactosidase was visualized better in slides with bone sections taken from PTHrP knock-in mice when bones were frozen and sectioned compared to bones that were embedded in plastic and sectioned using a microtome. Importantly, we were able to visualize β-galactosidase in plastic embedded bones when specimens were fixed, stained (X-gal), embedded in plastic, and then sectioned rather than being fixed, embedded in plastic, sectioned, then stained.     

Absorption of Proteoglycan via Clathrin-Mediated Endocytosis in the Small Intestine of Rats

An alginate was isolated from commercially cultured Nemacystus decipiens which had been harvested in Yonashiro Town (Okinawa, Japan). The yield of the alginate was 1.6% (w/w of wet alga), and the uronic acid, ash and moisture contents of the alginate were 86.0%, 12.0%, and 2.3% (w/w), respectively. The molecular mass of the alginate was estimated to be about 1.5×10⁵. The infrared spectrum and optical rotation of the alginate were in agreement with those of the standard alginate. D-Mannuronic acid and L-guluronic acid were identified by ¹H- and ¹³C-NMR spectroscopy, the molar ratio of both sugar residues being estimated to be 0.72:1.00.     

Cryostat sections for coexistence studies and preembedding electron microscopic immunocytochemistry of central and peripheral nervous system tissue

Perfusion-fixed tissue blocks were incubated in high molar sucrose solutions, shock frozen in melting isopentane, and sectioned on a conventional cryostat. Semithin sections (2-4 microns) alternatingly stained for parvalbumin and glutamate decarboxylase enabled us to demonstrate the coexistence of both antigens in the same cell. Thick sections (40 microns) of central and peripheral nervous system tissue were immunostained and processed for correlated light and electron microscopic studies. At the electron microscopic level, the preservation of ultrastructural features such as membranes and synaptic contacts was comparable to that normally seen in vibratome sectioned material. Hence, this technique can successfully be used for preembedding coexistence studies and electron microscopic preembedding immunocytochemistry when vibratome sectioning is problematic.     

A Device for Flat-Face, Epoxy Resin Embedding of Tissues and Coverslip Cultures

An easily constructed device permits the flat-face embedding of four specimens in epoxy resins. Either pieces of tissue or cells grown on cover slips can be used. After polymerization, the flat-surfaced capsules may be examined under high magnification for selection of areas to be sectioned. Specimens difficult to orient can be cut out and re-embedded in the proper position. The use of thick-walled BEEM capsules and the clamping action afforded by four screws prevent leakage of the resin.     

Histological Preparation of Large Vertebral Specimens

A histological method is described for processing human lumbosacral vertebral specimens through fixation, decalcification, dehydration and embedding in a mixture of low viscosity nitrocellulose and celloidin. The large blocks thus obtained were serially sectioned on a Jung Tetrander microtome at a section thickness of 100 microns. Shrinkage of 8.6% was observed for the mounted specimens by comparing the unprocessed specimens (by X-ray measurement) with the mounted sections (measured using a transparent ruler). A horizontally sectioned lumbosacral joint is used to illustrate the excellent histological detail obtained of the anatomy at this level of the spinal column.     

Periodate oxidation of sodium alginate in water and in ethanol-water mixture: a comparative study

Periodate oxidation of sodium alginate in aqueous solution as well as a dispersion in 1:1 ethanol-water was examined. The oxidation proceeded smoothly in both media, and the kinetics of oxidation was surprisingly similar. Polymer cleavage was observed in both media, but it was extensive in ethanol-water. The weight-average molar mass (Mw) of the oxidized product obtained from aqueous solution showed a gradual decrease with increase in the periodate concentration, whereas, except for very high periodate equivalent, the change in Mw was not reflected with increase in concentration of periodate in ethanol-water. The oxidized alginate obtained from the ethanol-water mixture was found to be more efficient in crosslinking proteins such as gelatin, leading to hydrogels. Oxidation of a dispersion has the advantage of generating large quantities of the oxidized alginate in higher yield with one reaction using less solvent.     

The plasticizing effect of alginate on the thermoplastic starch/glycerin blends

Corn starch and corn starch–alginate (5–15%) blends plasticized with 35% glycerin were prepared, water was intentionally excluded from the formulations. Torque rheometry measurements were carried out during the processing of the blends in a batch counterrotating twin screw mixer. A progressive decrease in the plasticization energy of the blends was observed as the alginate content was increased, with a 5-fold decrease for the blend with the higher alginate content (15%). The steady state torque of the plasticized melted blends also showed a decrease as alginate content was increased; with a drastic drop occurring for the formulation with higher alginate content. After mixing, test specimens for mechanical, thermal and microstructural testing were made by compression molding. A decrease in the elastic properties and an increase in elongation at break and impact resistance was observed when alginate content was increased in the blends. The transition of the materials towards a more viscous behavior, as alginate content was increased, was confirmed by differential scan calorimetric analysis. For the corn starch–alginate blends glass transitions were detected in the temperature range −60 to −90 °C. Scanning electron microscopy was used to examine the morphology of cryofractured surfaces of the molded test specimens. A reduction of the granular crystalline structures typical of corn starch was observed as alginate content was increased in the blends. The experimental evidence presented in this work indicates that, when water is excluded from thermoplastic corn starch preparation, alginate acts synergistically with glycerin increasing the degree and efficiency of the plasticization process.     

Purification and chemical and physical characterisation of an antitumour polysaccharide from the brown seaweed Sargassum fulvellum

A polysaccharide isolated from a hot-water extract of Sargassum fulvellum and purified by gel-filtration chromatography on Sepharose 4B inhibited the growth of subcutaneous Sarcoma-180 in mice. The purified, active substance appeared to be sodium alginate having a mol. wt. of 33,400 and a molar ratio of mannuronic acid to guluronic acid of 2.78.     

Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

Fluid transport and dimensions of epithelial cells and intercellular spaces in frog gallbladder

Summary Morphologic findings of widely dilated intercellular spaces in fluid transporting epithelia have been claimed as evidence for the existence of an epithelial compartment in which the coupling between solute and water fluxes takes place. The validity of using epithelial geometry in sectioned material as an argument can be questioned. The present report describes the morphological appearance of frog gallbladder epithelium — normal and ouabain-treated — in the living state in vitro and after fixation, dehydration and embedding. Gallbladder segments were photographed in the living state and at the end of each step of the preparative procedure. Direct observations of whole-mounted gallbladder segments were carried out, taking advantage of the possibility of optical sectioning and high resolution by Nomarski-microscopy. The same specimens were then sectioned and examined by conventional light and electron microscopy. The observations were quantitated and showed that the epithelial cells of normal and ouabain-treated gallbladders experienced an average linear shrinkage down to 70% of their length in Ringer's solution, which corresponds to a volume shrinkage down to 35%. Moreover, dilated lateral intercellular spaces appeared during the dehydration and embedding procedure in normal but only very moderately or not at all in ouabain-treated gallbladder specimens.     

Immobilization of cells by electrostatic droplet generation: a model system for potential application in medicine

The process of electrostatic extrusion as a method for cell immobilization was investigated that could be used for potential applications in medicine. An attempt was made to assess the effects of cell addition and polymer concentration on the overall entrapment procedure, ie, on each stage of immobilization: polymer-cell suspension rheological characteristics, electrostatic extrusion process, and the process ofgelation. The findings should contribute to a better understanding of polymer-cell interactions, which could be crucial in possible medical treatments. Alginate-yeast was used as a model system for carrier-cells. The electrostatic extrusion was considered as a complex two-phase flow system and the effects of cell and alginate concentrations on the resulting microbead size and uniformity were assessed. Under investigated conditions, microbeads 50-600 microm in diameter were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with higher standard deviations in size. We attempted to rationalize the findings by rheological characterization of the cell-alginate suspensions. Rheological characterization revealed non-Newtonian, pseudoplastic behavior of cell-alginate suspensions with higher viscosities at higher alginate concentrations. However, the presence of cells even at high concentrations (5x10(8) and 1x10(9) cells/mL) did not significantly affect the rheological properties of Na-alginate solution. Lastly, we investigated the kinetics of alginate gelation with respect to the quantity of Ca2+ ions and cell presence. The gelation kinetics were examined under conditions of limited supply with Ca2+ ions, which can be essential for immobilization of highly sensitive mammalian cells that require minimal exposure to CaCl2 solution. The molar ratio of G units to Ca2+ ions of 3.8:1 provided complete crosslinking, while the increase in alginate concentration resulted in prolonged gelation times but higher strength of the resulting gel. The cell presence decreased the rate of network formation as well as the strength of the obtained Ca-alginate hydrogel.     

A Rapid Technique for Preparing Microorganisms for Transmission Electron Microscopy

A rapid and efficient method of preparing microorganisms for transmission electors microscopy is reported. In developing the method Salmonella, streptococcal, ad protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

A rapid technique for preparing microorganisms for transmission electron microscopy.

A rapid and efficient method of preparing microorganisms for transmission electron microscopy is reported. In developing the method Salmonella, streptococcal, and protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

Mechanical properties of hydrocolloid gels filled with internally produced CO2 gas bubbles.

Agar (2%), alginate (1% algin), and kappa-carrageenan (1.5%) gel specimens were prepared from mother solutions that contained 0-2.5% sodium bicarbonate (agar and carrageenan) or calcium carbonate (alginate). Upon immersion in a citric acid bath (0-2%), the carbonate reacted with the diffusing acid to produce numerous carbon dioxide bubbles. The compressive strength and deformability of the gas-filled gels so produced were determined using a Universal testing machine and compared with those of pure gels and gels containing the carbonate but not subjected to the process after various immersion times. While the agar and alginate gels retained considerable mechanical integrity even after several hours, the carrageenan gels disintegrated after about 2-5 h. Under similar conditions, the number of bubbles produced in the agar gels was about twice that in the alginate gels, an observation that cannot be explained solely by stoichiometric considerations.     

Notes and queries

An instrument for the precise trimming of flat or cylindrical embedded specimens to be sectioned in an ultramicrotome is described. It will produce the required truncated pyramid accurately by hand slicing. A simple modification to the LKB specimen holder allows it to be used with the Reichert ultramicrotome as well.