Light-Polymerizing Plastics as Slide Mounting Media

Currently available mounting media require solvent evaporation for hardening. This process is slow and often is complicated by the formation of air spaces under the coverslip. Light-polymerizing plastics are introduced as histologic mounting media that eliminate these problems because they harden quickly when exposed to ultraviolet light and, therefore, do not depend upon solvent evaporation. These light-polymerizing plastics are easy to use, permit rapid and controlled hardening, and eliminate the formation of air spaces under the coverslip. The optical characteristics are satisfactory for light and fluorescence microscopy.     

On the Composition of “Viscol” and the Employment of Gum Arabic for Mounting Media

“Viscol”, a water soluble permanent mounting medium hardening through evaporation of water under the cover glass, has been analyzed. It proves to consist of a mixture of gum arabic, glycerol, phenol and water and is especially suitable for simple botanical preparations. The use of gum arabic for hardening permanent mounting media is reviewed. Instead of a glycerol-phenol-water mixture a lactophenol, potassium acetate or zinc chloride solution mixed with gum arabic may be used for a permanent mounting medium. However, gum arabic contains calcium, magnesium and potassium ions giving crystals with the solutions mentioned. In the case of lactophenol and potassium acetate, the calcium and magnesium ions must be removed beforehand, which is done by precipitation with sodium or potassium carbonate. In the case of zinc chloride the potassium ions must be removed, which is done by dialysis with zinc chloride. It is pointed out that the same principles may be used for a great variety of different mounting media.     

Notes on Technic: Simple, Reliable Detection of Latex Microspheres in High Quality Tissue Sections

Latex microspheres used in biological research have been visualized by light microscopy in mounts of cell suspensions, disrupted cells, or cleared tissues (Mishima et al 1987, Koonce et al 1986, LeFevre et al 1978); in unembedded coverslip monolayers (Koerten et al 1980); in fixed (Cornwall and Phillipson 1988) or unfixed (Wells et al 1988) frozen sections; in paraffin sections cleared and deparaffinized with n-butyl alcohol (Callebaut and Meeussen 1989); and in tissues embedded in resins suitable for transmission electron microscopy, such as Spurr's (Hampton et al 1987), Epon (Herzog and Miller 1979), or Ladd Low Viscosity Epon (LeFevre et al 1985). Paraffin embedding, and some plastic embedments, are impractical for demonstration of latex beads because the beads are dissolved by such organic solvents as xylene, dioxane, or chloroform (Van Furth and Diesselhoff-Den Dulk 1980), propylene oxide (Lentzen et al 1984), amyl acetate (Okada et al 1981), or toluene, the solvent in commonly used mounting media such as Fisher Permount (personal observation). The space remaining after dissolution of a bead is not maintained with paraffin embedding as it is with resin embedding. Even after plastic embedding, the resolving power of light microscopes may be inadequate to distinguish such spaces from other spaces found in and between cells. Latex beads are stable in methanol (Van Furth and Diesselhoff-Den Dulk 1980), ethanol. and n-butyl alcohol (Callebaut and Meeussen 1989).     

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.     

A technique for preserving pigmentation in some capsalid monogeneans for taxonomic purposes

A technique is described to preserve the pigment found in the bodies and the intestine of some brightly coloured and darkly pigmented benedeniine capsalid monogeneans. Previous studies of these pigmented capsalids have proven difficult because the pigmentation usually disappears when the worms are fixed using preservatives containing concentrations of formalin over 5% and/or ethanol, acetic acid, chromic acid, picric acid and mercuric chloride. The technique developed here uses a fixative comprising glycerol, acetone and formalin (GAF). After fixation under light coverslip compression for three minutes, specimens are transferred to absolute acetone for three minutes and cleared in a mixture of nine parts cedar wood oil and one part absolute acetone before mounting in Canada balsam. Processing must be carried out quickly, as these chemicals will cause the pigments to fade if the specimens are exposed to them for too long. Pigmented benedeniines processed using this technique retain the distribution, intensity and colour observed in live worms. The colour and distribution of pigmentation in monogeneans may be of taxonomic importance and this technique aids preparation of whole-mounts suitable for registration as type-material.     

Liquid crystallinity in condensed type I collagen solutions. A clue to the packing of collagen in extracellular matrices.

We recently described a new type of assembly of collagen molecules, forming typical liquid crystalline phases in highly concentrated solutions after sonication. The present work shows that intact 300 nm long collagen molecules also form cholesteric liquid crystalline domains, but the time required is much longer, several weeks instead of several days. Differential calorimetry and X-ray diffraction show that sonication does not alter the triple-helical structure of the collagen fragments. In the viscous solutions, observed between crossed polars in optical microscopy, the textures vary as a function of the concentration. Molecules first align near the air interface at the coverslip edge, then as the concentration increases by slow evaporation of the solvent, the birefringence extends inwards and liquid crystalline domains progressively appear. For concentrations estimated to be above 100 mg/ml, typical textures and defects of cholesteric phases are obtained, at lower concentrations zig-zag extinction patterns and banded patterns are observed; all these textures are described and interpreted. The cholesteric packing of collagen fibrils in various extracellular matrices is known, and the relationship that can be made between the ordered phases obtained with collagen molecules in vitro and the related geometrical structures observed between fibrils in vivo is thoroughly discussed.     

Influence of light and mounting medium on the fading of Feulgen stain.

Serial microspectrophotometric estimations of the absorbance of Feulgen-stained interphase nuclei of human fibroblasts were made on slides mounted in a variety of mounting media and stored in light or in darkness. All preparations showed some fading, at rates which varied with different mountants, were greater in the light than in the dark, and were greater in the monochromatic illumination of the microspectrophotometer under working conditions than in normal laboratory light conditions. Fading was minimised by using XAM improved white neutral mounting medium (G.T. Gurr), storing slides in darkenss, and by reading samples as quickly as possible.     

Vinylpyrrolidone-Vinyl Acetate Copolymers as Mounting Media for Azo and Other Dyes

Vinylpyrrolidone-vinyl acetate copolymers (Antara Chemicals, General Aniline and Film Corp., New York N. Y., PVP/VA, E-735 and E-635) were employed as mounting media by adition of ethanol-water to the concentrated ethanolic solution of these plastics (25% plastic in 50% ethanol). The E-735 copolymer was frequently employed and is specifically recommended because it exhibits the highest degree of water tolerance. This type of mounting medium was found to be especially satisfactory in the preservation of azo, oxidation, and other histochemically derived dyes. The medium is useful also in the preservation of stains for fat and certain metachromatic dyes. The inexpensive nature of this mountant and its ease of application recommend it as a useful substitute for glycerol-gelatin.     

Optically eliminating the visible outlines of pores in intact polycarbonate (Nuclepore) filters

Under the microscope, Nuclepore filters display pore outlines. The polycarbonate material has two refractive indices (n = 1.584 and 1.616), which polarize transmitted light into two sets of rays at right angles to one another. This birefringence was used to eliminate the image of the pore outlines by the use of a specially made mounting medium with n20D = 1.584 in combination with polarized light: in a filter preparation of human leukocytes mounted in a medium with one matching refractive index and focused in polarized light, pore outlines were not visible. The preparation of the matching mounting medium, its use and its properties are described and discussed.     

A method for the preparation of serial thick sections of plastic-embedded plant tissues.

A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Dry Ice Fixation of Myofibrils for Scanning Electron Microscopy

A rapid method of fixation of myofibrils using dry ice is reported. A glass slide or coverslip containing a drop of glutaraldehyde-fixed suspension of myofibrils is placed on dry ice causing the myofibrils to adhere to the glass surface. The specimens are then dehydrated through the alcohols, air dried and metal coated. This technique gives the myofibrils a corrugated appearance under the scanning electron microscope corresponding to the sarcomere banding.     

A method of comparing spermatozoa with light and scanning electron microscopy

A new method of comparing light microscopy and scanning electron microscopy in the study of small cells, such as spermatozoa, that must be examined under oil immersion is described. A grid is etched on the corner of a microscope glass slide, and its inner edges are incised. Its surface area is calculated as a function f the chamber of the critical-point drying apparatus. This method dispenses with the need for any special coverslip and enables the cells to be observed under oil immersion.     

Dissection, Staining, and Mounting of the Embryos of Conifers

A statement is given of the advantages of this special technic and its place in embryological investigations, including directions for selecting the proper stages in collecting conifer cones and ovules, their methods of dissection from living material and their preservation for later dissection. The choice of dissecting microscopes and dissecting instruments, as well as directions for staining embryos with phloxine which may be combined with slow dehydration in glycerin, or for staining with Delafield's or Heidenhain's hematoxylin which may be followed by the glycerin dehydration are described. Glycerin affords a convenient break for a temporary stopping place in this technic.

Directions are given for transfer from glycerin thru 95% and absolute ethyl alcohol into other solvents such as diaphane solvent, essence of euparel or an easily prepared sandarac solvent. Other mounting media which have been used for conifer embryos are discussed—glycerin jelly, Venetian turpentine and Canada balsam—emphasizing the special advantages found in the media employing sandarac.     

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans¹. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system^2,3. However, solid-state pulse lasers of modest costs (<$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (<1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers⁴). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.     

Advantages of Epoxy Resin as a Mounting Medium for Light Microscopy

The need for very durable mounting is especially felt in the teaching of parasitology and mycology; otherwise, the availability of microscope slides may depend on the use of fresh specimens. Resinous mounts and those in aqueous media sealed with fingernail lacquer, paraffin or asphalt do not preserve specimens satisfactorily. Polyvinylpyrrolidone (pvp), a water-soluble mounting medium described by Burstone (1962), cannot be applied directly for mounting of insects and certain other parasites which have water-repelling integuments; moreover, pvp bleaches eosin. Grimley et al. (1965) prepared large epoxy sections of tissues from which areas for electron microscopy could be selected. This procedure however is designed for electron microscopic techniques whereas the present paper describes a direct epoxy mounting method to produce permanent mounts for light microscopy.     

上海园林绿地植被结构与温湿度关系浅析

通过对上海市区园林绿地29个样方的植被结构与微环境及土壤湿度、温度的调查,结果表明,绿地具有明显的增湿降温作用,影响这一作用的主要因素是绿地的乔木植物盖度。土壤湿度与草本植物盖度呈极显著正相关。土壤湿度、温度在一定区间内,呈极显著正相关。复杂完善的植被结构对防止土壤水分蒸发具有较好的效果。     

Use of Dimethylsulfoxide to Prevent Clumping during Feulgen Staining of Ceratocystis ulmi

The dimorphic fungus Ceratocystis ulmi is the causative agent of Dutch Elm Disease. As part of a study on the regulation of this developmental phenomenon, we attempted to stain the nuclei of cells growing vegetatively in the yeast phase by a modification of the Feulgen technique described by Gauger (1975). The cells were harvested by centrifugation, washed twice, and resuspended in 0.05 M phosphate buffer (pH 6.5). A small portion of this cell suspension was placed on a clean No. 2 glass coverslip (22 ± 22 mm) and allowed to air dry. The coverslip was flamed briefly to heat fix the cells whereupon they were fixed in glacial acetic acid: 95% ethanol (1:3 v/v) for one hour, hydrolyzed in 1 N hydrochloric acid at 60 C for 5 minutes, and stained for 30 minutes. The Feulgen stain was prepared according to Stevens (1974). Subsequently, the coverslip was rinsed briefly with distilled water and dehydrated for 30 seconds in 70% ethanol. After air drying, the coverslip was mounted on a glass microscope slide with Permount (Fisher Scientific Co.) and examined.     

A method for determining the toxigenicity of micromycetes]

A method is suggested to determine toxigenicity of microscopic fungi from genera Aspergillus, Penicillium, Fusarium, etc. which contaminate fodder, food products and environment objects. The method is based on the property of micromycetes isolated from fodder, food products and environment objects to produce mycotoxins under optimal conditions of their cultivation on the nutrient media. Their further extraction by the organic solvent, evaporation and emulsification of the extract and its peroral introduction to young female rats cause a disease and death of animals. The suggested method may be used for recognizing and preventing mycotoxicoses of farm animals and poultry.     

Feulgen stain stability in relation to three mounting media and exposure to light

Fading of Feulgen-stained rat liver nuclei was studied with sections mounted in Eukitt, Clearmount, and Permount, each under conditions of light and dark storage. Repeated microdensitometric measurements were made on selected nuclei over a 160 day post-staining period. In all three media the stain faded significantly when exposed to daily laboratory lighting, but faded least in Eukitt. Slides stored in darkness faded less than those exposed to light but, again, Eukitt-mounted preparations showed the least fading. Eukitt's low refractive index, however, imparts a high refractility to the cytoplasm which may be undesirable for scanning microdensitometry, and its apparently long stabilizing period may be disadvantageous if care is not taken to measure slides at comparable times during the period following mounting.