A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

Selection for Electron Microscopy of Specific Areas in Large Epoxy Tissue Sections

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 6% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and rinsed in phosphate buffer for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO₄. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 4-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphalate to the standard epoxy mixture. The sections were spread on water and attached to coverslips by drying, then heating to 80 C for 1 min. Staining 2 min with 1-3% KMnO₄ and temporary mounting in glycerol on a slide allowed the desired area for electron microscopy to be selected and marked. This area was then cemented to the facet of a conventional epoxy casting with a drop of epoxy resin (without added dibutylphthalate). After polymerization, the coverslip was removed by quick cooling leaving a flat re-embedded portion of the original section. This portion was viewed by transillumination in a dissecting microscope and trimmed of surplus tissue. Ultrathin sections for electron microscopy were obtained in the usual manner.     

A Method for Preparing and Staining Histological Sections Containing Titanium Implants for Light Microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 μm thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

Improved diethylene glycol distearate embedding wax

Diethylene glycol distearate wax and cellulose caprate resin, 4:1 respectively by weight, were melted together at 75 C for five hours with occasional stirring. The resin tempered the extreme brittleness of the wax without softening it, and raised the melting point only one degree to 50 C. Fixed plant tissues were dehydrated in ethanol, cleared in xylene, and infiltrated with wax. Modified diethylene glycol distearate was easier to trim and shape, and formed flat sections more consistently than the pure wax. Sections were cut singly on Ralph knives with attached water pools on an ultramicrotome. Sectionability was excellent at 2-3 micrometers, variable at 1.0 micrometer, but impossible at 0.5 micrometer. Sections were transferred onto water drops on slides, dried, dewaxed, stained, and coverglasses applied as in the paraffin method. Histological feature of plant tissues were much sharper in modified diethylene glycol distearate sections than in paraffin sections, and were similar to plastic sections.     

Whole Mount Preparation of Chick Blastodermal Edge Tissue for Microscopic Examination

Three methods of preparation of chick blastodermal edge tissue for conventional microscopy were attempted: (1) Sections were cut from Water Wax (E. Gurr) embedded material. This method was unsatisfactory due to loss of morphological relationships produced by the inability to attach the tissue to a slide. (2) Frozen sections were cut from embryonic marginal tissue with its underlying white and yellow yolk which was embedded in a 25% gelatin solution. This method retained morphological relationships prior to 24 hr of egg incubation, but was technically impractical in excess of this period of incubation. Also, the gelatin could not be removed from the sections and cellular detail was obscured by its subsequent staining. (3) The blastoderm was removed from the yolk and adherent vitelline membrane, and the yolk dissected from a small piece of this blastodermal edge tissue under a dissecting microscope. The dissected tissue, primarily monocellular and dicellular in thickness, was transferred to a slide and attached to it by allowing a few drops of ether-alcohol (1:1) to flow over it. The plane of the tissue was, therefore, parallel with the plane of the slide. Most fixatives and staining techniques could subsequently be used. Fixing for 30-120 min in ether-alcohol (1:1) containing 0.5% glacial acetic acid gave excellent results with the staining techniques attempted. Fixation with Zenker's fluid for 24 hr followed by a 6 min hydrolysis with 1 N HC1 was best prior to the Feulgen technique.     

Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads

A simple photolithographic technique has been developed which can be used to produce microscopic grid patterns on glass coverslips. The grid pattern is first photo-reduced onto film, and the resulting photographic negative is then used as a mask. A glass slide or coverslip, coated with a layer of photoresist, is then exposed to tungsten light through the mask. After developing and etching, the grid pattern is transferred permanently onto glass. This simple and rapid procedure allows one to mass-produce very small, high resolution grids which are useful for monitoring individual microinjected cells or chromosomal spreads under the microscope.     

A method for preparing and staining histological sections containing titanium implants for light microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 microns thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

The application of polyethylene glycol (PEG) to electron microscopy

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.     

Quantitative Autoradiography of Isolated Thyroid-Gland Colloid

Deparaffinized and dehydrated sections (on slides) of human, dog, pig, guinea pig and rat thyroid were coated with 1% CHCl₃ solution of Plexiglas, dried thoroughly and immersed in water at 15°C for 2-10 min. When the Plexiglas film loosened, it was stripped from the slide; the colloid remained on the slide while the rest of the gland was removed in the film. Each glandular component was then subjected to separate autoradiographic processing.     

Sandwich Embedding of Monolayers of Cells in Epoxy Resin for Ultrathin Sectioning

In this procedure for embedding monolayers of cells, the usual glass slides are replaced by plates of resin 1-1.5 mm thick. Unlike the open-face embedding technique, the present procedure uses only a few drops of unpolymerized resin, which are applied to the fixed and dehydrated cells. During polymerization this small amount of liquid resin spreads across a relative large area, leaves the cells covered by a very thin layer, and permits phase contrast observations through it. Ultrathin sections of a particular cell encircled by a rotary scriber can be obtained by sectioning the resin slide, which has been trimmed and mounted directly in the specimen holder of the ultramicrotome.     

Reduced Methylene Blue as a Stain for Crustacean Nerves

Effective in situ staining of crustacean nerves was achieved with leuco methylene blue reduced with either ascorbic acid or sodium hydrosulfite (Na₂S₂O₄). A stock solution of methylene blue, 0.4% (ca. 0.001 M), and the reductants, ascorbic acid or sodium hydrosulfite (0.01 M), were prepared in van Harreveld's crayfish physiological solution. Methylene blue stock solution was mixed with either of the reductants in the approximate ratio of 1:10, v/v, and titrated to the end point. Ascorbic acid reduction is light catalyzed and requires intense illumination during titration. The cleared or leucomethylene blue stock solution is suitable for immediate use as a working nerve stain. With either reductant, the working solution oxidizes on standing in air, but can be titrated repeatedly without loss of staining properties. Dissected nerve trunks or tissue were immersed in the working stain for 20 min at room temperature and the staining process observed until suitable contrast developed. Excess dye was decanted and the tissues flooded with crayfish physiological solution. Contrast could sometimes be enhanced by flooding the stained area with 1% hydrogen peroxide in van Harreveld's solution. When permanent mounts were prepared, tissues were dehydrated with tertiary butyl alcohol in preference to ethyl alcohol series. For anatomical and neurophysiological studies of nerve distribution in crustaceans, the alternative use of either ascorbic acid or sodium hydrosulfite, as reductants for methylene blue, was preferable to the more complicated rongalit-technique and characterization of neural elements was fully as satisfactory.     

Handling Germinated Pollen on Millipore Membrane During the Feulgen and Autoradiographic Procedures

To prevent loss of pollen during the Feulgen's procedure, the pollen was grown on an autoclaved membrane filter (Millipore AA WP 025 00) in contact with a sterilized medium containing agar 0.5-1%, sucrose according to the genus (Malus 0.3-0.5 M; Persica and Tulipa 0.4 M), and H₃BO₃, 0.01%. To fix the germinated pollen of most species, the membrane was placed for 2 hr to overnight at 2-4 C on filter paper wet with the following mixture: OsO₄, 1 gm; CrO₃, 1.66 gm; and distilled water, 233 ml. To fix Persica pollen, 10% of glacial acetic acid had to be added to the fixative. Washing with distilled water and bleaching with a mixture of 3% H₂O₂ and sat. aq. ammonium oxalate, 1:1, were performed also on filter paper. Similarly, the preparation was processed for Feulgen staining by use of pieces of filter paper wet with the required fluids. Hydrolysis preceding the Schiff's reagent was performed at room temperature with 5 N HCl for 18 min. The differentiation after the Schiff's action was with 2% K₂S₂O₅ buffered to pH 2.3 with 9 ml of phosphate buffer (KH₂PO₄, 1.4 gm; conc. HCl, 0.35 ml and distilled water to make 100 ml). The stained pollen was floated off the membrane with a drop of glacial acetic acid to a gelatinized or an albumenized slide, and squashed. When the coverslip is removed the preparation may be either dehydrated and mounted or coated with autoradiographic film.     

Pressure Resin Infiltration of Aphids for Electron Microscopy

Fixed, dehydrated pea aphids, Acyrthosiphon pisum Harris, were partially infiltrated with epoxy resins by standard procedures, then placed in a pressure chamber at up to 1000 psi for varying lengths of time. Insects so treated were found to be more suitable than nonpressurized insects for ultrathin sectioning and electron microscopy.

It was necessary to remove one or more legs from the insects to obtain adequate infiltration even where high pressures were employed. Little damage was evident at light or electron microscope levels of examination.     

Embedding Stained Mammary Glands in Plastic

A simplified method of embedding stained mammary spreads of small mammals in plastic (Selection) is presented. Two standard 50 × 75 × 1 mm. glass slides, separated by 2 narrow glass strips of similar thickness, are used to form the embedding chamber. The glands are stained in toto in alum-carmine, dehydrated, defatted, infiltrated with uncatalyzed plastic and embedded in catalyzed plastic. After baking and cooling, the glass chamber separates readily and provides a thin square slide of plastic suitable for low-power microscopic examination, projection, and filing.     

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 morphological and immunolabeling study of freeze-substituted Bacteroides forsythus

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO₄ fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.     

松属针叶角质层微形态特征在分类学中的应用

角质层是表皮细胞壁表面的一层不透水的脂肪性物质。角质层与表皮细胞紧密结合,植物表皮细胞形态和排列方式、气孔器的形态结构等微形态特征均能在角质层上反映出来。利用光学显微镜和扫描电镜对松属（Pinus）12种植物针叶角质层微形态特征进行观察和比较,详细描述20个性状,其中12个性状来自角质层内表面,8个性状来自角质层外表面。结果表明,这些特征可为该属属下分类和相似种的鉴别提供有用信息,具有重要的分类学意义：①表皮细胞长度、表皮毛长度、角质层外表面起伏程度、表皮细胞轮廓、有无气孔塞和针絮状物质等角质层微形态特征具有自身特异性,在属下可作为松属组级水平上的分类依据。角质层微形态 特征不支持将五针松组（P. Section Cembra）和白皮松组（P. Section Parrya）合并为P. Section Quinquefolius的观点,亦不支持将油松组（P. Section Pinus）分成P. Section Pinus和P. Section Trifolius的看法。②白皮松（P. bungeana）针叶角质层微形态特征既与五针松组有相同之处,又与油松组有相似之处,还有部分特征显示出不同于松属其他种类的独特性,可为白皮松亚属（P. Subgenus Parrya）的建立提供新依据。③扫描电镜下表皮细胞垂周壁纹路,气孔塞有无和外表面气孔形状等特征可为形态相似种火炬松（P. taeda）和湿地松（P. elliottii）提供种间界定依据。     

A comparison of three methods for determining the stomatal density of pine needles

Alternative methods were compared for determining the stomatal density of needles from two pine species. Densities estimated from air-dried, whole needles using a binocular dissecting scope were compared to densities estimated from vacuum-dried, intact needles using a scanning electron microscope and expanded peels (or macerated cuticles) using a compound light microscope. Differences among methods were expected from two sources: (1) expansion and shrinkage as a function of water content, and (2) differences in geometry of the measured surface. Estimates from the dissecting scope were similar to those from scanning electron microscopy (t=0.509, n=21, P:=0.62), presumably because both used dried, but otherwise intact whole needles. Light microscopy estimates, however, were lower than dissecting scope estimates (t=-2.307, n=13, P:=0.04). After adjusting for expansion due to hydration and changes in needle geometry, differences disappeared (t=-1.205, n=13, P:=0.25). These results are an important consideration for researchers reconstructing palaeo-atmospheric conditions and assessing plant response to environmental change.     

A novel, stable bioradical

Several cellular systems have shown a novel EPR spectrum at 77 K, comprising a slightly anisotropic doublet centred on the free-spin g-value, with a hyperfine splitting of ca. 120 G. It is suggested that this species is an occluded protein radical centred on a tryptophan unit.