Modification of the anal tape method for detection of pinworms in rodents

A modification of Graham's anal tape technique for detection of oxyurid worm eggs involves a coverslip or microscope slide covered with a thin layer of adhesive. The worm eggs stick to the adhesive and can be directly observed under the microscope.     

A Method for Embedding Thin Membranes in Historesin

A method is described for flat-embedding thin membranous tissues in Historesin. It allows easy orientation for sectioning large areas parallel to the surface. Selected fields can be monitored from the unfixed specimen, throughout preparation, to mounting on the microscope slide. For cross-sectioning, the flat-embedded tissue can be stacked and re-embedded to increase the amount of material examined per section.     

Sticky Wax Infiltration in the Preparation of Sawed Undecalcified Bone Sections

The undecalcified bone specimen was surfaced by an ordinary motor-driven circular saw. After thorough drying in air, the specimen was infiltrated with melted Caulk sticky wax (L. D. Caulk Co., Milford, Del., 19963) without casting in a block. The specimen was affixed to the Gillings-Hamco thin-sectioning machine with cut surface parallel to the circular diamond blade. Prior to sawing each section, the specimen surface was blown dry and coated with a thin supporting layer of stick wax. The section was then attached to an albumen-coated glass slide with the newly cut surface facing the slide. After drying in room temperature, the slide was soaked in xylene to partially dissolve the sticky wax, and the loosened residue was removed subsequently by gentle brushing. The section was mounted and covered with a coverglass. Sections 50-100 μ thick were prepared satisfactorily by this method. The advantages of using sticky wax as an infiltration medium depend on its physical properties: it is gluey when melted, and holds the bony trabeculae together; it becomes hard and nonsticky at room tempperature, and can be sawed together with bone tissue. Since a new layer of wax blends readily with the old wax surface, it allows the important supportive coating of wax to be added to the sawing surface for each section cutting     

Preparation of Drosophila mitotic chromosomes for electron-microscopic studies]

A method of chromosome spreading on microscopic slides was modified for electron microscopy of metaphase chromosomes in Drosophila tissues. The slides covered with an electron transparent film were plasmochemically modified to make them hydrophilic. A piece of fixed tissue was macerated in 60% propionic acid before spreading chromosomes over the slide. The parts of preparation selected under light microscope for electron microscopic examination were cut and peeled of the slide to the top of a water drop. It was shown that the resolution of chromosomal structures was significantly higher than seen under optical microscope, but lower than in serial sections.     

X-ray micrography and imaging of Escherichia coli cell shape using laser plasma pulsed point x-ray sources.

High-resolution x-ray microscopy is a relatively new technique and is performed mostly at a few large synchrotron x-ray sources that use exposure times of seconds. We utilized a bench-top source of single-shot laser (ns) plasma to generate x-rays similar to synchrotron facilities. A 5 microlitres suspension of Escherichia coli ATCC 25922 in 0.9% phosphate buffered saline was placed on polymethylmethyacrylate coated photoresist, covered with a thin (100 nm) SiN window and positioned in a vacuum chamber close to the x-ray source. The emission spectrum was tuned for optimal absorption by carbon-rich material. Atomic force microscope scans provided a surface and topographical image of differential x-ray absorption corresponding to specimen properties. By using this technique we observed a distinct layer around whole cells, possibly representing the Gram-negative envelope, darker stained areas inside the cell corresponding to chromosomal DNA as seen by thin section electron microscopy, and dent(s) midway through one cell, and 1/3- and 2/3-lengths in another cell, possibly representing one or more division septa. This quick and high resolution with depth-of-field microscopy technique is unmatched to image live hydrated ultrastructure, and has much potential for application in the study of fragile biological specimens.     

Cryostat Sectioning; Modification of the Antiroll Plate

A replaceable antiroll plate and holder have been designed for use in the Ames Lab-Tek cryostat which replace the plastic plate supplied with the instrument and insure a flawless, properly aligned plate for maximum efficiency in thin section cutting. A metal plate holder is attached to the existing screw-driven bracket provided with the instrument by the manufacturer. Glass plates made from one half of a 1.5 × 3 inch microscope slide are coated on the leading edge with spray-on Teflon and provided with tape spacers. These plates slip into the holder and can be adjusted for angular inclination and alignment with the cutting edge by movement within the holder or manipulation of the adjustment screw.     

Fine structure of the knobby spore type of Streptomyces torulosus

The type strain of Streptomyces torulosus Lyons and Pridham (1971) was studied by scanning- and transmission electron microscope. Spore chains were formed in spirals by aerial mycelium. The spores were connected by nozzles in which small channels could be observed. The knobby ornamentations of the spores arised on a thin fibrous sheath, enveloping the spore chains. These irregular blunt projections, called knobs, had varying diameters of 100 to 250 nm. The base of the knob, consisting of globose to flattened electron dense material, was sitting directly on the sheath. It was covered by several small vesicles of the same material. Each hollow vesicle beared a thin bowlshaped shell of electron transparent material. In general, the cupular bowls and their supporting vesicles became easily depressed on their base, but not detached from the surface of the spores. This type of knobby spore ornamentation was suggested to be designated as a verrucate spore type.     

A Serum-Film Technic for Bone-Marrow Smears

A chemically clean slide was covered with a thin film of serum by using a glass spreader. A drop of marrow suspension (bone-marrow in autogenous serum, about 1 to 20 dilution) was placed upon this film and spread by blowing gently on it until a central area of about 2 cm in diameter became dry. The peripheral rim of the smear was then wiped off and the slide stained. A statistical analysis showed that the variation in size of cells did not influence their distribution within the area and that improved cell morphology with fewer damaged cells favored quantitative studies.     

Measurement of live bacteria by Nomarski interference microscopy and stereologic methods as tested with macroscopic rod-shaped models

A new method is proposed to measure bacterial cells under growth conditions. Bacterial cells, suspended in their growth medium, were attached to a cover slip with poly-L-lysine. The cover slip was inverted and placed on a glass microscope slide. To prevent dehydration of the medium, the edges of the cover slip were sealed to the microscope slide with clear fingernail polish. The bacteria on the slide were then quickly photographed with a Leitz light microscope, using Nomarski optics. The photographic negatives were then projected at a standard distance through a lens system, and the projected images of the whole cells were outlined by hand onto graph paper. The profile images so transcribed onto the graph paper were in effect transverse sections of each of the cells. Using stereologic grid and point counting techniques, the area of the cell transverse section as well as the perimeter or circumference of the transverse section were estimated. Formulae were developed so that both the volume and surface area of the whole cell could be ascertained from these area and circumference measurements. Since the efficacy of any measurements of surface area and volume of microscopic rod-shaped bacterial cells could be questioned, macroscopic rod-shaped models were used to test the theory and formulae and to compare this method with other commonly used cell-sizing techniques. This technique could be used in any study of bacterial cell size or changes in cell size (e.g., osmotic shifts).     

Measurement of live bacteria by Nomarski interference microscopy and stereologic methods as tested with macroscopic rod-shaped models.

A new method is proposed to measure bacterial cells under growth conditions. Bacterial cells, suspended in their growth medium, were attached to a cover slip with poly-L-lysine. The cover slip was inverted and placed on a glass microscope slide. To prevent dehydration of the medium, the edges of the cover slip were sealed to the microscope slide with clear fingernail polish. The bacteria on the slide were then quickly photographed with a Leitz light microscope, using Nomarski optics. The photographic negatives were then projected at a standard distance through a lens system, and the projected images of the whole cells were outlined by hand onto graph paper. The profile images so transcribed onto the graph paper were in effect transverse sections of each of the cells. Using stereologic grid and point counting techniques, the area of the cell transverse section as well as the perimeter or circumference of the transverse section were estimated. Formulae were developed so that both the volume and surface area of the whole cell could be ascertained from these area and circumference measurements. Since the efficacy of any measurements of surface area and volume of microscopic rod-shaped bacterial cells could be questioned, macroscopic rod-shaped models were used to test the theory and formulae and to compare this method with other commonly used cell-sizing techniques. This technique could be used in any study of bacterial cell size or changes in cell size (e.g., osmotic shifts).     

Erratum

A solution of plastic consisting of toluene, 720 ml; methanol, 180 ml; ethyl cellulose (Ethocel, standard 7 CPS), 250 gm; and Dow resin 276 V-2, 75 gm is applied to a leaf surface which has been dampened with toluene. The plastic is spread to a thin film with the edge of a card and allowed to dry. After drying, the plastic may be peeled from the leaf surface and either stored dry in a small envelope or mounted permanently on a microscope slide. Permanent mounts are prepared by placing a small section of the peel from the upper and lower surfaces of the leaf on a microscope slide and covering with a No. 1 cover glass. A small spot of balsam on each corner of the cover glass secures the glass in position. This air mount has proved to be superior to water, glycerol or balsam mounts. Fresh leaves are washed with a mild detergent before application of the plastic. Herbarium specimens are soaked in water overnight to restore the leaf to a semiturgid condition. Five species of different plant families have been illustrated to show the diagnostic features of the surface of the cuticle. An isolated layer of epidermal cells obtained by chemical maceration permitted cell and imprint comparison. The remarkable amount of detail shown by the prints is an aid for phylogenetic studies and may make the recognition of fossil cuticles possible.     

Surface Printing of Plant Leaves for Phylogenetic Studies

A solution of plastic consisting of toluene, 720 ml; methanol, 180 ml; ethyl cellulose (Ethocel, standard 7 CPS), 250 gm; and Dow resin 276 V-2, 75 gm is applied to a leaf surface which has been dampened with toluene. The plastic is spread to a thin film with the edge of a card and allowed to dry. After drying, the plastic may be peeled from the leaf surface and either stored dry in a small envelope or mounted permanently on a microscope slide. Permanent mounts are prepared by placing a small section of the peel from the upper and lower surfaces of the leaf on a microscope slide and covering with a No. 1 cover glass. A small spot of balsam on each corner of the cover glass secures the glass in position. This air mount has proved to be superior to water, glycerol or balsam mounts. Fresh leaves are washed with a mild detergent before application of the plastic. Herbarium specimens are soaked in water overnight to restore the leaf to a semiturgid condition. Five species of different plant families have been illustrated to show the diagnostic features of the surface of the cuticle. An isolated layer of epidermal cells obtained by chemical maceration permitted cell and imprint comparison. The remarkable amount of detail shown by the prints is an aid for phylogenetic studies and may make the recognition of fossil cuticles possible.     

Direct Bacterial Count as a Rapid Freshness Test for Fish Fillets

Comparison of various indices of deterioration of refrigerated fish fillets showed that the direct bacterial count can be used to predict the storage life of the foodstuff. For direct counts, a thin film made from fillet surface material was spread on a microscope slide, stained, and read. Initial counts were found to correlate well with keeping quality; a period of freshness of 24 or 48 hr at 5 C could be reliably predicted. Preliminary data indicated that hypoxanthine estimation could probably also be used for the prediction of shelf life but that the relative complexity of the procedure detracted from its usefulness.     

Affixing Plant Sections without Protein Based Adhesives for Protease Histochemistry

To submit a section of plant tissue to histochemical analysis using protease, the protein based adhesives which keep the slices attached to the slides must be replaced because they are attacked by the enzyme and the slices are washed off the slides. We devised a method to keep the slices attached to the slides during histochemical extractions and subsequent staining. Slides are frosted on two lateral zones by spreading on them a fluoride paste composed of 15 g barium sulfate, 15 g ammonium difluoride, 8 g oxalic acid, 40 ml glycerine and 12 ml deionized water using a thin paint brush. After removing the paste with tap water and drying the slides, the sections are placed on the central clear zone of the slide and covered with an ethyl-cellulose film that keeps the slices in place and allows the reagents to act through it. To do this, the slides are dipped into 0.5% ethyl cellulose (ETC) prepared in a 4:1 mixture of toluene and absolute ethanol. The ETC coating is layered three times to improve its firmness and its ability to retain the slices on the slides. To obtain perfect adhesion, the slide should be oven dried (40-50 C for 10-15 min) to remove any trace of humidity before applying each layer of ETC. Subsequently the sections can be extracted and stained without undue loss of material.     

Semipermanent microscope slides of microfungi using a sticky tape technique

A technique is described for making semipermanent microscope slides of fungi using sticky tape. After being touched to a fungal colony, a modified segment of sticky tape is touched to ethyl alcohol and then immersed in a 50% glycerine solution containing cotton blue stain. Finally, it is transferred (sticky side up) to a microscope slide, covered with a cover with a cover glass, and sealed.     

Collodion membrane secures cells sorted by flow cytofluorometry onto microscope slides

Quantitative microscopic cytology of cells previously sorted by flow cytofluorometry has been hindered by the loss of cells from the microscope slide during staining procedures. The simple application of a semi-permeable membrane of collodion over fixed or unfixed cells sorted directly onto a microscope slide secured virtually 100% of the cells onto the slide. Cells covered with the collodion membrane studied with Papanicolaou's stain as well as routine clinical cervical cytologic preparations. In contrast, fewer than one half of the cells sorted onto uncoated or albumin coated slides were retained after staining.     

嫁[虫戚]的齿舌带及其齿片的形态差异分析

在光镜和电镜下对嫁[虫戚]（Cellana toreuma）的齿舌形态进行观察研究。嫁[虫戚]的齿舌带每1横列具有2枚侧齿和2枚缘齿,缺乏中央齿,齿式为1.1.0.1.1。齿舌带前端弯曲,齿片排列松散且存在明显的磨损现象;中段齿片排列紧密、整齐;后端齿片无色且宽度有略微的缩小。侧齿呈镰刀型,具1个齿尖,基部呈三角形且具突起,尖齿部分细长;缘齿具3个齿尖,第2尖齿靠近第3尖齿。采用多个比例参数来比较嫁齿舌带及其前、中、后3段上的齿片形态,发现嫁齿舌带前、中、后3段各比例参数的值存在一定的关系,即中段大于前段、中段大于后段。     

Serial Sections of Smears for Electron Microscopy

Ultrathin sections for electron microscopy may be prepared from smears or squashes embedded in methacrylate. The cover slip or glass slide with the attached fixed cellular material is passed through alcohols to methacrylate monomer and finally to monomer containing a catalyst. The portion of the smear to be sectioned is covered with a gelatin capsule containing partially polymerized methacrylate. When polymerization is completed at 47°C, the hardened block is separated from the cover slip and trimmed under the compound microscope so as to encompass the desired area. Photographs are made of the intact smear to afford a basis for identification of cellular materials in electron micrographs of the individual ultrathin sections.     

Serial Sections; Simplified Handling of Paraffin Ribbons on a Floating-Out Bath

Pieces of para & ribbon containing serial sections are arranged in overlapping rows on a microscope slide coated with albumen or glycerol. The assembly of sections is then floated free by immersing the slide in a bath of warm water. The rows of sections forming the assembled unit adhere to each other along their overlapping edges. After the sections have softened and expanded, the unit is picked up on a slide, covered with wet filter paper, rolled flat with a photographic print roller, and allowed to dry.     

REDUCTION OF HEATING ARTIFACTS IN THIN SECTIONS EXAMINED IN THE ELECTRON MICROSCOPE

Certain phenomena affecting contrast obtained from tissue sections with the electron microscope have been investigated and a technique is described for reducing destruction by the electron beam of fine details in sections. It has been concluded that loss of embedding material is slightly higher at exposed surfaces of sections than it is at surfaces covered by substrate film. Covering of both surfaces of sections with thin films of formvar, collodion, or carbon materially improves the general appearance, reduces distortion, and sometimes reduces loss of tissue mass from the section as result of exposure to the electron beam. This improvement is considered to result from the relatively high melting-point of the covering films which serve to eliminate or reduce surface-tension or other forces operating in methacrylate softened by the electron beam.