Grid staining--a useful device.

A simple and disposable holder for electron microscope grids can be constructed from discs of silicon rubber. The discs (10 mm thick X 25 mm diameter) are scored 0.4 mm deep with a razor. Flexing the discs opens the scores for insertion of grids. Staining wells can be made from polyethylene snap-in vial tops by cutting away the nipple-like portion. After impaling the holders on the tips of a pair of forceps the grids can be washed by immersion. This holder is rigid, inert and inexpensive. It is of particular advantage for handling single-slotted grids.     

Production and Cleaning of Freeze-Etch Replicas which Show Complementary Surfaces of Fractured Fungus Spores and Hyphae

London finder grids (H-2) and 3 mm copper discs were flared on opposite sides by use of a die and punch. The grids were registered on the discs and attached with vaseline. The specimen was squeezed through the grid apertures by the 2 copper discs. The complete sandwich was frozen in freon 22 and placed in a hinged specimen holder which fractured the layer of the specimen between the 2 finder grids. Platinum-carbon and carbon replicas were made immediately after fracturing. After drying the replicas, the vaseline was removed by chloroform, and replicas were cleaned with 70% H₂SO₄ for 1-18 hr at 52 C, followed by 6% commercial sodium hypochlorite (Chlorox) for 1-2 hr at 23-25 C.     

A Holder for Simultaneous Fluid Processing or Carbon Coating of Electron Microscope Grids in Lots of 10 or More

A piece of polyethylene tubing about 14 cm long, ID 3 mm and with a 1.5 mm wall is shaped to hold grids in a linear series as follows. A series of pairs of transverse cuts I mm apart are made across and about $3/4 through the tubing at intervals of 1 cm. After making diagonal cuts on either side of each pair of cuts, wedge-shaped pieces of tubing are removed to leave free-standing 1 mm widths of the tubing wall. Each arch of wall is then bisected by a cut in the longitudinal direction of the tubing, thus providing the jaws of a clamp for a grid. By spreading the jaws with forceps, the grid can be inserted. On removal of the forceps, the grid is held firmly and when the holder is filled, the grids face in a direction perpendicular to its long dimension. By inserting a piece of glass tubing into the lumen of the holder, it is held straight and can be placed in a test tube for fluid processing. Stains or other fluids can be introduced through the glass tube, thus allowing fluid changes without exposure to air. Carbon coating can be achieved without removing the grids, since they all face the same way. For convenience in loading and unloading, the holder can be temporarily attached to a base with double-coated adhesive tape.     

Modification of a scanning transmission electron microscope specimen holder for large section viewing.

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter "giant" grids is explained and the procedure for sample preparation is outlined. The modification aids the microscopist in his evaluation of tissue structural relationships by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm2 electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Staining and Washing Ultrathin Sections; a Device for Handling Various Materials Simultaneously

To eliminate individual manipulation, as many as 10 grids, each held firmly by a small notched bar of polyethylene plastic, are simultaneously stained, then washed. If the stain used is reactive with atmospheric CO₂ it can be forced through a Millipore filter into a small chamber made of glass tubing which contains the grid holder. The stain, cleared of any solid particles, has very little contact with air and remains free of lead carbonate contamination. Washing is carried out by submerging the chamber and removing the grid holder under water (Feldman, D. G., J. Cell Biol., 15: 592-5, 1962). Washing is minimized because there is not the risk of contaminating grids and wash water with stain trapped between the points of forceps. The polyethylene is nonadherent to the wash water, and the grids can therefore be dried quickly on the holder. With this method, the relative stainability of different materials may be observed because each grid within a batch receives identical treatment.     

Formation of a new fibrous attachment to human dental roots

Summary This study was performed to improve currently employed in vitro models for the study of periodontal regeneration by using a porous filter upon which periodontal ligament cells were grown. Periodontal ligament cells were harvested and 0.3 mm root discs cut from three partially erupted and extracted third molar teeth of one patient. Experimental culturing was performed by seeding periodontal ligament cell suspensions on Puropor-200 filters supported by wire-mesh grids in Grobstein Petri dishes. The following day, an interdental space of 0.1 to 0.3 mm was created by gently placing two dental root discs upon the filter. Cultures were terminated after 42, 56, 112 and 124 days, and processed for light- and electron microscopy. Collagen fibril diameters were measured. Adjacent and often attached to large areas of cementum-lined root discs, a dense fiber fringe developed. This fiber fringe was not found on dentinlined root discs. Although less organized, older cultures demonstrated a similar disc-culture interface, which depended upon the presence or absence of original root cementum. Collagen fibrils of early cultures had a mean diameter of about 42 nm, while in older cultures the diameters ranged from 47 to 68 nm. It is concluded that the fibrous matrix attached to cementum-lined root discs somewhat resembles the initial stages of the formation of dental root cementum in vivo.     

A multiple filtration device for glass fibre filters

A practical multiple filter holder is described for collection of particles on glass fibre filters. The device utilizes large diameter discs and the ends of individual filter tubes compress against the filter so that, after filtration, the separate filtering areas can easily be taken from the large disc with forceps. If analyses of collected particulate matter are to be made at a later time, the filtering areas are left attached to the original filter. Precise volumes of water can be filtered by attaching volumetric pipettes to individual filter tubes.     

A Simple Device for Staining Many Single-Hole Grids Without Individual Manipulation

In serial sectioning for electron microscopy one of the greatest problems encountered is that the Formvar support film may break when grids are being mounted in or removed from a holder used for staining, or during staining. The latter is particularly troublesome when grids are stained individually. We describe here a device that conveniently eliminates this problem.     

Electron Microscopic Study of a Slime Layer

Slime layers are being studied in our laboratories in an attempt to understand their functions in the control of pollution in natural streams. A method for fixing, staining, and embedding microorganisms in the intact slime has been developed. In this method, epoxy resin discs are placed in a holder and are introduced into a simulated stream. After various periods of time the discs are punched out of the holder into the fixative. The disc with the attached slime is fixed, stained (4% osmium tetroxide plus ruthenium red), dehydrated, and embedded in epoxy resin so that thin sections can be cut through the vertical plane of the slime mass. Such thin sections permit detailed examination of the attached layer, the surface-slime interface, the spatial relationships between cells in the vertical slime structure, and the strands of extracellular material between and around cells. No special attachment structures were noted as the cells appeared to be attached to the surface by extracellular material alone. This material was observed in strands and netlike forms between cells which are positioned 1 to 4 mum apart in the slime.     

Modification of a Scanning Transmission Electron Microscope Specimen Holder for Large Section Viewing

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter “giant” grids is explained and the procedure for sample preparation is outlined The modification aids the microscopist in his evaluation of tissue structural relationship by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm² electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

通过膜片钳玻璃微电极内插管进行胞内透析

本文介绍一种膜片钳微电极内插管进行胞内透析的方法。利用通用的微电极夹持器在其抽吸负压的侧管上方钻一斜孔直通夹持器中央管腔。插入微量移液管头拉制成的细管（外径约0.1mm）,后者与Ag-AgCl电极一起伸出夹持器口端。通过相连的注射器,可以很方便地进行电极内液置换及胞内药物透析,此法和二次钳压技术及国外介绍的微插管电极内液置换相比,更加简便易行,结果更可靠。     

Contributions to semithin sectioning on a conventional rotary microtome.

Procedures for obtaining sections 1 micrometer thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen holder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections 1 micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

A Filter Method for Processing Small Samples of Marine Ova Through Fixation, Dehydration, and Embedding Without Changing Containers

Free-floating cells can be fixed, dehydrated, and embedded in a single container. The container was constructed from stainless steel, and the paraffin block formed by the shape and size of a container was perfect for microtoming. Eight containers were embedded in a fiberglass holder. This holder was designed so that it could be used with a 47 mm Millipore filter. Cells were pipetted into the top of a container while the Millipore filter sealed the bottom; thus the cells were retained on the filter while fluids were allowed to pass through it. The exposure of the cells to histological reagents was regulated by applying a vacuum to control the rate of flow through the filter.     

Micro- and nanopatterning by lithographically controlled wetting

This protocol describes how to perform lithographically controlled wetting (LCW). LCW enables large-area patterning of microstructures and nanostructures of soluble materials, either organic or inorganic, including biological compounds in buffer solutions or compounds for cell guidance. LCW exploits the capillary forces of menisci established under the protrusions of a stamp placed in contact with a liquid film. In the space confined by each meniscus, the self-organization of the deposited solute yields highly ordered structures that replicate the motif of the stamp protrusions. The method does not require any particular infrastructure and can be accomplished by using simple tools such as compact discs or microscopy grids. Compared with other printing methods, LCW is universal for soluble materials, as it does not require chemical binding or other specific interactions between the solute and the surface. A process cycle takes from 2 to 36 h to be completed, depending on the choice of materials.     

Growth Inhibition Test for Identification of Mycoplasma Species Utilizing Dried Antiserum-Impregnated Paper Discs

The growth inhibition test for identifying Mycoplasma species has been modified by drying antibody-impregnated paper discs at 5 C. When stored at -20 C, these discs have been found to retain their inhibitory activity for longer than 7 months. Since these discs can be stored for long period of time, significant advantages over present methods result. When, for example, discs are arranged on a ring, a single test can be used for the identification of an unknown human species. Valuable antisera can be distributed to other laboratories on paper discs in much less volume than can fluid antiserum. Considerable savings of time result from prior preparation of many discs that can then be stored and used over a long period of time. The growth-inhibiting antibody is stable, and the activity is not enhanced by a heat-labile accessory factor from fresh guinea pig serum which increases the antibody titer in the metabolic inhibition test.     

Evaluation of sorting grids for deepwater rose shrimp (Parapenaeus longirostris) in the Eastern Mediterranean demersal trawl fishery

The study aimed to evaluate sorting grids of 10 and 15 mm bar spacing specifically for separation of deep water rose shrimp, but including other species, in a Mediterranean multispecies demersal trawl fishery. Data were collected 15–25 October 2008 in S??ac?k Bay, Eastern Aegean Sea with the commercial trawler, ‘Hapulo?lu’, using a modified bottom trawl net. A total of 22 valid hauls (12 with 10 mm, ten with 15 mm grids) were obtained. The separation rate of anglerfish was highest, with 100% for weight and number in both codends among all species. Grid elimination of broadtail short‐fin squid showed differences between 95.3 and 80.3% in terms of weight, and 89.7 and 66.2% in terms of number for 10 and 15 mm, respectively. Separation ratios for hake, silver scabbard fish, and horse mackerel were between 96.3 and 100% in terms of weight, and 92.2 and 100% in terms of number in both codends. Shrimp separation was in total calculated as 60.8 and 37.0% by number, and 70.7 and 44.4% by weight in 10 and 15 mm bar spacing trawl grids, respectively, demonstrating that substantial improvement in species selectivity (deep water rose shrimp from others) is possible to achieve by adding sorting grids in the Mediterranean demersal trawl fishery. To optimize the overall selection performance in such fisheries, a variety of grid systems, bar spacings and materials as well as the economic consequences need to be evaluated.     

Signal-to-noise ratio improvements using anti-scatter grids with different object thicknesses and tube voltages

PurposeThe appropriate object thickness to start using anti-scatter grids (grids) has not sufficiently investigated in previous studies, and thus we rigorously investigated the effectiveness of two generally used grids with grid ratios of 6 and 10 (G6 and G10) for different 50–200 mm thicknesses at tube voltages of 60–100 kV.MethodsAcrylic phantoms with 30 × 30 cm² and different thicknesses were used to measure the signal-to-noise ratio improvement factors (SIFs) of grids. To evaluate the infants’ conditions, field sizes of 225, 400, and 625 cm² were also evaluated at 60–80 kV. In addition, the signal difference-to-noise ratio (SDNR) was used to evaluate tube voltage dependencies of grids for each thickness.ResultsSIF values exceeded 1.0 for ≥70 mm thicknesses and mostly exceeded 1.07 for the 100 mm thickness with 400 cm² field size corresponding to a 1-year-old infant abdomen. The estimated dose reduction capabilities for a 1-year-old infant were approximately 15% using G10 at 70 and 80 kV. The tube voltage dependencies for grid use was almost not prominent for all conditions tested, except for some conditions that are not clinically realistic.ConclusionsG6 and G10 can improve SNR for  ≥100 mm thickness. The results from this work demonstrate approximately 15% dose reduction or image quality improvements at the same dose level for the use of G6 and G10 grids for 100 mm thickness, traditionally excluded from the recommended grid use conditions.     

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.     

A leaf disc method for determination of tobacco mosaic virus and potato x-virus in leaves by indirect serological reaction using35s

A method for determination of potato X-virus and tobacco mosaic virus in infected tobacco leaves was tested. The leaves are rubbed with isolated antibodies against homologous viruses and after 30 minutes incubation in a humid chamber at room temperature are washed with fresh distilled water 3 times. The leaves are afterwards rubbed with³⁵S-labeled pig gamma globulin against rabbit gamma globulin. The most convenient radioactivity of the labeled pig gamma globulin was 100 (µCi. Leaf discs 5 mm in diameter are punched out after half an hour incubation and their radioactivity is measured on a 27π window methane flow counter. The leaves from healthy plants are treated in the same way. The virus presence is presumed from the differences of radioactivity between healthy and infected leaves. If the mean radioactivity of the discs from healthy leaves equaled 100, the discs from potato X-virus infected leaves showed a moan value of 382.3%. This method can be used for estimation of virus distribution in the plant, and/or tracing its translocation.