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.     

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.     

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 × 25 mm diameter) arc scored 0.4 mm deep with a razor. Flexing the discs opens the scorns 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.     

A glass drinking tube for small birds and mammals

A drinking tube was fabricated with a piece of 8 or 10 mm O.D. glass tubing. It gives accurate measurement of the volume of liquid consumed by small birds and mammals. The tube can be readily cleaned and autoclaved.     

A simple instrument for achieving accurate alignment and pre-insertion stability with mandibular bone plates

The modified Dingman forceps provides several advantages over currently designed plate-holding forceps. The rubber tubing prevents metal-to-metal contact of the forceps on the plate and provides increased stability to the plate. The long lingual jaws of the Dingman forceps are applied to the lingual bone at a point remote from the screw canal, and this prevents damage to the prepared canals. The design of the forceps' jaws provides three-point rigidity to stabilize the plate and, therefore, ensures proper alignment, adaptation, and pre-insertion fixation.     

Metabolism of hybridoma cells and antibody secretion at high cell densities in dialysis tubing.

The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter [10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness] inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation. The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily. The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks. The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions. Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities. In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Silicone-tubing aerated bioreactors for somatic embryo production

Bioreactors equipped with silicone tubings for bubble free oxygen supply are suitable for culture of embryogenic cell suspensions. The advantages of bubble free aeration systems over various devices for dispersion of air bubbles are the lack of foam formation and the possibilities of precise control of the desired oxygen set point. The specification of silicone tubing (length, diameter, wall thickness) has to be adapted according to the amount of embryogenic biomass to be produced in the bioreactor. Cell suspensions of Euphorbia pulcherrima were cultured --2 l bioreactor at 60% pO₂, supplied by a silicone tubing system of 155 cm length, 4.0 mm diameter and 0.4 mm wall thickness. The oxygen concentration decreased when the packed cell volume exceeded 14% (=3.7 g l^-1 cell dry weight), indicating the upper limit of oxygen supply by the silicone tubing. Mathematical considerations for membrane aerated bioreactors are presented with the intention of enabling a more precise definition for the configuration of silicone tube systems in different bioreactor types.     

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.     

Your “Third Hand” in Mounting Serial Sections on Grids for Electron Microscopy

A heavy base 12 cm in diameter supports a vertical rod 40 × 3 cm. To this rod is clamped a horizontal rod 24 × 1.5 an, which bears on its outer end a rack-and-pinion mechanism that carries a clamp. Slow motion can be imparted to the clamp by a knurled pinion knob. For use, a grid is grasped at its edge by a watchmaker's forceps, and the forceps secured in the clamp. The rack and pinion is adjusted to give motion at about 45° and thus allow the grid to be lowered at an angle into the flotation water. While observed under magnification, the ribbon of sections can be manipulated into proper alignment with the grid, the end section fastened to the grid, and the grid raised mechanically to cause the remainder of the ribbon to adhere to it.     

An Improved Method of Making Tissue Carriers for TEM and SEM Fixation

Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

A simple apparatus for sampling epiphytic communities associated with emergent macrophytes

The apparatus allows the sampling of both algal and animal components of the epiphyton. It consists of a perspex tube, 100 cm long with an internal diameter of 4 cm, and of an epiphytic sieve. The latter is made of two 2.5 cm cross-sections of perspex tubing with a piece of circular mesh glued between them. While sampling, a randomly chosen shoot is isolated from the ambient water by means of the tube, cut at the bottom sediment and removed together with epiphyton. The water, taken together with the shoot is then removed using the epiphytic sieve, joined with the tube under the water surface. The detached organisms, associated with the stem and those scraped from the stem with a stiff brush afterwards, concentrate on the mesh.     

A PLastic MIcrostrainer for HAndling MAmmalian BLastocysts

Friend (1963) has described a microstrainer for handling microscopic marine ova which permits direct transfer of batches of ova through various histological solutions without centrifugation or decanting. This strainer consists of a length of glass tubing with an electron microscope grid cemented over one end. In our laboratory, several modifications of this device have been made for use in handling mammalian ova and blastocysts. The most satisfactory model, which can be made easily, was contructed from BEEM plastic capsules (Better Equipment for Electron Microscopy, Inc., P.O. Box 132 Jerome Avenue Station, Bronx, New York 10468) and circular electron microscope grids of suitable mesh size.     

A Plexiglas Isolation-Transfer Chamber for Use with the Fonbrune Micromanipulator

The chamber is made with two pieces of clear Plexiglas, both 1 inch wide and 3 inches long; the top piece, 1/16 inch thick; the bottom, 1/4 inch. A recess 14 mm wide, 37 mm long and 5 mm deep is cut into the bottom piece leaving a margin 1.5 mm wide, this margin is then cut down to a height of 2.5 mm for the length of the recess; a piece of moistened filter paper I1/2 inches long and 5 mm deep is attached to the rear wall, leaving the bottom clear for the transmitted light; two notches 13 × 15 mm and 12 mm apart are cut from the top piece. The top is superimposed on the bottom and held by two screws to form a chamber that is accessible to microdissection instruments through its open side. Two cover glasses, one bearing the specimen and the other, the medium to which the transfer is to be made, are placed over the notches in the top (agar side down). The actual transfer of material is made by shifting the position of the cover glasses with the mechanical stage of the microscope while the specimen is held by the micromanipulator.     

A Mechanical Device for Retrieving Ribbons of Ultrathin Sections Without Folds

An improved version of a mechanical device for retrieving ribbons of ultrathin sections free of wrinkles is described. The device is simple and easy to use and can be used by both right- and left-handed operators. A grid is grasped by forceps which are held in position by a magnet in such a way that it can be lowered into the water well behind the floating sections. After a ribbon has been properly aligned, moved to the grid and fastened by an end-section, the grid with the sections is slowly drawn up and out of the water with a rack-and-pinion mechanism.     

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 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 Plexiglas Isolation-Transfer Chamber for Use with the Fonbrune Micromanipulator

The chamber is made with two pieces of clear Plexiglas, both 1 inch wide and 3 inches long; the top piece, 1/16 inch thick; the bottom, 1/4 inch. A recess 14 mm wide, 37 mm long and 5 mm deep is cut into the bottom piece leaving a margin 1.5 mm wide, this margin is then cut down to a height of 2.5 mm for the length of the recess; a piece of moistened filter paper I1/2 inches long and 5 mm deep is attached to the rear wall, leaving the bottom clear for the transmitted light; two notches 13 × 15 mm and 12 mm apart are cut from the top piece. The top is superimposed on the bottom and held by two screws to form a chamber that is accessible to microdissection instruments through its open side. Two cover glasses, one bearing the specimen and the other, the medium to which the transfer is to be made, are placed over the notches in the top (agar side down). The actual transfer of material is made by shifting the position of the cover glasses with the mechanical stage of the microscope while the specimen is held by the micromanipulator.     

Items from the Literature

Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

Porohyperelastic finite element modeling of abdominal aortic aneurysms

Abdominal aortic aneurysm (AAA) is the gradual weakening and dilation of the infrarenal aorta. This disease is progressive, asymptomatic, and can eventually lead to rupture--a catastrophic event leading to massive internal bleeding and possibly death. The mechanical environment present in AAA is currently thought to be important in disease initiation, progression, and diagnosis. In this study, we utilize porohyperelastic (PHE) finite element models (FEMs) to investigate how such modeling can be used to better understand the local biomechanical environment in AAA. A 3D hypothetical AAA was constructed with a preferential anterior bulge assuming both the intraluminal thrombus (ILT) and the AAA wall act as porous materials. A parametric study was performed to investigate how physiologically meaningful variations in AAA wall and ILT hydraulic permeabilities affect luminal interstitial fluid velocities and wall stresses within an AAA. A corresponding hyperelastic (HE) simulation was also run in order to be able to compare stress values between PHE and HE simulations. The effect of AAA size on local interstitial fluid velocity was also investigated by simulating maximum diameters (5.5 cm, 4.5 cm, and 3.5 cm) at the baseline values of ILT and AAA wall permeability. Finally, a cyclic PHE simulation was utilized to study the variation in local fluid velocities as a result of a physiologic pulsatile blood pressure. While the ILT hydraulic permeability was found to have minimal affect on interstitial velocities, our simulations demonstrated a 28% increase and a 20% decrease in luminal interstitial fluid velocity as a result of a 1 standard deviation increase and decrease in AAA wall hydraulic permeability, respectively. Peak interstitial velocities in all simulations occurred on the luminal surface adjacent to the region of maximum diameter. These values increased with increasing AAA size. PHE simulations resulted in 19.4%, 40.1%, and 81.0% increases in peak maximum principal wall stresses in comparison to HE simulations for maximum diameters of 35 mm, 45 mm, and 55 mm, respectively. The pulsatile AAA PHE FEM demonstrated a complex interstitial fluid velocity field the direction of which alternated in to and out of the luminal layer of the ILT. The biomechanical environment within both the aneurysmal wall and the ILT is involved in AAA pathogenesis and rupture. Assuming these tissues to be porohyperelastic materials may provide additional insight into the complex solid and fluid forces acting on the cells responsible for aneurysmal remodeling and weakening.