Preparing Single or Serial Sections of Calcified Bones and Teeth

An apparatus for cutting single or serial sections of calcified bone and teeth consists of a motor-driven shaft on which is mounted one saw (for single section cutting) or a gang (for serial sectioning at one cutting operation). The plastic-embedded specimen is attached to a cylindrical plastic holder which is in turn mounted on the machine and fed into the saw. Prior to cutting the specimen may be oriented in two planes, as well as rotated, with respect to the cutting edge. Single or serial sections made by means of repeated cuts with a single saw, may be 0.3 mm or more thick as determined by the setting of a micrometer screw. For serially sectioning a tooth or bone specimen at one cutting operation, the thickness of the separators between adjacent saws (0.5 mm or more) determines the section thickness. After sectioning, specimens may be ground and polished, with or without reimbedding in fresh plastic.     

Automatic grinding of undecalcified bone sections with exact adjustment of thickness]

A new grinding machine for preparing thin undecalcified bone sections after methylmethacrylate embedding is described. About 20 rather small bone sections can be ground at the same time; bigger specimens, up to 8 cm of length, are allowed. Bone sections are mounted on a cylindrical specimen holder by an adhesive film. Then the final thickness of the sections is exactly adjusted by screwing three rubies out of the holder's bottom. Now the prepared holder is set in a guide ring on a turntable carrying a rough ended glass plate. The desired thickness of the sections is reached as soon as the three rubies touch the glass surface. The variation in the thickness of the sections is less than +/- 3 micron. The machine is simply constructed, easily to handle and rapidly to clean.     

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     

X-ray microtomography of biological tissues using laboratory and synchrotron sources

X-ray microradiography is a well established technique for the study of biological structures in which the projected absorption is measured, usually with photographic film or resist. If scanning X-ray microradiography with a 15-μm beam, 2-D scanning, and photon counting is used, more accurate results can be obtained and real-time experiments undertaken. Addition of a rotation axis allows computerized axial tomography to be done at a resolution of 15 μm. This technique overcomes the inherent difficulty of microradiography that all detail perpendicular to the plane of the specimen is superimposed. This method has been applied to the study of the 3-D mineral distribution in a 0.8×0.8 mm column of human cortical bone with a laboratory X-ray source. Calculation of the wavelength dependence of the linear absorption coefficient for liver and bone shows that, for a choice of wavelength in the range of 3–0.4 Å (4–30 keV), the specimen thickness can be from 100μm–2 cm and 10 μm–3 mm, respectively. Synchrotron X-radiation has the potential for better resolution because of the higher intensity, which allows the use of a narrower beam. There is also the possibility of determining individual element 3-D distributions from measurements on either side of the absorption edges because of the continuous nature of the spectrum and also the possibility of doing this from X-ray fluorescence measurements. To investigate these possibilities, a tomographic apparatus has been built based on the availability of accurately ground, tungsten carbide balls. Metrological assessment shows that the specimen remains within <1 μm of the required position during translation and rotation. Preliminary X-ray tomographic studies with a 4-μm diameter beam have been started at the Daresbury laboratory synchrotron source.     

Preparing Bone Sections for Radioautography

The method eliminates all contact of bone with aqueous solutions. One hundred to 200µ sections are first cut with a guided circular saw. These sections are simultaneously embedded and mounted on glass slides by means of a clear thermoplastic cement, Gelva. The mounted bone sections are then ground to 5-25µ on fine silicon carbide paper. The sections are finally polished with levigated alumina on a cloth pad. A procedure for preparing contact radioautograms of ground bone sections is given and the results obtained are illustrated.     

Distortion-Free Mounting of Frozen Sections by Handling on Paper Supports

Frozen sections are cut from the specimen until the level of interest is reached. A strip of paper (bond or similar writing paper) 5 cm long and about 1 cm wider than the specimen is moistened with water, closely applied to the surface of the specimen and frozen onto it. As the section is cut, the end of the paper strip above the knife is grasped and turned backward toward the other end of the strip. The section is then applied to an albumenized glass slide, firmed and thawed by finger pressure, and the paper removed. After thorough drying, the preparation is ready for further processing. When properly performed, mounted sections whose details coincide to those of the uncut block can be obtained. If thawing on the knife is prevented by cooling the knife, the technic can be performed without a cryostat, but it is also feasible to use a cryostat if a favorable temperature is maintained. The authors obtained 30 μ serial sections, suitable for stereotaxic mapping, from rabbit brain by this method.     

Sectioning of Undecalcified Bone; with Special Reference to Radioautographic Applications

A rapid technic for the preparation of 6 μ serial sections of undecalcified bone is described. The specimen is fixed and dehydrated in acetone and ether. It is then treated with a 1:1 mixture of the monomers of ethyl and n-butyl methacrylate catalyzed with benzoyl peroxide. The monomers are removed with ether and the ether is removed under vacuum. Infiltration is accomplished under vacuum using a partial polymer of the same mixture of monomers. Polymerization is completed in 36 hours under pressure at 50°C. The tissue is sectioned with a heavy-duty microtome, the sections are mounted on glass slides and nuclear emulsions applied. Young and adult bone of laboratory animals and man have been cut successfully. Microscopic structural detail is preserved and there is no evidence of translocation of the radioactivity.     

Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 μm, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

A technique for section thickness evaluation for microphotometry and image analysis of sectioned nuclei.

The exact knowledge of the section thickness is a requisite for making the necessary corrections on DNA measurements in tissue sections. Several methods have been proposed to evaluate section thickness, each of them with advantages and disadvantages depending on the type of specimen and equipment available. We herein report another method based on preparation of standard material whose optical density varies as a function of its thickness and is sectioned and measured alongside the tissue specimen. The standards consist of celloidin cylinders stained with the PAS reaction and embedded in paraffin. For prior characterization of the cylinders, sections of different thickness were obtained and mounted. The optical density of each section was measured by direct microphotometry or image analysis. The actual thickness of each section was evaluated following re-embedding of piled groups of sections in a paraffin block and transversal sectioning. The thickness was then measured with a micrometric eye-piece. Optical density and actual thickness of each section were plotted on a normogram curve. Once a given tissue is sectioned alongside with the reference cylinder, the actual thickness is determined by its optical density on the normogram curve.     

Cement line staining in undecalcified thin sections of cortical bone

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

The Membrane Method of Electron Microscope Autoradiography

Thin sections of methacrylate and Araldite embedded tissues labelled with radioactive isotopes were transferred with a wire loop or brush from the knife edge onto thin formvar membranes which covered 7 mm holes in 76 × 25 × 1.5 mm or 76 × 38 × 1.5 mm plastic slides. To facilitate the mounting of sections, a platform supported the plastic slides close to the ultramicrotome knife. Photographic emulsion diluted 1:5 or 1:10 with water was applied with a pipette to the upper surface of each formvar membrane to cover the mounted sections. Excess emulsion was drained off and the remaining thin film was dried on a warm plate at 45 C to produce a uniform layer over the sections. After storing in the dark for several weeks, preparations were processed in photographic solutions and washed, and sometimes stained, before applying electron microscope grids to the underside of each formvar membrane. To detach each grid with its adherent formvar, section and emulsion, the membrane was pierced around the perimeter of the grid. Grain counts made over nuclei of cells labelled with tritiated thymidine indicate that emulsion is uniformly distributed over each section and that quantitative comparison is possible between labelled areas.     

A simplified procedure for preparation of undecalcified human bone sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 micron +/- 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.     

A Simplified Procedure for Preparation of Undecalcified Human Bone Sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 μm ± 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.     

Diametral compression of non-circular diaphyseal bone sections

Many research endeavors involve strength testing of long bones, frequently using whole-bone four-point bending models. Recently, diametral compression of short sections has been used to quantify local mechanical parameters and effects of treatment, but testing of biologically derived samples entails a number of added complications, such as the non-circularity of bone sections, ambiguity of load orientation during testing, thickness variation in a section, and size and shape variation between sections in a single sample. In order to quantify the effects of these confounding factors, finite element diametral compression models of a number of bone sections were compared with simplified circular and elliptical sections. Each anatomic section was tested in all rotationally stable load configurations. A high degree of correlation was observed between the anatomic sections and their circular and elliptic analogs, indicating that meaningful comparisons may be made between bone sections of disparate geometry. The aspect ratio and shape of the bone sections did not have a significant impact on the maximum in-plane principal stresses, whereas stresses were strongly dependant on the mean thickness and spatial thickness variation. Some variation due to load orientation was observed. These results indicate that diametral ring compression testing of anatomic sections can be used effectively to measure structural and material parameters of long bones, and that anatomic variation can be successfully accommodated. The ability to use diametral compression testing should allow researchers to obtain many more samples from each specimen than whole-bone bending without the difficulty of extracting solid core or dog-bone samples.     

An Abrasive Paper Technique for Preparing Sections of Wood for Microscopic Study

Dry wood specimens are sawed to 2mm thickness and impregnated with resin such as Lewisol 28 (Hercules Powder Co.). One side of the specimen is ground by hand on abrasive papers of grades #100, #180, #240, and #320. This side is then cemented to a petrographic glass slide with stick shellac and the other side similarly ground. Scratches can be eliminated by scraping the ground surface with the sharp edge of an ordinary glass microscopic slide. The section is removed by heating the slide, dissolving the shellac with alcohol and the resin of the embedding matrix with xylene. The sections can be stained in a hot saturated alcoholic solution of safranin O, counterstained with 0.01% fast green in an equal parts mixture of clove oil, methyl cellosolve, and absolute alcohol, and mounted in balsam.     

Preparation of Thin Undecalcified Bone Sections by Rapid Manual Method

Sections from 3 μ to over 100 μ thick of fresh, unfixed, unembedded, unde-calcified and undehydrated bone are made by grinding 1 to 2 mm slabs of the desired orientation on waterproof carborundum abrasive paper, grit No. 320, 360 or 400. The manner of controlling the section is the crux of the technique. The section is held by wrapping a fresh strip of sandpaper around a 3' × 1' slide and accomplishing the grinding on a used piece of paper. The abrasive points on the fresh paper effectively prevent the section from sliding off the slide. The specimen is kept wet with water during the entire procedure. Sections are then stained, and excess surface stain can be ground off in similar fashion. After washing in dilute detergent solution to remove adherent derbis, the section is air dried and mounted in any nonacidifying resinous media. The method is suitable for wood and for fruit pits also.     

Preparation of Thin Undecalcified Bone Sections by Rapid Manual Method

Sections from 3 μ to over 100 μ thick of fresh, unfixed, unembedded, unde-calcified and undehydrated bone are made by grinding 1 to 2 mm slabs of the desired orientation on waterproof carborundum abrasive paper, grit No. 320, 360 or 400. The manner of controlling the section is the crux of the technique. The section is held by wrapping a fresh strip of sandpaper around a 3″ × 1″ slide and accomplishing the grinding on a used piece of paper. The abrasive points on the fresh paper effectively prevent the section from sliding off the slide. The specimen is kept wet with water during the entire procedure. Sections are then stained, and excess surface stain can be ground off in similar fashion. After washing in dilute detergent solution to remove adherent derbis, the section is air dried and mounted in any nonacidifying resinous media. The method is suitable for wood and for fruit pits also.