Notes and queries

An instrument for the precise trimming of flat or cylindrical embedded specimens to be sectioned in an ultramicrotome is described. It will produce the required truncated pyramid accurately by hand slicing. A simple modification to the LKB specimen holder allows it to be used with the Reichert ultramicrotome as well.     

Methods for Precisely Trimming Block Faces for Ultramicrotomy

Two devices are described to aid in trimming block faces of embedded tissue for ultramicrotomy. The first, a reticle to fit the ocular of a stereomicroscope, can be manufactured by the ultramicrotomist and is designed to outline the edges of the block face so that it can be trimmed to a standard size and shape with the area of interest centered in it. The second, a rectangular “trim-align” block mounted in the knife holder of the uitramicrotome, is, with the block face, aligned to the plane of sectioning, and aids in retrimming the top and bottom edges of the block face. This is the simplest trimming device yet described and the first which will, from any sort of embedded material, produce a block face with parallel top and bottom edges even if the block face is not perpendicular to the axis of the specimen holder. If the edge of the diamond knife used for sectioning is parallel to the axis of rotation of the knife holder, the block face has also been automatically aligned to the knife as a consequence of this aligning and trimming procedure. As a result, sectioning can begin immediately without further adjustments.     

Grinding Thin Sections of Plastic-Embedded Bone

The method describes an adaptation of a metallurgical procedure whereby dry, calcined bone may be simultaneously infiltrated and embedded in a transparent plastic, Transoptic, and then ground to the desired thinness for microscopic observation with transmitted light. A 2 mm.-thick specimen of bone, ground smooth on one side, is placed ground side up in a 1' mold assembly of a metallurgical specimen mount press. About 5 ml. of the plastic medium is added, the temperature raised to 130° C, and the pressure raised to 100 pounds. When 130° C. is reached, the heater is disconnected, the pressure immediately raised to 3500 pounds and maintained at that level until the mold cools to 68° C. The pressure is then released and the 5 mm.-thick plastic disc, with the embedded specimen therein, expressed from the mold. Grinding, as well as polishing, is dry; abrasives in fluid media are not used. The disc and specimen are coarse ground on #340 grit dry silicon carbide paper until histological details begin to appear. Final fine grinding is done on #600 grit dry silicon carbide paper. The disc is then polished and may be mounted on a 1 × 3 Plexiglas slide.     

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.     

Monitoring of Epoxy Block Trimming by the Use of a Laterally Mounted Mirror

Mounting a small mirror on the side of the knife holder of an ultramicrotome permits the monitoring of selected areas during epoxy block trimming without removing the block from the chuck. This technique is particularly useful for trimming toward preselected areas of in situ embedded cell cultures for subsequent sectioning. When the visibility of the embedded cells is improved by staining with Paragon-1301 or toluidine blue before embedment, such mirror monitoring can be carried out at magnifications up to 40 times during the trimming procedure.     

An Instrument for the Controlled Trimming of Plastic Specimen Blocks for Light and Electron Microscopy

A device for the controlled trimming of plastic specimen blocks for light and electron microscopy is described. Many advantages of previously reported instruments together with 1) a rack and pinion control of knife movement, and 2) a control of rotation of the specimen block at 90°, 180°, 270°, or 360° are incorporated. In conjunction with an ocular micrometer, the device allows accurate removal of thin slices during trimming.     

Improved technique for electron microscopy of cultured cells.

A number of techniques are presented which precise selection and efficient preparation of individual cultured cells for electron microscopy. Techniques described include marking of the living and embedded cells, drilling and mounting cores of embedded material, and improved technique for marking of the selected area on the block face before trimming.     

Tenascin in the human intervertebral disc: alterations with aging and disc degeneration

Our objective for this study was to determine the presence and distribution of tenascin in the human intervertebral disc. The tenascins are a family of extracellular matrix proteins with repeated structural domains homologous to epidermal growth factor, fibronectin type III and the fibrinogens. Little is known about the presence of this protein in the disc. Ten normal human discs donated from subjects newborn to 15 years old, 10 control discs from adult donors aged 24-41 years, and 11 surgical disc specimens from patients aged 26-76 years were examined for immunolocalization of tenascin. In young discs, tenascin was localized throughout the annulus; in the nucleus, localization was confined to pericellular matrix. In adult control and degenerating disc specimens, tenascin in the annulus was localized primarily in pericellular matrix regions encircling either single cells or clusters of disc cells; in rare instances localization was more diffuse in the intraterritorial matrix. In young, healthy disc, tenascin was abundant throughout the annulus. In contrast, degenerating discs in adults showed a localization restricted to the pericellular, and rarely, more restricted intraterritorial matrix. These observations indicate that changes in the amount and distribution of tenascin may have a role in disc aging and degeneration, possibly by modulating fibronectin-disc-cell interactions, and causing alterations in the shape of disc cells.     

A Rigid Illuminator Giving Transmitted Light to Facilitate Microhardness Testing of Calcified Tissues

The structural form of calcified tissues necessitates their examination with transmitted light to identify various morphological features which are not evident with incident illumination alone. To achieve stability for indentation, the specimens must be rigidly mounted. A Leitz Model 514095 base illuminator was fitted with a bottom plate of 3 mm brass for attaching it to the graduated stage and a perforated, 3 mm thick brass top plate, surmounted by a 6 mm thick piece of plate glass. The 25 mm hole in the top plate coincided with a ground glass disc to transmit diffused light through the plate glass to the specimen. Thus, the specimen could be examined on a rigid microscope stage, morphological features identified, and subsequent interpretation of indents made with the usual incident illumination.     

Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening

Summary The protein-based technologies used to screen newborns for sickle cell disease require confirmation with a liquid blood specimen. We have developed a strategy for rapid and specific genotypic diagnosis using DNA extracted from a dried blood spot on the filter paper blotter used to screen newborns. DNA could be microextracted from a specimen as small as a 1/8 inch diameter punched disc representing the dried equivalent of approximately 3 μl of whole blood. We utilized the DNA from a 1/4 inch diameter specimen (12 μl equivalent) for polymerase chain reaction amplification of the β-globin region spanning the sickle cell mutation with detection by allele-specific oligonucleotide probes. Molecular confirmation of genotype from the original blotter would reduce the personnel costs associated with obtaining follow-up liquid blood specimens and would provide information to the family in a more timely and less equivocal manner.     

Improved Methods for Molding, Facing and Sectioning Glycol Methacrylate Embedded Tissue Blocks

Improvements in glycol methacrylate embedding, block facing, trimming, and sectioning are described. The improvements are derived from a novel molding system, a multipurpose instrument for rapid block facing, trimming and examination, and a device for removing unwanted sections from the microtome knife while sectioning is in progress. Together, these methods facilitate specimen preparation and result in a significant reduction of the time required to prepare high resolution, very thin sections for light microscopy.     

The preparation of microtome sections of unaltered soil for the study of soil organisms in situ

Summary A new technique for study of small soil organisms in situ in unaltered soil is described.The soil samples are cooled in a refrigerator at — 10°C to kill the animals. A small portion taken from a frozen soil sample, is slowly immersed in a solution of gelatin. When the specimen is infiltrated with gelatin and the whole cooled it is fixed in formalin to enable it to withstand treatment with hydro-fluoric acid for removal of sand grains. Subsequently the specimens are immersed in gelatin solution for a second time after which the specimens are affixed to wooden blocks which can be clamped in the microtome. Before sectioning, the embedded specimen affixed to the wooden block is hardened in methylalcohol after which it is possible to cut sections 7,5–10µ thick.The most satisfactory staining procedure proved to be the quadruple staining method of Johansen. By this method nematodes, fungi, bacteria and amoebae are easily distinguishable from the soil particles.     

A New Preparation Method For Several Histopathological Examinations On A Single Block

A new method of specimen preparation is described permitting several studies such as routine staining, histochemistry, enzyme histochemistry, immunohistochemistry, and electron microscopy on a single block of biopsy specimens. Tissues are immersed in the fixative, which primarily stabilizes carbohydrate moieties, and embedded in the mixture of JB-4, methylmethacrylate and divinylbenzcne. The resin is polymerized at 4 C Thin sections (1-2 microns) are obtained with a sliding microtome, and ultrathin sections (60-90 millimicrons) with a ultramicrotome. The sections are stained directly with various conventional procedures without removing the embedding resin. This preparation method offers a potentially useful tool for histopathological studies on biopsy specimens.     

A new preparation method for several histopathological examinations on a single block.

A new method of specimen preparation is described permitting several studies such as routine staining, histochemistry, enzyme histochemistry, immunohistochemistry, and electron microscopy on a single block of biopsy specimens. Tissues are immersed in the fixative, which primarily stabilizes carbohydrate moieties, and embedded in the mixture of JB-4, methylmethacrylate and divinylbenzene. The resin is polymerized at 4 C. Thin sections (1-2 microns) are obtained with a sliding microtome, and ultrathin sections (60-90 millimicrons) with a ultramicrotome. The sections are stained directly with various conventional procedures without removing the embedding resin. This preparation method offers a potentially useful tool for histopathological studies on biopsy specimens.     

植物模式标本数据整合新的机遇与挑战

命名模式是分类群名称永久依附的成分,在分类学研究中有不可替代的价值。中国复杂的植物采集历史以及对中国植物标本开展研究的单位各异,导致我国植物模式标本零散分布于全球各大标本馆,给分类工作的开展带来了极大的困难。标本数字化的开展为模式标本数据整合提供了新的机遇,同时也给我们带来了人名和地名标准化以及模式类型确认等方面的挑战。我国于2006年开始对模式标本数据进行收集和整理,迄今已完成国内外20余家标本馆9万余条标本数据的收集。模式考证和模式类型清理是我们下一步亟须开展的工作,同时我们应将地名变更资料、人名考证资料进行整合并建立相应的数据库以推动模式标本数据的标准化。这将在很大程度上有助于我们摸清中国模式标本的家底。     

Stain Historadiography

Fresh bone specimens were dehydrated in acetone and embedded in Ward's Bio-Plastic. Sections 10-15μ thick were cut with a special bone-cutting microtome and subjected first to radiography and then stained with safranin-fast green. The radiograph was made with a Machlett AEG-50-A X-ray tube on a fine-grained, spectroscopic plate; which, after processing, was mounted (dry) on a glass slide adjacent to the stained specimen. By these means, it was possible to make a correlated study of the calcium-bearing parts of the tissue and determine accurately their relationship to the bone itself. The method serves well for investigations of growing bone and for bone that is undergoing pathological involution. It has been named “Stain Historadiography” because it combines a stained specimen with its radiograph.     

The influence of specimen length on the tensile failure properties of tendon collagen

Connective tissues such as ligament, tendon and skin are composites of strength-bearing collagen fibers embedded in a hydrated matrix. The tensile response and failure properties of rat-tail tendon are thought to represent those of the collagen fiber itself. In this study, the tensile failure properties of rat-tail tendon (tendon collagen) were determined for specimens of various test length. The experimental results indicated that failure strain, based on the test grip-to-grip dimension, and failure strain energy density decreased as specimen length increased. The failure stress, on the other hand, did not change appreciably with specimen length. Thus, tensile failure data cannot simply be normalized by the grip-to-grip length of the test specimen. Experimental data from various laboratories must clearly document the length of the test specimen.     

Scanning electron microscopy of plasma etched implant specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 microns histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using epipolarization microscopy.

We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.     

Scanning Electron Microscopy of Plasma Etched Implant Specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 µl;m histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.