Modification of the Carmen-Faull-Pullar Racks for Free-Floating Serial Sections

The original staining racks designed by Carmen et al. (Stain Techn., 43: 157-60) have been redesigned to use inside plates of 0.06 inch thickness and outside (top and bottom) ones of 3/16 inch. The greater thicknesses permit freer circulation about the sections and avoid the need for outer clamps to hold the assembly together. As in the original racks acrylic sheeting has been used, but with 25 × 45 mm holes for sections in each plate. Ordinary fiberglass window screening was cemented to one side of each plate. The assembly of 12 inner and the 2 outer plates was held together by 2 bolts made of 1/4 inch acrylic rod. Since clamps on the edges of the assembly were not needed, smaller staining dishes could be used, with coincident economy in volume of staining solutions.     

Success of a low‐sloping rack for improving downstream passage of silver eels at a hydroelectric plant

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A Staining Rack for Handling Cover-Glass Preparations

A porcelain staining rack (Fig. 1) has been devised for handling cover-glass preparations. The design is along the general lines of staining racks for slides commonly sold by commercial houses. It consists of three parallel rods—one at the bottom, two on the sides. These three rods are held together by two end pieces. Each of the rods has twelve slots, thus the rack holds twelve cover glasses. The slots have been arranged to hold the cover glasses some distance apart. Circular cover glasses having a diameter of 22 mm. are most desirable. Each of the two end pieces has a hole near the top for inserting one end of the wire tongs or handle (see Fig. 1) which is used for removing the rack from the stender dish. The staining rack is 40 mm. long and 35 mm. high and is designed, as shown in the figure, to fit the ordinary Stender dishes commonly used in many laboratories.     

The Barrnett-Seligman Sulfhydryl Reaction for Plant Meristems

Onion root tip meristems, fixed in 14 different fixatives representing ingredients and mixtures used in plant cytology, were tested with the Barrnett and Seligman histochemical procedure for protein-bound sulfhydryl groups. The relative intensity of staining was measured photometrically and the distribution of stain after each type of fixation described. Measurements indicated that conditions governing the staining of SH and S—S are not fully predictable; for example, fixation in saturated HgCl₂ enhances staining although inhibition was expected through mercaptide formation. Specificity of the reaction was further checked by treating fixed sections with known SH reagents. Partial blocking by such reagents as p-chloromercuribenzoate, and N-ethyl maleimide is apparently reversed by lengthy incubation in the 2,2'-dihydroxy-6,6-dinaphthyl-disulfide (DDD) reagent. Sulfhydryl oxidizing agents such as I-KI or chromic acid were either ineffective in blocking or could be reversed. For this reason and because previously reduced sections were proportionately better blocked than untreated ones it is suggested that the sulfhydryl reagent may open and then react with S—S bonds. Parallel runs indicate no difference in specificity between animal and plant tissues.     

A Staining Rack for Handling Cover-Glass Preparations

A porcelain staining rack (Fig. 1) has been devised for handling cover-glass preparations. The design is along the general lines of staining racks for slides commonly sold by commercial houses. It consists of three parallel rods—one at the bottom, two on the sides. These three rods are held together by two end pieces. Each of the rods has twelve slots, thus the rack holds twelve cover glasses. The slots have been arranged to hold the cover glasses some distance apart. Circular cover glasses having a diameter of 22 mm. are most desirable. Each of the two end pieces has a hole near the top for inserting one end of the wire tongs or handle (see Fig. 1) which is used for removing the rack from the stender dish. The staining rack is 40 mm. long and 35 mm. high and is designed, as shown in the figure, to fit the ordinary Stender dishes commonly used in many laboratories.     

Apparatus for Bone Sectioning

A new type of apparatus for cutting 100-200µ sections of bone is described. This apparatus consists of an especially designed bone vise and a mounted circular saw with three directional movement controls. The saw is driven by one D.C. motor and its vertical movement assembly is driven by another. Their speeds can be regulated individually. The bone sections may be made serially at intervals of 0.3 to 0.5 mm. and bones as large as 2 cm. in diameter or longitudinal slabs of larger bones may be sectioned.     

The Production of Large, Epoxy-Embedded, 50 μ Sections by Precision Sawing; A Preliminary to Survey for Ultrathin Sectioning

Tissue slices not more than 4 mm thick—including those containing mineralized bone—were fixed in buffered 6.25% glutaraldehyde. Parts of the specimen to be used for subsequent electron microscopy had to be within 1 mm of the surface to permit proper penetration of the second fixation in 1% OsO₄, therefore, if such parts were not originally so exposed, superfluous tissue was dissected away. After fixation and washing, a regular dehydration and embedding sequence was used, but with the time in individual fluids tripled, and 2 changes of absolute ethanol and 2 of undiluted resin added to insure thorough permeation. The resin mixture (containing Epon 812) was the hardest one normally used for ultra-thin sectioning. The blocks were cut at 50 μ on a Gillings-Bronwill thin-sectioning, hardtissue saw, which had been modified by the addition of supplementary water jets for cooling the diamond blade and the tissue block. Sections were examined microscopically in glycerol; a selected area was punched out with a 1 mm diameter leather punch, and the resulting disk was cemented to a preformed blank casting adapted to the chuck of the ultramicrotome. Tissue specimens presenting surfaces of 1.5 × 3 cm can be surveyed rapidly with this method. The use of a punch to obtain the disks for ultramicrotomy leaves the rest of the section undamaged.     

Differential Impregnation of Degenerating Nerve Fibers in Paraffin-Embedded Material

The silver method of Nauta and Gygax (1951) has been used on paraffin embedded material to give a result closely comparable to that obtained by Nauta and Gygax (1954) on frozen sections. It has been found that pyridine plays an important part in suppressing the impregnation of normal fibers in paraffin embedded material and that this sup pression can be augmented by the use of some of the higher methylated derivatives of pyridine, particularly 2,4,6-trimethyl pyridine (collidine).     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

Preparation of large temporomandibular joint specimens for pathological study

Ten fresh temporomandibular joint (TMJ) specimens about 5 X 4.5 X 3.5 cm in size were removed at autopsy by 5 cuts according to appropriate anatomical landmarks. After routine formalin fixation, the whole-TMJ specimens were wrapped with a thin layer of self-curing resin and then cut with a low speed bone saw along the parasagittal plane predetermined by x-ray guidance. Each specimen was serially cut into 4 to 5 parallel slices of 3 mm thickness, which were then decalcified with 14% EDTA and embedded in paraffin. Histological sections of 5 microns were stained with hematoxylin and eosin. The procedures were accomplished within 20 to 24 days after autopsy. With this technique, the anatomical interrelationships among the various joint components could be maintained and the macroscopic and microscopic topography of the TMJ could be studied in the desired reference plane. Therefore, the corresponding changes among the joint components in a diseased TMJ could be thoroughly examined. This technique was also applicable for the study of large specimens containing both hard and soft tissues.     

Length-weight relationships of six fish species from the Xiangjiang River in Guangxi region,China

This study aimed to estimate the parameters of length-weight relationships (LWRs) for six freshwater fish species that inhabit in the Xiangjiang River of Guangxi region, China. The specimens were sampled in April, July, and October 2014 and in January 2015 using electrofishing (CWB-2000P, China; 12 V import, 250 V export) and traps (mesh size 8.5 mm; length 200 m) at five sampling sites. For each sampling site, a trap was settled on one nearshore zone of the river for 24 hr, and the electrofishing was applied within 30 min from downstream to upstream with 200 m long. Standard length was measured (be accurate to 0.1 cm) and total weight determined (be accurate to 0.1 g) after preservation in formaldehyde (10%). The values for the parameter b of the length-weight relationships were estimated together with the coefficient of determination scores. In addition, this study provides a new tentative LWRs for the six species.     

Formic Acid Corrosion for the Demonstration of Pulmonary Elastic Tissue

By a revised technique, human pulmonary elastic tissue can be isolated in a form suitable for examination under the stereoscopic microscope. Fresh human lungs from autopsy are fixed by intrabronchial infusion with 10% formalin for 24 hr. Slabs 1.5 cm thick are cut and the formalin removed in running water. One such slab is embedded under intermittent vacuum in an aqueous mixture containing 15% gelatin, 10% glycerol, and 1% phenol; then allowed to gel. Frozen sections 2 mm thick are cut on a large-section MSE sledge microtome. Squares 3 × 3 cm from such a section are corroded for 4-5 days in 88% formic acid at 45 C, washed once with distilled water, and mounted in glychrogel containing 6% gelatin. The elastic tissue network of the lung will have been freed from surrounding elements. The preparation should be stored in a refrigerator. Blocks for thin sections and large thick un-corroded sections can be prepared from the same lung as part of an over-all procedure.     

Histoautoradiographic Localization of Calcium in Oat Plant Tissues

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

Preparation of Large Epoxy Sections for Light Microscopy as an Adjunct to Fine-Structure Studies

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 5% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and soaked in 0.1 phosphate-buffer (pH 7.3) for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO₄. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 1-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphthalate to the standard epoxy mixture. The sections were spread on warm 1% gelatin and attached to glass slides by drying, baking at 60 C, fixing in 10% formalin or 5% glutaraldehyde and baking again. Sections were mordanted in 5% KMnO₄ (5 min), bleached with 5% oxalic acid (5 min) and neutralized in 1% Li₂CO₃ (1 min). Several stains could then be applied: azure B, toluidine blue, azure B-malachite green, Stirling's gentian violet, MacCallum's stain (modified), tribasic stain (modified) and phosphotungstic acid-hematoxylin. Nuclei, mitochondria, specific granules, elastic tissue or collagen were selectively emphasized by appropriate choice of staining procedures, and cytologic detail in 1-3 μ sections was superior to that shown by conventional methods. Selected areas from adjacent 4-8 μ sections could be re-embedded for ultramicrotomy and electron microscopy.     

Periodic Acid-Schiff-Toluidine Blue-Aurantia; A Stain for the Gland Cells of the Stomach

By a revised technique, human pulmonary elastic tissue can be isolated in a form suitable for examination under the stereoscopic microscope. Fresh human lungs from autopsy are fixed by intrabronchial infusion with 10% formalin for 24 hr. Slabs 1.5 cm thick are cut and the formalin removed in running water. One such slab is embedded under intermittent vacuum in an aqueous mixture containing 15% gelatin, 10% glycerol, and 1% phenol; then allowed to gel. Frozen sections 2 mm thick are cut on a large-section MSE sledge microtome. Squares 3 × 3 cm from such a section are corroded for 4-5 days in 88% formic acid at 45 C, washed once with distilled water, and mounted in glychrogel containing 6% gelatin. The elastic tissue network of the lung will have been freed from surrounding elements. The preparation should be stored in a refrigerator. Blocks for thin sections and large thick un-corroded sections can be prepared from the same lung as part of an over-all procedure.     

祁连山青海云杉林地土壤水分特征研究

通过对祁连山青海云杉林地土壤水分含量及主要影响因子的测定,结果表明,青海云杉林地土壤具有容重低、入渗率高、土壤水分含量垂向分布受降水影响明显等特征,灌木-青海云杉林、苔藓-青海云杉林和草地(对照)平均容重分别为0．522、0．641、0．874g·cm^-3．土壤初渗率分别为6．0、21．0、2．5mm·min^-1,稳渗率分别为3．98、11．2、0．5mm·min^-1．苔藓-青海云杉林地土壤可划分为4层：水分活跃层(0～30cm)、水分调节层(30～60cm)、水分传输层(60～80cm)和蓄水层(80ClTI以下),各层土壤贮水量分别为3．30、3．17、2．80和2．83mm·cm^-1．灌木-青海云杉林可分为3层：水分稳定层(0～30cm)、水分活跃层(30～60cm)和水分传输层(60cm以下),各层土壤贮水量分别为3．98、3．65、3．48mm·cm^-1．两种林地土壤水分含量的季节变化受降水影响较大,相比而言,苔藓．青海云杉林地入渗率更高,有较明显的蓄水层,水分传输能力更强,而灌木-青海云杉林地贮水能力更强．     

Optimization of sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees

Aims: To develop fast and reliable sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees. Methods and Results: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4‐mm‐diameter, 43‐cm‐long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analysing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1–4 drillings were either analysed separately, and the results were combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56·6% for the scheme based on a single drill to 96·8% for the scheme based on four drillings analysed separately. Both schemes significantly differ (P < 0·05) from those based on two and three drillings, whose efficiency was 72·6% and 83·9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. Conclusions: Highest diagnostic efficiency was obtained by analysing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. Significance and Impact of the Study: Fast and reliable sampling procedures make DNA‐based diagnosis more suitable for tree inspection procedures.     

Maize nodal root ramification: Absence of dormant primordia,root classification using histological parameters and consequences on sap conduction

Maize plants grown in field conditions were used to describe the histological organisation of the nodal roots, those of their laterals, and also to test the presence of critical stages where the subsequent capability for growth and development of young laterals was determined irreversibly. The absence of undeveloped primordia, which stop their development before boring through the nodal mother-root epidermis, proved that the number of laterals could not be regulated between the differentiation and the emission stage. Cross sections performed on nodal roots beared by the internodes 2, 4 and 6 and their long (>3 cm) and short (<3 cm) laterals showed that: u     

Bromobimanes — fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections

Summary Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient.In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons—not stainable by the classical methods—which can be identified as peptidergic ones by the bromobimane technique.A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.     

Affinity purification of two populations of antibodies against format determinants of synthetic myelin basic protein peptide S82 from S82-AH- and S82-CH-Sepharose 4B columns

Two different kinds of immunosorbents were prepared that contained the synthetic myelin basic protein didecapeptide S82 (TTHYGSLPQKAQGHRDQDEG)—one coupled with AH-Sepharose 4B through hexanoate spacers to the C-terminal glycyl residue; the other, with CH-Sepharose 4B through hexanoate spacers to the N-terminal threonine residue. An antiserum rich in antibodies to a format determinant of S82 was passed through each column, and, by means of affinity purification, two homogeneous populations of anti-format antibodies were obtained, each with a binding affinity of 1×10⁸M^–1 for S82. The population recovered from S82-AH-Sepharose 4B cross-reacted to a considerable extent with synthetic peptide S8 (GSLPQKAQGHRPQDENG) but only to a limited extent with S79 (AQGHRPQDEG). The population recovered from S82-CH-Sepharose 4B crossreacted poorly, if at all, with S8. An equimoler mixture of S8+S79, however, reacted well with either population of anti-format antibodies, thus showing that the mixture could mimic the format of S82. It was concluded that secondary structural conformation of S82 could be preserved during the coupling procedure and that the resulting immunosorbents could be used for the affinity purification of anti-S82 antibodies to the format determinants.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.Supported by Research Grants NS-10237 (Duke) and NS-15322 (St. Luke's) from the National Institutes of Health and by RG1197-B7 from the National Multiple Sclerosis Society.