Microscopica acta最新文献

A method is described for staining semithin sections of epoxy-embedded cells and tissues. PAS-positive structures are specifically demonstrated by a red staining providing a vivid contrast to the other tissue components differentially stained in shades of blue, using methylene blue/azure II. Typical staining conditions for 1 to 2 micron thick Epon sections include 30 min oxidation with 5% periodic acid at 50 degrees C, 30 min incubation with Shiff's reagent at 50 degrees C, 20 min counter-staining with 0.5% methylene blue/0.5% azure II in 0.5% aqueous borax solution at room temperature. For 10 years, this method has provided excellent differential staining with a variety of tissues. Stained sections showed no signs of fading during this period of time. Therefore, this procedure is recommended as a simple method of staining semithin sections both for tissue orientation in electron microscopy and for brillant representation of cells and tissues, required for microphotography in color or black-and-white.

A new method for the preparation of azure A-SO2 and safranine-SO2 For use in Feulgen procedure has been described herein. The method involves the use of m-phosphoric acid or tartaric acid in place of N HCl in the preparation of these eye-reagents which exhibit enhanced pH producing increased staining intensity of the nuclei as compared with those of the controls, prepared with N HCl. Possible explanation for the increased staining intensity as well as the reason for the shorter shelf-life of these eye-reagents have been offered.

Porous silicone microvascular prostheses with a 1 mm inside diameter were inserted in the infrarenal part of the abdominal aorta of 35 rats. By modifying the usual histotechnical procedures like using paraffin with high consistence, embedding the specimen under vacuum and icing the sectional plane it was mde possible to produce perfect histological sections which guaranteed an exact histological evaluation of the implanted microvascular prostheses.

Stevenel's Blue is a reliable, rapid, and clean, one-step polychromatic stain for 1 micron thick epoxy sections. The staining solution, originally used by L. Stevenel (1918) to stain human parasites, is made by adding diluted potassium permanganate (2%) to an aqueous solution of the methylene blue (1.3%) and redissolving the precipitate thoroughly, by boiling in water bath and filtering. Staining is carried out in a Coplin jar at 60 degrees C for approximately 10 minutes for tissues embedded in Epon 812 or Poly 812, or 20 minutes for tissues embedded in Spurr's medium. The sections are rinsed, air dried, and mouted in Permount. The staining solution is very stable, and does not tend to form precipitates on the tissue. The stain brings excellent histological differentiation to nuclear, cytoplasmic, and extracellular components. Incorporation of the stain by elements within each tissue varies from intense to light with a subtle gradation of intermediate shades of purple and blue tones. For most cell structures the density of the stain parallels the electron density of that structure as seen under the electron microscope. For example, nucleoli and heterochromatin stain in dark purple while euterochromatin appear in a light blue shade. In all cases, the embedding media remains unstained. The bond between Stevenel's Blue and the tissues is stable, remaining unaltered by the mounting medium. It is also resistant to time-fading.

Histologic work-up of tissue specimens, especially from endoscopic biopsies, requires the use of fine forceps for orientation and dressing in fluid paraffin during the embedding procedure. These forceps are usually preheated over an open flame. If, however, smaller tissue particles are handled with overheated forceps, arteficial alterations may occur which are apt to hamper or falsify the histologic evaluation of the prepared section. Some typical tissue artefacts due to the handling with overheated forceps, are demonstrated with slides from liver biopsies. Recent experiences with an auxiliary instrument for paraffin embedding (Histostat of Vogel, designed by Ciplea) are reported. The forceps are kept at stable temperatures by immersion in fluid paraffin during the embedding procedure, thus excluding almost completely the risk of tissue artefacts by handling with overheated forceps.

The authors describe a new preparation technique for scanning electron microscopic study of the internal structure of skeletal muscle cells. The specimens were fixed, dehydrated and imbibed with Epon or Durcupan--without polymerisation--and thereafter they were freeze-fractured.

Naturally and artificially devitalized cells of various origin can be loaded with heparin and subsequently stained with DAPI, a newer fluorochrome (4',6-diamidino-2-phenylindole), leading to a bright yellow fluorescence of the nuclear envelope. Depending on the cell type it is also possible to coat the outer cell membrane with heparin or other sulphated mucopolysaccharides and to achieve similar staining phenomena with DAPI.

Microscopic observations of weak fluorescence from living cells can be achieved by using image intensification techniques in situations where conventional film recording is not feasible. A brief description is given of experimental arrangements that have been used, involving recording the intensifier output alternately on film, or TV vidicons. References are given to more detailed descriptions of particular systems, and an example is presented of the detection of Ca++ in Haemanthus by means of the fluorescence of chlorotetracycline.

The freezing velocity, the most important parameter for the quality of cryofixation of biological objects, was measured in frog liver specimens. The cooling course was found to depend on the size of the specimen, the specimen support and the cooling medium used (liquid nitrogen, supercooled nitrogen, Freon 12 and propane). The results were compared with scanning electron micrographs of freeze fractures cryofixed in the same manner: Propane yielded the highest cooling rates and, consequently, the best structural preservation. Morphologically similar results were obtained by combining Freon 12 and very small specimen supports. Generally, it can be said that the smaller both specimen and specimen support are, the higher is the freezing rate and the better the structural preservation. The findings are discussed with regard to further possibilities of improving the cryofixation of biological tissue.

By means of polarized ultraviolet fluorescence microscopy the structural changes of F-actin and myosin were discovered at the changing a functional state of a living muscle fibre and during spreading degeneration (Zenker's necrosis). The character of conformational changes of F-actin and myosin at activation, contraction, contracture and rigor is similar, but the number of changed macromolecules depends on a fibre state. At fibre local damage in its morphological intact parts there was found an alternation of zones, reflecting two states unusual for a fibre. During spreading degeneration these states transform into irreversible contracture and then into rigor. Similar changes were observed in muscle fibres obtained from denervated muscles.