Microscopica acta最新文献

This communication presents highly satisfactory methods for the demonstration of DNA in animal materials. The method involves selective extraction of RNA with concentrated, 90% or 75% phosphoric acid of 5 degrees C for 20, 40 and 120 min, respectively, followed by staining with aqueous solutions of basic dyes, such as setoglaucine, setocyanine, pinakryptol green 2) and alcoholic aniline blue without SO2. Perfect blue nuclei were seen when staining was performed with aqueous solutions of setoglaucine and setocyanine at pHs 3.5 and 4.0 to 4.5, respectively. Sections of tissues from which RNA has been extracted and then hydrolysed in 6 N HCl at 28 degrees C for 15 min followed by staining with these dyes also revealed perfect colouration of the nuclei. Acid-hydrolysed sections when stained with alcoholic aniline blue-SW2 prepared with N HCl and sodium thiosulphate revealed nuclei of magenta colour, and sections from whcih RNA has been extracted and then hydrolysed in hydrochloric acid and stained with this dye-reagent revealed nuclei of purplish colour. Sections of tissues fixed in Carnoy, 10% buffered neutral formalin as well as paraformaldehyde were found to be most suitable for staining with these dyes. The in situ absorption spectra of nuclei stained with aqueous solutions of setoglaucine, setocyanine and alcoholic aniline blue without SO2, after extraction of RNA as well as those of nuclei in tissue sections from which RNA has been extracted and then acid-hydrolysed and stained with alcoholic aniline blue-SO2 have been presented. Also presented herein are absorption data of nuclei in tissue sections which wee hydrolysed in hydrochloric acid and then stained with alcoholic aniline blue-SO2. Some implications of these findings have been discussed.

We have designed a controlled-temperature stage for the observations of live microorganisms under all magnifications of the compound microscope. The use of water-immersion objectives eliminates the need for a cover-slip and permits interventions such as liquid medium changes, microsurgery or the insertion of microelectrodes. Simple in design and relatively inexpensive this stage has an observation area of 50 X 75 mm.

The influence of shape and size of structures as well as the individual effect on accurate results and the speed of evaluation in point-counting and automated planimetry is investigated. The following statements can be made: 1) Planimetry is with the factor 2 to 5 more exact than point-counting. 2) The individual effect on the results is small in planimetry and therefore can be neglected in most cases. However, this is impossible in point-counting. 3) The evaluation-error increases with the decrease of size. It goes over 5% below an area of 40 mm2 in planimetry. The comparable border-values of point-counting are much higher. 4) The shape has little influence on the results in structures with smaller deviations from the form of a circle (stretching-factor below 2.7), but with increasing complexity of the borders, the results show more variability. 5) The border-line examination in planimetry is 5 to 10 times more exact than in point-counting. 6) The time used for equal samplings including calculation of MW and standard deviation is similar for both procedures, but planimetry only needs few measuring steps for small confidence-limits and is able to estimate more parameters in one estimation. 7) The orientation of structures has a planimetry no influence of the results obtained for the single section, in contrast to point-counting of intersections. 8) The psychic condition has influence on both procedures.

By combining an incident light microscope (C. Zeiss) with an inverted camera microscope (ICM 405, C. Zeiss) having a common optic axis, it was possible to present simultaneously two microscope images of one object. These images were recorded by two television cameras and displayed on a single monitor with the aid of a video mixer (Pieper, Schwerte). This method was applied to intravital microscopy of mesenterial capillaries (rabbit and frog). It allowed the choice of a vascular network as an overview with the incident light microscope from which a suitable capillary segment for an electrical stimulation experiment could be selected and magnified with the transmitted light microscope. The results of its application provided proof that electrically induced constrictions in capillaries are independent of proximally or distally occurring vascular reactions. Capillaries of mesenteries in rabbits and frogs were stimulated with direct currents ranging from +0.015 micro A to +20 micro A. The constrictions were found to be reversible and reproducible. In addition capillary constrictions could also be produced by topical application of histamine and serotonin droplets applied to the surface of the mesentery. These observations may help to resolve the question of whether capillary contractility in the sense of Stricker (1865, 1876) is a biological mechanism.

This communication presents a new quick method for selective extraction of RNA from formalin-fixed tissues, such as kidney, intestine, ovary and testis of white rat and liver of rodents, Tatera indica and Millardia meltada and of the frog, Rana tigrina as well as of human wart. Sections of pancreas and kidney fixed in acetic acid-alcohol were also tried. The method is to treat deparaffinised sections in 20% and 15% meta-phosphoric acid at 5 degrees C for 10-20 and 20-30 min, respectively, and then to stain nuclear DNA with 0.5% aqueous solution of methyl green, methylene green, Giemsa, toluidine blue O or 0.125% pyronin G for 2 min, rinsed with water, treated with n-butanol for 2-3 min, cleared in xylene and mounted. It has been found that liver sections of rodents require 24 hours of treatment in 15% cold meta-phosphoric acid for complete removal of RNA, whereas those of the frog require only 20 min at 20% acid. It has been concluded that following treatment of sections in cold meta-phosphoric acid, RNA is extracted selectively leaving DNA in a native state. Therefore, staining of DNA in tissue sections from which RNA has been extracted is due to binding of its negatively charged phosphate groups with the positively charged dye molecules.

One of the main concerns of dental research is the observation of the oral tissues and the materials applied to the dentition. The changes in composition and structure of the outer surfaces and the materials deposited on these surfaces are of special interest. In the literature, a variety of replica techniques for these purposes is described (Grundy in 1971 [12]; Saxton in 1973 [25]). The use of these techniques is limited because of artifacts in the samples, and a restricted resolution power resulting from useful magnifications in the order of 800x. An accurate and universal replica technique for the examination of specimens to be viewed under the SEM has been developed. The first impression is made by a light body silicone elastomer (President Coltene). The positive replica is made by electrodeposition of copper in an electro plating bath (Acru plat 5 electronic, Dr. Th. Wieland, D-7530 Pforzheim). The reliability and accuracy of this replica technique was verified by a scanning electron microscopic comparison of the replicas and the actual structures of etched enamel. To illustrate the applicability of the replica technique to structures with much lower hardness, also high resolution images of dental plaque were produced. The copper surface offers a perfect, original and proper electroconductive medium that withstands the bombardment of electrons and the relatively severe conditions in the scanning electron microscope. Reproducibility was accurate as judged by the duplication in position, size, and shape of the fine detail at magnifications of 7500x offering a resolution of 25 nm.

The influence of embedding media on the fading of FITC-labelled coverslip cultures was tested. It was found, that the value of the fading depend on the pH value of the embedding media. In glycerin-PBS pH 8.5 as embedding media and immediate measuring the fluorescence intensity, the decrease of the fluorescence intensity after 300 seconds blue light excitation was 5%.

This paper presents a very simple and reliable procedure for the staining of animal chromosomes employing dyes, such as pyronin G, acridine red, rhodamine B, rhodamine 3GO, belonging to aminoxanthene group and brilliant cresyl blue and methylene violet 3RD, belonging to quinone-imine group. The procedure has been tried on sections of the grasshopper and mouse testes fixed in Dutt's modification of Nawaschin mixture. The method is to deparaffinise sections and then to stain with aqueous solution of these dyes for 2--3 minutes, rinsed with water and dehydrated through grades of ethanol, keeping for 15--30 seconds in each grade with several dips. Preparations are then cleared in xylene and mounted. Stained preparations following this procedure revealed excellent colouration of the chromosomes at all the various stages of mitosis and meiosis, particularly in the case of the grasshopper. Mouse chromosomes stained with these dyes following the same method revealed perfect colouration of the fully condensed chromosomes at all stages of mitosis and meiosis but not of the very early stages, except the sex chromosome. Moreover, grasshopper testis sections when treated with cold concentrated phosphoric acid for varying time-periods and then stained with these dyes also revealed excellent colouration of the chromosomes. The implications of these findings have been discussed.

The subcommissural organ (SCO) of vertebrates is located in the roof of the third brain ventricle, and secretes into this ventricle a glycoproteinaceous, fibre-like structure, the liquor fibre or Reissner's fibre. A method is described for the densitometric measurement by means of computer-controlled scanning cytophotometry of two cellular parameters directly related to the secretory activity of the subcommissural organ in European green frogs. These parameters are: i) the amount of stained secretory material in the SCO, and ii) the amount of secretory material in the SCO labelled by a radioactive precursor. It appears that scanning cytophotometry offers a fairly rapid, accurate, objective and reproducible method to measure these parameters in stained sections and in autoradiographs of the SCO if this histological material fulfils certain conditions.

The total protein and total protein-SH content of both the cytoplasmic fraction and the nuclei of Ehrlich ascites tumor cells (EATC) were determined macroscopically and microspectrometrically. The separation into cytoplasmic and nuclear fraction was performed by a modification of the method of Mamaril et al. [4]. The macroscopical determination of the total protein and the total protein-SH content were performed with the Folin method of Lowry et al. [2] and the DTNB method of Ellman [1], respectively. The quantitative microspectrometrical determinations of total protein content and of total protein-SH content were performed using the tetrazonium staining method of Nöhammer [5] and Nöhammer et al. [6] and the mercurochromcyanide (MCN) method of Nöhammer et al. [7], respectively. Within the intact cells, fixed and stained histochemically, the total protein and the total protein-SH content of the nuclei were determined microspectrometrically. The so called "nuclear extinctions", measured as the sum of the extinctions of the nucleus and of the parts of the cytoplasm above and below the nucleus, were calculated into the true nuclear extinctions, which then show a good correspondence to the values measured both microspectrometrically and macroscopically on isolated nuclei. The calculation for the true nuclear extinctions is based on a special preparation of spherically shaped cells and nuclei.