Showing papers in "Microscopica acta. Supplement in 1976"

Preparation and counts of particles in electron microscopy: application of negative stain in the agarfiltration method.

Abstract: The agarfiltration method, published by Kellenberger and Arber in 1957, has been adapted to negative stain with sodium phosphotungstate and uranyl acetate. The technique is described in detail including all recent improvements. The precision obtainable in particle counts is discussed for agarfiltration and sprayed droplets.

Staining mycobacteria with carbolfuchsin: properties of solutions prepared with different samples of basic fuchsin.

Abstract: Acid fast staining of mycobacteria in the form of beadings is obtained by means of a carbolfuchsin solution (Ziehl-Neelsen stain) prepared from pararosaniline or from certain kinds of basic fuchsin. After such acid-fast stains, the intensity of the bacilli's colouring was rather poor and unstable, so that some bacilli lost their acid-fast stain. In contrast, an acid-fast staining of mycobacteria in rod form results by using a carbolfuchsin prepared from rosaniline or from other basic fuchsins included new fuchsin. The spectrophotometric and thin-layer chromatographic data indicate that the main component of those basic fuchsins showing beady staining may be pararosaniline, whereas the main ingredient of basic fuchsin with staining the bacteria in rod form may be its higher homologues. Neither chloride nor acetate of the fuchsin could affect the appearance and number of stained bacilli. The commercially available "basic fuchsin" is either the chloride or acetate of pure pararosaniline or consists of variable mixtures of it with higher homologues. Consequently, only a basic fuchsin which has an absorption maximum at lambda greater than or equal to 552 nm could be employed for the acid-fast stain of mycobacteria in a stable manner. Pararosaniline included some basic fuchsins, composed mainly from pararosaniline, should not be selected for the preparation of the carbolfuchsin formula.

Methods of fixing, sectioning and staining amphibian eggs for cytological study.

Abstract: A combination of methods for fixation (sublimate, cobalt nitrate, formaldehyde, acetic acid in water), inclusion (celloidin dissolved in methyl salicylate, paraffin-paraplast) and staining was used to make serial sections easy, to avoid clefts and to give a good picture of segmentation mitoses, as well as a good contrast of yolk and cytoplasmic components. Four methods of staining were used concerning the Urodele eggs: Safranin-methyl blue-orange G, safranin-picro-blue black naphthol (Curtis), safranin-violet crystal-orange G (Flemming) and Feulgen-methyl blue-orange G. The achromatic apparatus of the normal segmentation mitoses is clearly delineated and the relationships between astral fibers and yolk are different at metaphase and anaphase. By these methods, particularly suitable for demonstration of nuclei, cytoplasm and achromatic apparatus, the cleaving egg may be used as a test for the inhibition of achromatic apparatus and chromosome damage by antimitotic substances. The contrast between vitelline cytoplasm and cytoplasmic non-vitelline abnormal fibrillar systems, produced by transformation of astral and diastematic fibres, is made particularly evident by these methods of staining.

[Staining the Nissl bodies in 4--10 microns thick Araldite sections with an area of about 2x2 cm (author's transl)].

Abstract: For light microscopy 4--10 microns thick sections with an area of 2x2 cm o Araldite-embedded tissue (human autopsy brain) are stained in 1% aqueous methylene blue at 65 degrees C for 4--12 hours and are differentiated in 98% ethanol for some seconds or very few minutes. The Nissl bodies, nuclei and nucleoli are stained dark blue, the neuropil is nearly colourless.

[Biological and medical applications of the cathodoluminescence in the scanning electron microscope (author's transl)].

Abstract: The light emission according to the excitation of solids and molecules by electrons--named cathodoluminescence--extends the analytical field of applications of the scanning electron microscope. This method can be applied in biological and medical research. It can be used for the investigation of marked specimens similar to the fluorescence microscopy. The autoluminescence of some substances can also be used for special applications. The cathodoluminescence in the scanning electron microscope has the advantage that more sensitive detector-systems can be used than with the fluorescence microscope. Moreover, in the scanning microscope it is possible to combine the cathodoluminescence mode with other imaging or analytical equipments.

Phosphotungstic acid-iron-haematoxylin staining method for osteoid, boundary bone and bone components in paraffin sections.

Abstract: A new staining technique which stains osteoid and bone tissue differentially and also demonstrates boundary bone, pathological osteoid and the changes in ageing, pathological and dead bone matrix in decalcified paraffin or low-viscosity-nitrocellulose bone sections was developed This phosphotungstic acid-iron-haematoxylin (PTAIH) method is based on pretreating the sections with phosphotungstic acid followed by an iron alum mordant and staining in haematoxylin with subsequent timed differentiation, at certain stages of which the features listed above appear Van Gieson's picrofuchsin is then used as a counterstain After standard differentiation osteoid appears red in sharp contrast with the black bone, young and woven bone, old and lamellar bone, and allows one to demonstrate changes in stainability of diseased osteoid and bone matrix, and dead bone With the differentiation done individually and interrupted at certain stages it is possible to distinguish between various bone components depending on the amount and quality of their in vivo mineralisation Comparison with controls showed that in this respect the method is more sensitive than the curremt staining techniques of undecalcified bone sections since it demonstrates not only unmineralised and fully mineralised tissues but also shows the poorly calcified, demineralised and ill-calcified bone components The advantages of the method compared with those using undecalcified sections are its simplicity, suitability for fixed and decalcified material in any unspecialised histological laboratory and the fact that osteoid and other bone components can be studied in sections of unlimited size and in undisturbed relationship to their surrounding soft tissues

A computerised microscope focusing technique.

Abstract: An automatic focusing technique for a computer-controlled optical microscope is described, which is based on an algorithm originally proposed by Mendelsohn and Mayall. The procedure is capable of attaining a focusing accuracy of less than 1/20 micron in the slide-objective distance. The dependence of focusing accuracy and time to focus on the scanner signal-to-noise ratio, and the change in the focus position with time, are discussed.

[The different procedures for value collection in biological morphometry and stereology (author's transl)].

Abstract: Each stereological or morphometrical investigation presumes a high experience in structure diagnosis. Therefore, a good contrast between the structures to be evaluated is necessary, especially when using automatic measuring systems. Furthermore, some suggestions are given to facilitate the object-evaluation. The stereological principles including pattern-recognition are mentioned briefly in order to understand some of the technical details. The techniques are divided into simple and advanced procedures. However, it is also possible to evaluate using a simple counter combined off- or on-line with a computer. The advanced system can be divided into tracing instruments and photometric- and television-scanning procedures. The first system which is manipulated by eye-control can also be called "optomanual devices". These are planimetry, a type of automatic point counting, and instruments which locate the coordinates by ultrasound or a magnetic tableau. With relatively inexpensive electronics the coordinates can be transformed into stereological values. The photometric instruments and the television devices "Scan-computers" discriminate only between gray-steps while measuring. The first group works by moving either the photometric spot (flying spot) or the microscopical object-stage. The second group is guided by its computer and the output immediately shows the stereological results. The discussion gives information about choosing the best instrument for investigation. The scientist should keep in mind that the size of the over-all procedure, the frequency of measuring, the time of object preparation, the possible difficulty in discrimination of the gray-steps and the extent of his scientific funds are important for his decision.

[A coordinate and area recording microscope for the topographic analysis of particle size distributions (author's transl)].

Abstract: A coordinate recording microscope equipped with a rolling disc planimeter is described. The application of the instrument for the analysis of cell size distribution in histologic preparations is demonstrated. The microscope stage is moved in x, and y directions by two digital micrometer spindles. The planimeter is equipped with a rotary encoder. The spindles and the rotary encoder are connected to digital counters which are input to a multiplexer. The multiplexer outputs the data to a teletype terminal both as hard copy and on paper tape. To avoid repeated measurements a video-acoustic monitoring system is provided. Processing of the data is carried out off-line with a IBM 1130 computer. Here the measured cells are displayed as points or circles on an oscilloscope. The distribution of cells of any size can be studied topographically in any region of the field of measurements.

A fluorescence staining method for the examination of microorganisms on natural substrates.

Abstract: A fluorescence staining technique is described which uses the magnesium salt of 1-anilino-8-naphthalene sulfonic acid as a protein stain. At a concentration of 2.5 or 3.0 mg ml-1 in distilled water or phosphate buffer the compound does not fluoresce under ultra-violet or far blue illumination until it is bound to a protein or similar compound. It can be applied to natural substrates such as soil, food materials, organic material in water, etc., and can be examined immediately without any washing procedures. The application of this staining method to detect microorganisms on dead green algae (detritus) is described.

Fast cell size distribution analysis by laser flow microphotometry-- applications to ciliate populations.

Abstract: The laser flow microphotometer for rapid cell size analysis is introduced. In contrast to known principles absorption is detected of a part of each cell only. Cell classification is achieved by pulse form analysis of absorption pulses. Cell lengths between 5 and 300 mum have been measured without changing the geometry of the flow system. Other ranges of size can be covered by changing the flow diameters. The principle can be applied to stained or unstained cells. No further preparation of samples is necessary. Examples of high speed measurements on different cell populations of unstained ciliated (Tetrahymena pyriformis), human blood and different pollen grains are given.

[A method of microphotometry with TV-image analysing systems (author's transl)].

Abstract: In scanning microphotometry only a small spot in the specimen is illuminated at the same time. However, lamp and optical system are designed to transfer 10(5) to 10(6) times more information at once. It is shown, that correct and reproducible results can be attained with stimultaneous illumination of the field received by a TV-camera. It is necessary to calibrate for stray light. Thus the high speed of measurement of TV-Systems can be used for microphotometry.

[Preparation of biological specimens for the scanning electron microscope (author's transl)].

Abstract: Three methods usually applied in preparing biological material for the scanning electron microscope were tested by the investigation of two species of amoebae with different content of water (Amoeba proteus, Vannella simplex). Air drying resulted in both the production of cell shrinkage and cell distortion. When the specimens were dryed from media with increasing vapour-pressure, more satisfactory preservation of surface structures could be obtained. The sequence of potency was: Ethanol, chloroform, isopentane, ethyl ether, freon 11 and freon 13. Short drying periods proved to be more favourable than long ones. Critical-point drying provided much better details of cell surface morphology in both amoebae species. However, some arteficial changes were still detectable as small breaks and destruction of the mucous layer. They must be attributed to the fixation and dehydration procedure. Freeze drying turned out to be superior to both air drying and critical-point drying. Specimens prepared by this method showed no visible differences in cell surface morphology compared to living cells. As a consequence of the relatively high content of water the preparation of A. proteus was more difficult than that of V. simplex.

Endocrine influences on peritoneal fluid cellular content.

Abstract: Since the cellular composition of peritoneal fluid is altered by sex, species, pregnancy, ovulation and the estrous as well as menstrual cycles we believe that the cellular content of peritoneal fluid is affected in a characteristic manner by the administration of hormones. Estrogen, progesterone, testosterone, chorionic gonadotropin, cortisone as well as various physiologic model situations were considered.