Microscopica acta最新文献

This paper describes a method for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2 for use in Feulgen procedure. The method involves replacement of the usual N HCl by N H2SO4 and the usual amount of potassium metabisulphite. Following this method of preparation, an extra-sensitive Schiff's reagent is obtained which requires only 4-5 min for optimum nuclear colouration even when staining is performed at 5 degrees C. This Schiff's reagent produces perfect Feulgen staining up to 6 months after preparation. Toluidine blue O-SO2, prepared with N H2SO4 and potassium metabisulphite, also produces perfect Feulgen type staining of the DNA-aldehyde molecules of acid-hydrolysed mammalian tissue sections. Toluidine blue O-SO2 when shaken with activated charcoal and filtered produces very satisfactory result. The shell-life of this dye-reagent is just a week. The suitability of the use of N H2SO4 for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2, has been discussed.

A new method employing phosphoric acid in place of hydrochloric acid for the preparation of azure A-SO2 and toluidine blue O-SO2 has been described in this communication. It has been found that o-phosphoric acid in a weak concentration can be a suitable substitute for hydrochloric acid in the preparation of these dye-SO2 reagents. These dye-reagents produce excellent blue nuclei in human uterine tumour. Toluidine blue O-SO2 fortified with an amino aicd, glycine also produces excellent blue chromosomes in the squash preparations of grasshopper testes fixed in acetic acid-alcohol. The shelf-life of these two dye-reagents has also been presented herein.

An esterification with isopropyl alcohol containing 0.2% periodic acid and 2% acetone (at 56 degrees C for 16 hours) followed by a treatment in a special physical developer, similarly, an acetylation with a 3:2 mixture of pyridine and acetic anhydride (at room temperature for 16 hours) followed by the same development, render the elastic fibres and membranes visible. Both pretreatments (esterification and acetylation) serve to make the catalytic points more active in the elastic elements than in the other components of tissue.

An esterification with n-butyl alcohol containing 0.5% sulphuric acid (at 56 degrees C, for 16 hours) followed by a treatment in a special physical developer renders smooth muscle cells as well as other contractile elements (striated muscle, cilia, flagella, myoepithel cells) visible in light microscope and birefringent in polarization microscope. A few kinds of non-contractile tissue components without oriented fibrillary structure also stain but do not display birefringence.

The area of contact between the macula densa and the terminal vascular components, the vas afferens, the vas efferens and the mesangium (Goormaghtigh cells) of five glomeruli of the rat has been determined morphometrically using serial sections. The surface, the filtration area, the volume and the total length of the capillaries of each glomeruli has been estimated, and these data have been correlated. There is evidence, that a contact between the macula densa and the mesangium always exists, a contact to the vas afferens is not obligatory.

A time-saving method is described for the preparation of cultivated cells (monolayer) for electron microscopy using a new test chamber system (TCSC-1). The versatile chamber is fitted with a screw-in PTFE-adapter which connects via a silicone ring gasket to the surface on which the cells grown. All procedures (fixation, staining, embedding) are performed directly in the chamber. The cells, in this particular communication epithelial liver cells, are grown as a monolayer on polyester foils. After embedding of the cells, the foil can be easily taken off. Removal of the foil does not in any way effect the quality of the first slice for electron microscopic examination. The resin block can also be easily pressed out from the PTFE-adapter.

Reflection contrast microscope methods are generally used for studies of those portions of the cell that are turned towards the glass coverslip, to comprehend the structure of the cytoskeleton and the dynamics of cell movement, as well as formation of cell-glass contacts. In incident illumination only reflected light contributes to picture formation. The intensity of which in the case of observation of unstained cells is small because of small refraction differences. To overcome this problem a reflection contrast system was developed by Leitz according to Ploem [49], in which by using contrast preserving measures the reflection becomes prominent in comparison with the lens reflexes. The emerging pictures are a result of interferences of reflections at glass-cell, cell-culture medium and culture medium-cell interfaces. According to Fresnel's equations the reflected intensity depends on the differences of the particular refractive indices and the thickness of the layers, which determine the phase of interfering beams. In idealized systems of thin films the reflected intensity is a measure for their optical constants. Relative reflection measurements from glass-cell areas is comparison with the known glass-medium reflection, can therefore be revealing as far as refraction index, cell-glass distance or cell thickness are concerned. The estimates by Bereiter-Hahn et al. [15] were made in the assumption of vertical illumination neglecting its actual conical shape: the comparison of two Fresnel functions of cytological relevant measurements show - in accordance with Gingell and Todd [24] - that this is only permitted under certain conditions, depending on the required accuracy of the measurements; an incidence angle of about 30 degrees leads to an error of about 10%, an angle of 50 degrees to more than 50%.

This paper reports on a new method for the use of acridine orange (AO) in an aqueous solution at pH 4.5 for staining DNA of rat tissue sections from which RNA has been extracted selectively with cold phosphoric acid. Not only this, AO can also be used as dye-SO2 reagent, prepared with NHCl and potassium metabisulphite, for staining DNA-aldehyde molecules of acid-hydrolysed tissue sections. AO samples, manufactured by the National Aniline Division as well as by G. T. Gurr have been used with equal success. Studies of stained sections under light microscope reveal the presence of specifically stained yellowish-orange nuclei. Those sections under fluorescent microscope with proper exciter and barrier filters reveal nuclei of maroon colour. The in situ absorption spectra of nuclei stained with AO-SO2 following acid-hydrolysis of tissue sections as well as those of nuclei stained with an aqueous solution of the dye following extraction of RNA have been presented herein. The mode of binding in the former case has been considered to be due to binding of the teritary amino group of the dye molecules with the DNA-aldehyde molecules and in the latter case to be due to electrostatic binding between the positively charged dye molecules with negatively charged phosphate groups of DNA. Implications of all these findings have been discussed.