Microscopy and photomicrography. Part IV.
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Isolated lymphocytes from peripheral blood of 22 healthy donors were examined with the help of the indirect membrane immunofluorescence (MIF) for immunoglobulin determinants on the surface of the cells. The quantity of MIF+-cells for IgM was on the average 8.7% (3-18) and for IgG 15.5%(8-18). Five different anti-IgM-sera were characterised with regard to their use in the MIF (precipitation titre, entire protein content, content of anti-IgM-antibodies). Differences in the quantity of the MIF+-lymphocytes were above all based on differing antibody concentrations of the sera. It was referred to further possible causes for these differences. The influence on the results of the examinations by protein addition and trypsination.
In an attempt to improve the assay of leukocidin from Staphylococcus aureus a combined vital fluorochrome staining procedure with acridine organe and ethidium bromide was developed. It proved to be more suitable for the demonstration of leukocidic effects on leukocytes than phasecontrastmicroscopy. Damaged leukocytes fluoresced distinctly red and undamaged leukocytes green. With the fluorochrome-method leukocidic changes became also evident in some lymphocytes. These were not clearly demonstrated by phasecontrastmicroscopy.
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Pneumocystis carinii pneumonia can only be diagnosed once the microorganism is demonstrated. The authors review and illustrate various staining technics, and discuss for each of them specific advantages. All samples must by systematically examined both by phase contrast microscopy and by light microscopy after Gomori-Grocott and Gram-Weigert stains. This morphological study is completed by ultrastructural photographs.
The emission anisotropy of selected fluorescent probes which interact with cells and their membranes is a sensitive parameter for studying the structural changes associated with different functional states. Such measurements can now be made on individual living cells at rates of up to 103 per second and the cells separated on the basis of the anisotropy function alone or combined with other physical signals using a multiparameter automated computer-controlled cell separator (MACCS). Thus, selection can be on the basis of simple or complex algorithms reflecting the size, macromolecular content, and rotational mobility of cellular components or liganded reporter molecules. Cells isolated in this manner are sterile, viable, and can be used for outgrowth or biochemical studies related to dynamic changes occurring during differentiation or malignant transformation.
The possibility of fluorescence microscopical examination of mitochondria in living animal cells using fluorochrome berberine sulphate is shown. At concentrations of 30--50 g per ml the chemical is accumulated selectively in mitochondria of living cells. The specificity of berberine sulphate accumulation in mitochondria was shown by comparative phase contrast and fluorescence microscopy. The advantages of the method is its high sensitivity and simplicity, especially when mitochondria can not be examined by the phase contrast microscopy.
This paper discusses some of the methods used by for making basic biological tests of stomatological materials. Although we do not dispense with the use of methods based upon the observation of fixed and, possibly, stained cells and allowing the states assumed by cells in a certain period of time to be noted, yet we believe that far better results may be obtained from tests enabling the development of changes of living cells cultivated in vitro to be followed. The results of the experiments conducted by the authors indicate that it is possible for the final effect to be modified in dependence upon the type of material used, its concentration, or the time allowed to elapse from the preparation of the particular material and that an observation of living cells procides much more information about the character of alterations and, more specifically about the time course of changes. If necessary, this method may be supplemented by an electronmicroscopical analysis of the changes produced.
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Reported in this paper are results obtained in morphological virus diagnosis by using the negative contrast technique on the basis of electron microscopy. The availability of high-efficiency electron microscopy as well as of perfectly improved techniques of preparation, knowledge of the latest virus model concepts, and indivudual skills in diagnosis are essential conditions for the above approach. Parvoviridae, picornaviridae, and togaviridae are identifiable only in high particle concentrations and by group representation. Papovaviridae, adenoviridae, herpetoviridae, poxviridae, and reoviridae, on the other hand, can be safely identified even as single particles. The diagnosis of orthomyxoviridae, paramyxoviridae, rhabdoviridae, and retroviridae is facilitated by their own dimensions and their characteristic helico-symmmetrical nucleocapside. Coronaviridae are of highly conspicuous morphology but, nevertheless, pose problems in differential diagnosis. Substantive improvement of morphological virus diagnosis, in terms of minute details, may be achieved by means of the negative contrast method on the basis of immune electron microscopy. Advantages implied in that morphological method include less time-consuming and quite uninvolved practicability and good dependability of diagnosis for more efficient decision-making in research and practice.
It is demonstrated that cytoplasmic laminar bodies (CLBs) can be identified in Nissl stained celloidin sections by means of phase contrast microscopy. Provided that the refractive index of the mounting medium is in the range of 1.56 to 1.65, CLBs appear as bright holes in the cytoplasm.
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Six specimens of human cadaveric pancreas were collagenase-dispersed and placed in tissue culture. Media were changed at 2 day intervals and assayed for insulin and amylase. Experiments were terminated after 8 days, and phase-contrast and light microscopy were performed on cultured tissue. Insulin content in media remained high for 6 days in five cases and for 8 days in four experiments; this correlated directly with morphologic viability of cultured tissue. Media amylase fell to zero after 4 days in four cases and after 6 days in two cases. These data support the concept that tissue culture may be an efficient method for (1) islet cell purification, since acinar tissue and amylase activity disappear, and (2) islet preservation.