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1.
Double staining techniques were devised to study Escherichia coli attachment to mucopolysaccharide-coated urinary tract epithelial cells. In addition, vital stains were used to distinguish between viable and nonviable epithelial cells. Alcian blue was used to confirm the presence of glycosaminoglycans and periodic acid Schiff was used to distinguish a further group of polysaccharides, proteoglycans, neutral mucosubstances and glycolipids. The staining methods enabled investigations to be carried out concerning the possible importance of mucopolysaccharides in the attachment of bacteria to mucosal surfaces. Staining techniques were also used to investigate whether the presence of a mucopolysaccharide coat is related to epithelial cell viability. The combinations of vital and mucopolysaccharide stains were found to give reproducible results when cell preparations were evaluated by three individuals. Both in vivo and in vitro certain populations of epithelial cells have been found with a large number of bacteria preferentially attached. The double staining techniques described here may help to reveal the nature of these target cells.  相似文献   

2.
New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: paradoxical concanavalin A staining (PCS) to identify mucous neck cells, periodic acid Schiff-concanavalin A staining to distinguish mucous neck cells from surface mucous cells, and a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: Feulgen hydrolysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; Feulgen hydrolysis-PAS-concanavalin A-Bowie staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.  相似文献   

3.
New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: A) paradoxical concanavalin A staining (PCS) to identify mucous neck cells, B) periodic acid Schiff-concana-valin A staining to distinguish mucous neck cells from surface mucous cells, and C) a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: 1) Feulgen hydroIysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; 2) Feulgen hydrolysis-PAS-concanavalin A-Bowic staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.  相似文献   

4.
D. L. Smith 《Protoplasma》1972,74(4):465-479
Summary The rhizoids of gametophytes ofPolypodium vulgare L. rapidly absorb vital stains whereas the protonemal cells are impermeable to these stains, which can only enter the cells from the rhizoids. The protonemal cells which bear rhizoids were found to have a slightly higher osmotic equivalent than did the rhizoids or the protonemal cells on either side. From the results of several staining procedures it was demonstrated that the rhizoid walls contain free carboxyl groups and thus possess cation exchange properties. Most of the carboxyl groups are probably present in a yellow-brown wall matrix substance, which shows high resistance to acid and alkali extraction. The precise nature of this substance has not been determined but it could be an acid mucopolysaccharide. Carboxyl groups are detectable in the protonemal cell walls only after saponification and are probably esterified in the untreated wall. Several other chemical and physiological differences were found between the rhizoids and the protonemal cells and it was concluded that the specific properties of the rhizoids are related to their function as organs of uptake.  相似文献   

5.
R G Garrison  F K Mirikitani  J W Lane 《Microbios》1983,36(145-46):183-190
Fine structural aspects of Rhodococcus rhodochrous and R. equi are described and illustrated by electron micrographs after staining of cells by a variety of electron cytochemical procedures. The cell contents of these actinomycetous bacteria were those of a typical prokaryotic cell and consistent with that observed for other species of the Actinomycetales. Fixation with either osmium tetroxide or permanganate indicated the presence of an electron opaque substance at the wall exterior of R. rhodochrous which is thought to be composed of protein. Ruthenium red and Alcian blue-lanthanum stains for mucosubstances revealed that both species possess a capsular substance thought to be composed of a mucopolysaccharide or mucopolysaccharide-protein complex. This substance was non-reactive toward the PATAg stain for polysaccharide macromolecules containing vicinal glycol groups.  相似文献   

6.
The role played by either of the two differentiated mammary epithelial cell types in human breast cancer progression is currently not defined. This work addresses the question of whether the mammary tumor suppressor gene product BRCA1 is localized in basal and/or luminal epithelial cells in noncancerous outgrowth cultured from breast organoids. Primary epithelial cell outgrowths from ductal and alveolar preparations were directly employed to facilitate small-scale analysis under conditions closely approximating intact tissue. BRCA1 immunofluorescence was detected for the most part in cell nuclei of the epithelial outgrowth when using confocal microscopy. Nuclear staining was punctate in the cells with higher labeling intensity. Only minimal nonspecific staining was observed with mouse IgG as a negative primary antibody control or with primary antibody against the cell membrane receptor ErbB2, reported to be expressed in breast cancer, but was either not detectable or weakly expressed in normal breast tissue. Dual labeling was used to distinguish which epithelial cell type(s) stains for BRCA1. Primary monoclonal antibody against vimentin was used to identify basal cells, while antibody against cytokeratin 19 was used to identify luminal cells. Monoclonal antibody against BRCA1 was used for colabeling with each of these markers. Epifluorescence microscopy revealed BRCA1 immunoreactivity in both basal and luminal interphase cells. BRCA1 immunofluorescence was diffusely located about the chromosome mass during mitosis.  相似文献   

7.
From patients with bacterial vaginosis motile, anaerobic, comma-shaped bacteria can be isolated, which have recently been placed into the new genus Mobiluncus. In this study, electron microscopy was used to examine the in situ adherence of these motile curved rods to detached epithelial cells (comma cells) in vaginal fluid from two patients with bacterial vaginosis. Thin sections showed that the curved rods attached both directly to the epithelial cell surface and at various distances from it. It is concluded that after initial attachment these motile bacteria can grow at the epithelial cell surface in sessile microcolonies. Ruthenium red staining demonstrated a coating of precipitated glycocalyx material both on the surface of the curved rods and on their flagella. This may indicate that in situ the adherent curved rods were enclosed in a very hydrated matrix of exopolysaccharides. Conspicuous was the ability of the curved rods to attach to the epithelial cell surface via their cell tips. However, in situ no specialized bacteria cell surface structures were seen that might explain this polar attachment. Electron microscopy of pure cultures demonstrated that both Mobiluncus curtisii subsp. curtisii and Mobiluncus mulieris can produce a glycocalyx in vitro.  相似文献   

8.
The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.  相似文献   

9.
Functional in vitro studies with isolated gastric mucosal cells require cytological identification of different cell types in suspension or primary culture. Since suitable techniques have not been well established, different staining methods for the discrimination of dispersed pig and guinea pig gastric cells have been developed on the basis of modified previous protocols for enzymatic cell dispersion. Chief and parietal cells were visualized by combined periodic acid-Schiff stains. Surface mucous and mucous neck cells were identified by affinity-labelling, using lectins with selective staining properties in situ. Two of the lectins were found to be specific markers for gastric polymorphonuclear cells. The following vital tests were found to be useful: succinic dehydrogenase for parietal cells, Nile blue/brilliant cresyl blue stains for chief cells, and different phagocytosis assays for endothelial cells and gastric phagocytes. Endocrine cells were characterized by immunocytochemistry using specific antibodies against gastrin, somatostatin, histamine and serotonin. The same technique using a vimentin antibody was performed for the identification of fibroblasts. Proliferation of mucosal cells in primary culture was monitored by the incorporation of bromo-deoxyuridine, which was subsequently detected by a monoclonal antibody.Some of these results were presented at the 7th International Conference on Experimental Ulcer, Berlin, Germany 1991 (see Giebel and Schwenk 1991)  相似文献   

10.
Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.  相似文献   

11.
The Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy, 5-[125I]iodophenyl)propionate, was used as a vital stain for developing amphibian and tunicate embryos and for isolated cells (human erythrocytes and cultured chick limb mesenchymal cells). We found that the Bolton-Hunter reagent can be used on living cells at room temperature with techniques that are quite similar to the techniques routinely used to label isolated macromolecules in vitro. At concentrations of vital stain that were sufficient to label intracellular proteins in intact-cells, labeled cells underwent normal developmental sequences. Under these conditions, vital staining with the Bolton-Hunter reagent disproportionately labeled exterior proteins, and it seems likely that the Bolton-Hunter reagent is an especially good vital stain for cell surface and cell membrane proteins. The Bolton-Hunter stain is covalently bound, is not reutilizable, and appears not to disrupt natural physiological and developmental processes. Thus, we used the Bolton-Hunter reagent to follow the natural life spans of proteins in vivo and we were able to distinguish particularly long-lived proteins in Xenopus embryos.  相似文献   

12.
DNA-binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei.These were:the cytoplasmic streaming in pollen tubes whose nuclei were stined,the simultaneous visualization of fluorochromatic reaction and nucleus staining in isolated generative cells,and the capability of isolated.prestained generative or sperm cells to fuse with other protoplasts.The results confirmed that 4,6-diamidino-2-phenylindole(DAPI),Hoechst 33258 and mithramycin could be used as real vital stains,though their efficiency varied from case to case;among them DAPI showed best effect.The fluo rescent vital staining technique offered a useful means foridentification and selection of heterokaryons in gametoplast manipulation studies.  相似文献   

13.
The Dyer, Chance, alcian blue, and tannic acid-crystal violet wall stains for bacteria were compared. All gave apparent cell wall widths which were considerably greater than those demonstrated by electron microscope methods. The exaggerated width, as seen by the light microscope, was apparently due to staining of a portion of the cytoplasmic material adjacent to the wall, and in addition, to a precipitate on the surface of the cell produced by the Dyer and the Chance stains. Heat-fixed cells on slides were shown to retain considerable height or third dimension, being about half as high as wide. Moreover, such cells had their walls flattened against the slide to form a collar-like projection around the periphery of the shrunken protoplasm. This latter effect alone was not sufficient to explain the exaggerated wall thickness shown by staining. For obtaining reliable measurements of cell widths, the nigrosin negative stain was found to be as good as any, provided that the thickness of its film were controlled. Negative stains were used so that comparisons of cell widths shown by them and by positive stains could be made. This, in turn, facilitated the detection of the apparent widening of cell bodies caused by dye precipitates on their surfaces.  相似文献   

14.
Histochemical identification of cultured cells from human endometrium   总被引:1,自引:0,他引:1  
Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, gamma-glutamyltranspeptidase, peroxidase, and beta-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelia in sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma.  相似文献   

15.
Rapid assessment of bacterial viability by flow cytometry   总被引:8,自引:0,他引:8  
The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDAthan with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.Correspondence to: J. P. Diaper  相似文献   

16.
Abstract Computerised image analysis was utilised to enumerate the attachment of Staphylococcus epidermidis to HEp2 cell monolayers. A differential staining technique was employed such that individual staphylococcal cells stood out in sharp contrast against the uneven cell surface and granular contents of the epithelial cells. The primary image analysis operation involved subtracting an out-of-focus image from an in-focus image of the bacteria on the monolayer, thereby accentuating the bacterial image. Enumeration, using a particle counting routine, was rapid and reproducible, facilitating counting in excess of 700 bacteria per field at ×500 magnification. The computerised programme compared favourably with manual counting and would provide a rapid, objective and morphologically discriminatory method for evaluating bacterial attachment to various tissues.  相似文献   

17.
Legionella pneumophila attached to, penetrated and replicated within the three eukaryotic cell lines, MRC-5, HEp-2 and Vero. Multiplication occurred rapidly in these cells for an initial 48 h after inoculation and declined thereafter. Infected MRC-5 cell monolayers developed lytic-type cytopathic changes, with organisms being readily released. HEp-2 cells showed a more chronic infection, with slowly developing granular changes in the monolayers, and slow release of intracellular bacteria. In Vero cells, organisms were released rapidly along with a more progressively developing granular cytopathic effect in the monolayers. L. pneumophila was unable to grow in cell-free culture fluids. Uptake and intracellular development was similar for each cell type, and was initiated by 'bacteriopexis', a process in which the organisms bound via receptors and were surrounded by cellular microvilli which eventually fused, leading to bacterial engulfment. Replication of organisms in vacuoles within the cytoplasm of infected cells was confirmed by thorium labelling. These vacuoles were lined with ribosomes and, at the early stages of intracellular development, were found in close proximity to mitochondria, cytoplasmic filaments and banded enclosures. Ruthenium red staining showed that acid mucopolysaccharide capsular material was not present on these organisms during the attachment process or intracellular phase. Organism release was by lysis of the infected cells.  相似文献   

18.
Few fluorescent stains specific for cell constituents other than DNA are available. To assess their potential use as fluorescent stains for flow cytometry, the cell staining specificity of 55 compounds, originally synthesized for use as textile dyes and fluorescent brighteners, was explored and their excitation and emission wavebands determined. From these, six dyes were chosen for more detailed analysis. All six are vital stains, with excitation wavelengths allowing their use with an argon ion laser, and specific for a range of cell structures including mitochondria, Golgi bodies, lipid droplets, nuclear membrane, and endoplasmic reticulum. Concentrations as low as 0.01-0.25 microM were found to be adequate for most purposes, and high background fluorescence was not a problem. Their specificity allows differentiation between non-cycling and cycling cells. The properties of two of the stains allows their combination with propidium iodide or ethidium bromide for simultaneous determination of DNA content profiles. Being vital stains, usable at very low concentrations, and specific for a range of cell organelles, these six stains may be of considerable utility in flow cytofluorometry. We suggest that other textile dyes may be of use in flow cytofluorometry, or that their structures may form a starting point for the synthesis of further fluorescent stains of enhanced specificity.  相似文献   

19.
During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.  相似文献   

20.
Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.  相似文献   

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