ELECTRON-OPAQUE FIBRILS AND GRANULES IN AND BETWEEN THE CELL WALLS OF HIGHER PLANTS

The components of higher-plant cell walls which become electron-opaque after staining with ruthenium-osmium were studied by electron microscopy. A fibrillar material which absorbs this stain is a major wall constituent in the root epidermal cells of carrot and morning glory. In both form and size, these fibrils resemble those found on the surface of suspension-cultured cells of the same species Some cells of woody species show an irregular distribution of electron-opaque material in the cell wall matrix and middle lamella. This material, which has an amorphous appearance with many electron stains, is shown by ruthenium-osmium staining to be an aggregate of discrete granules, 150–220 A in diameter. These observations are not consistent with the concept of the cell wall matrix and middle lamella as an amorphous, uniform gel     

The use of alizarin red S to detect and localize calcium in gametophyte cells of ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca2+ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KCl to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca2+. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca2+ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

Fluorescence of yeast vitally stained with ethidium bromide and propidium iodide

Both ethidium bromide and propidium iodide stain growing yeast. As visualized in the fluorescence microscope, ethidium stains the nucleus and cytoplasm in wild type yeast and in those grown in 10% dextrose, with brightly fluorescent cytoplasmic granules being present in both. Under the latter conditions, the mitochondria are repressed but not absent. In rho 0 cells, in which the mitochondrial DNA is absent, ethidium appears to bind to the cell wall or membrane preferentially with no cytoplasmic granules being visible. In all cell types, propidium appears to bind the cell wall or membrane with no cytoplasmic granules being visible in any cell. The staining patterns thus suggest greater differences in the binding of these two types to mitochondrial DNA in situ than is suggested by their in vitro behavior. These differences in binding could explain their different mutagenic capacities..     

Characterization of fibrous compound of Golgi vesicles in tapetal cells ofDelphinium

Summary It was shown that Golgi structures abundantly appearing in tapetal cells ofDelphinium Ajacis L. developing anthers, prior to meiocytes meiosis, show a fine fibrous material within their vesicles. At the time of the formation of tapetal cell wall this fibrous component, released by an exocytotic process, is incorporated into the cell wall. The membrane of dictyosomes derived vesicles participates in the development of plasma membrane. Fibrous material appears to be morphologically similar to the fibrils of tapetal cell wall; this cell wall gives a positive reaction for cellulose and pectins, as visible in the light microscope. Moreover, the fibrous and pectinase resistant compound of dictyosomes derived vesicles and the fibrils of cell wall disappear partly after cellulase digestion which proves their cellulosic character. On the other hand pectinase treatment as well as ruthenium red staining suggest associated with cellulose pectins within Golgi vesicles.     

The effects of tracheid dimensions on variations in maximum density of Picea glehnii and relationships to climatic factors

An investigation was made of the effects of tracheid dimensions on variations in the maximum density of Picea glehnii Mast., which were associated with climatic changes. Radial cell diameter and the thickness of the tangential cell walls of the last-formed cells in 90 annual rings of nine trees with different annual ring widths were analyzed by image analysis. Correlations between maximum density and tracheid dimensions indicated that changes in maximum density were due mainly to changes in cell wall thickness of the last-formed cells in annual rings and were not due to changes in radial cell diameter. The effects of climatic factors on tracheid dimensions were examined by application of dendroclimatological techniques. A chronology of cell wall thickness that represented common signals among trees was established. Simple correlation and response function analyses of the chronology revealed that cell wall thickness was influenced positively by summer temperature and negatively by precipitation in August, and these responses were similar to those of maximum density. The study demonstrated that variations in maximum density were due to variations in the cell wall thickness of the last-formed cells, which varied depending on the weather in summer. Received: 8 February 1999 / Accepted: 7 October 1999     

The Use of Alizarin Red S To Detect and Localize Calcium in Gametophyte Cells of Ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca²⁺ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KC1 to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca²⁺. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca²⁺ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled beta-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS : I. The Structure of the Cell Wall of Cylindrotheca fusiformis Reimann and Lewin

An electron microscope study on the cell wall of the diatom Cylindrotheca fusiformis was carried out using stereoscopic and sectioning techniques. Material prepared by an enzyme treatment or by a mechanical method showed that the wall consists of two major components: a silica shell and organic material. Vapor of hydrofluoric acid was employed to remove the silica and thereby reveal the arrangement of the organic material. An attempt was made to increase the contrast of the organic component by "staining." Uranylacetate not only increased the electron opacity of the organic material but also apparently decreased the electron opacity of the silica shell. In ultrathin sections of complete cells, the structure as revealed by stereoscopy could be confirmed and extended. Every part of the silica shell is tightly enclosed by organic material. In the valve region the silica enclosed in this way is located between other layers of organic material. The whole cell wall is surrounded by a mucilaginous substance which stains with ruthenium red.     

Staining and osmotic properties of young gametophytes ofPolypodium vulgare L. and their bearing on rhizoid function

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.     

The Detection of Tension Wood with Fluorescent Dyes

Sections of a thickness of 15 β cut from unfixed material by means of a freezing microtome were treated for 1 hr with 0.2% aqueous solutions of a number of fluorochromes. Under normal microscopical conditions no uniform staining pattern could be observed. With the aid of an ultraviolet microscope, however, one type of fluorescence pattern became apparent. In all cases observed the thickened gelatinous secondary cell walls of tension wood showed a remarkable lack of fluorescence; this in marked contrast with a very distinct fluorescence exhibited by the primary cell wall and by both primary and secondary cell walls of normal wood.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled β-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.     

卵巢囊性颗粒细胞瘤的临床病理学特征、诊断及鉴别诊断

目的:探讨卵巢囊性颗粒细胞瘤的临床病理学特征、诊断和鉴别诊断要点。方法:复习1例卵巢囊性颗粒细胞瘤患者的临床资料、肿物的大体以及镜下病理组织学特征、免疫组化染色特征。结果:患者左侧单房囊性颗粒细胞瘤,大小为14.6 cm×18.9 cm×9.8 cm,囊壁厚薄不均一;镜下可见内壁由颗粒细胞组成,细胞层数不一,可见典型的Call-Exner小体;免疫组化染色可见α-inhibin,Vimentin,CD99均阳性。结论:卵巢囊性颗粒细胞瘤可依据镜下发现不典型增生的颗粒细胞、典型的Call-Exner小体和核沟等特征性的组织形态学作出诊断。     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

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.     

Effect of Yeast Cell Morphology,Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.     

Feulgen, iron-propionocarmine and cupra-ammonium in preparing algal chromosomes for light microscopy.

A method for obtaining algal chromosomal preparations is described employing the Feulgen method for DNA staining, Fe-propionocarmine as an enhancing stain, and cupra-ammonium to remove cell wall material. Fe-propionocarmine applied as a gradient to the side provides cells stained with the Feulgen stain alone or with the Feulgen Fe-propionocarmine stain, thereby facilitating useful comparison. Where dilute the Fe-propionocarmine enhances nuclear staining without staining orthe organelles; where more concentration it also stains the nucleolus, spindle, spindle polar bodies, pyrenoid and protoplast. Treatment with cupra-ammonium, to remove polysaccharide wall material, followed by neutralization with propionocarmine, enables thinner squashes and better chromosome spreads without loss of differential staining. Preparations mounted in euparal are long-lasting.     

Light microscopic detection of PAS-positive substances with thiosemicarbazide in freeze-substituted ovaries of Paspalum longifolium before pollination

Summary Localization of polysaccharides in the freeze-substituted, Eponembedded ovaries of Paspalum longifolium prior to pollination was carried out by periodic acid-Schiff's (PAS), periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-AgPr) and periodic acid-thiosemicarbazide-osmium (PA-TSC-OsO₄) reactions. The specificities of these three reactions were also studied. These three reactions are all effective for light microscopic demonstration of polysaccharides in the filiform apparatus, starch grains in the cells and PAS substance in the micropylar region. Nonspecific staining of the nucleoli of the egg and polar nuclei was observed in the PAS reaction. The PA-TSC-AgPr reaction is very specific for polysaccharides but its overall reaction takes a much longer period of time than the PAS reaction. The PA-TSC-OsO₄ reaction colors the cytoplasm and nuclei of most cells and therefore stains of the cell walls, especially those of the egg cell and synergids, do not stand out clearly. The synergid cytoplasm contains some amorphous polysaccharides and thus it colors even in PAS and PA-TSC-AgPr preparations. In the mature embryo sac, the egg and central cell as well as antipodals are vacuolated but the two synergids have no visible vacuoles under light microscope. Each synergid has a prominent filiform apparatus at the micropylar end, which stains intensely in all three preparations. The walls of the central cell and antipodals adjacent to the nucellar cells have many inward papillae which are also intensely stained in all three preparations. Starch grains are abundant in the ovary wall and usually absent in the nucellus and integuments. They are present in the egg, central cell and antipodals, but not in the two synergids.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Early death at medium acidification and survival after low pH adaptation inCryptococcus neoformans

WhenCryptococcus neoformans was grown in yeast nitrogen base (YNB) supplemented with 0.5% glucose, the medium was acidified to below pH 3 during the exponential growth phase, which caused early growth-phase death in susceptible strains. Even in resistant strains, 30–70% cells died if incubated for 2 d in YNB supplemented with 1.5% glucose, whereas the remaining cells survived long. Two types of fatal alterations have been observed in dead cells. In the first type, release of cytoplasm occurred through weakened parts of the cell wall; structures attached to cell walls of dead cells were shown to be rich in proteins by FITC staining, indicating their cytoplasmic origin. In the second type, cells shrank distinctly with no sign of wall rupture. The shrinkage may be due to dysfunction of the plasma membrane at low pH. The mechanism of cell survival in medium below pH 3 was also examined. Aniline blue alone, or calcofluor together with methylene blue, allowed cell wall glucan or chitin and dead cell cytoplasm to be stained simultaneously. In the later stages of incubation, cells showing bright staining for cell wall glucan and chitin emerged. These changes in cell wall synthesis could be considered as an adaptation mechanism to acidification of the medium, because such cells survived longer than cells showing no change in the cell wall staining pattern.     

Feulgen, Iron-Propionocarmine and Cupra-Ammonium in Preparing Algal Chromosomes for Light Microscopy

A method for obtaining algal chromosomal preparations is described employing the Feulgen method for DNA staining, Fe-propionocarmine as an enhancing stain, and cupra-ammonium to remove cell wall material. Fe-propionocarmine applied as a gradient to the slide provides cells stained with the Feulgen stain alum or with the Feulgen Fe-propionocarmine stain, thereby facilitating useful comparison.

Where dilute the Fc-propionocarmine enhances nuclear staining without staining other organelles; where more concentrated it also stains the nucleolus, spindle, spindle polar bodies, pyrenoid and protoplast. Treatment with cupra-ammonium, to remove polysaccharide wall material, followed by neutralization with propionocarmine, enables thinner squashes and better chromosome spreads without IOU of differential staining. Preparations mounted in euparal are long-lasting.     

Assessment of bacterial viability status by flow cytometry and single cell sorting

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished : reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g. enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by : active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite^®) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.