伦敦白胶和茶：一种既快又安全的用于透射电子显微镜的细菌样品制备方法

【目的】检测乌龙茶提取物是否可作为电子染色剂取代醋酸双氧铀用于细菌细胞染色,使其能在透射电子显微镜下进行观察。【方法】利用伦敦白胶对细菌样品(大肠杆菌和金黄色葡萄球菌)进行胶块的制备,再在复染铅与不复染铅这两种情况下对超薄切片样品进行3种不同染色剂的电子染色,之后在透射电子显微镜下观察比较其不同之处。这3种不同的染色剂分别是醋酸双氧铀、0.05%乌龙茶提取物以及0.1%乌龙茶提取物。首先将带有超薄切片样品的铜网悬浮于不同的待比较染液中10?15 min,若需进一步用柠檬酸铅复染,则将经3次蒸馏水冲洗过后的铜网再次悬浮于柠檬酸铅染液中8?10 min。【结果】复染铅的情况下,在透射电子显微镜下无论是大肠杆菌还是金黄色葡萄球菌,利用3种电子染色剂进行染色的结果均非常相似。【结论】实验结果表明,在观察细菌结构中,乌龙茶提取物可以替代醋酸双氧铀进行透射电子显微镜样品的电子染色。     

Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.     

The correlation between bismuth and uranyl staining and phosphorus content of intracellular structures as determined by electron spectroscopic imaging

Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.     

Contrasting of Lowicryl K4M thin sections.

A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Contrasting of Lowicryl K4M thin sections

Summary A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferase from Streptococcus sobrinus

Abstract Oolong tea extract (OTE) and the purified polymeric polyphenols from OTE have been found to inhibit glucosyltransferase (GTase) of mutans streptococci. In view of the partial fermentation characteristic of oolong tea, we describe here an in vitro model reaction system to produce partially fermented products of d-(+)-catechin or green tea extract (GTE) using horseradish peroxidase. A dimeric catechin molecule was identified as dehydro-dicatechin A by instrumental analyses. The molecular size of some oligomeric catechins was estimated by the elution profile with HPLC. These catechin oligomers markedly inhibited GTase from Streptococcus sobrinus 6715. As the degree of polymerization of catechin or GTE increased, GTase was inhibited more effectively. These results suggest that polymeric polyphenols found in OTE are synthesized by partial fermentation due to oxidases/peroxidases present in tea leaves.     

Selective staining of nucleic acid containing structures by uranyl acetate-lead citrate

The conventional uranyl acetate-lead citrate staining procedure has been modified to obtain selective staining of nucleic acid structures in glutaraldehyde fixed and epon embedded material. The selectivity of the stain is dependent on the concentration of uranyl acetate in the staining solution, the pH of the staining solution, the staining time and the washing conditions.     

INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : I. Fixation and Electron Staining Reactions

Chick embryo mitochondria, studied with the electron microscope, show crista-free areas of low electron opacity. These areas are observable after fixation with osmium tetroxide, calcium permanganate, potassium permanganate, formaldehyde, acrolein, acrolein followed by osmium tetroxide, uranyl acetate followed by calcium permanganate, and acetic acid-alcohol. Staining of sections with lead hydroxide or uranyl acetate, or with both, resulted in an increased density of a fibrous material within these areas. The appearance of the fibrous structures varied with the fixative employed; after fixation with osmium tetroxide the material was clumped and bar-like (up to 400 A in diameter), whereas after treatment of osmium tetroxide-fixed tissues with uranyl acetate before dehydration the fibrous structures could be visualized as 15 to 30 A fibrils. Treatment with ethylenediaminetetraacetate (EDTA) in place of uranyl acetate coarsened the mitochondrial fibrils. After fixation with calcium permanganate or potassium permanganate, or a double fixation by uranyl acetate followed by calcium permanganate, the fibers appeared to have a pattern and ultrastructure similar to that observed after the osmium tetroxide-uranyl acetate technique, except that some of them had a slightly greater diameter (up to 50 A). Other fixatives did not preserve the fibers so well. The fibers appeared strongly clumped by formaldehyde fixation, and were difficult to identify after fixation with acrolein or acetic acid-alcohol. The staining of nucleic acid-containing structures by uranyl acetate and lead hydroxide was improved by treatment of osmium tetroxide-fixed sections with hydrogen peroxide, and the mitochondrial fibers also had an increased density in the electron beam after this procedure. The staining characteristics suggest the fibrous material of chick embryo mitochondria to be a nucleic acid-containing structure, and its variable appearance after different fixations parallels that previously reported, or described in this paper, for the nucleoplasm of bacteria and blue-green algae. The results, in addition to those described in the accompanying communication, indicate that these mitochondria contain DNA.     

Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy

Summary Specimens infused with or suspended in a mixture of 10–30% poly(vinylpyrrolidone) and 2.07–1.61m sucrose can often be more easily frozen-sectioned than those infused with sucrose alone. The pH of such a mixture can be efficiently adjusted to neutrality by using Na₂CO₃. Use of poly(vinylpyrrolidone) causes little or no increase in the background level of immunolabelling. Adsorption staining of ultrathin frozen sections with a mixture of uranyl acetate and poly(vinyl alcohol), i.e. a simple thin-embedding of the sections in such a mixture, produces positive staining effects that are often enough to delineate structures of many organelles. When OsO₄-treated frozen sections are stained with uranyl acetate and further adsorption-stained with a mixture of lead citrate and poly(vinyl alcohol), the overall staining effects are similar to those observed in double-stained conventional sections.A large portion of the findings was reported as a part of the author's presentation in the 11th International Congress on Electron Microscopy, held in Kyoto, Japan, in 1986.     

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)     

Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.     

Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans Streptococci.

Oolong tea extract (OTE) was found to inhibit the water-insoluble glucan-synthesizing enzyme, glucosyltransferase I (GTase-I), of Streptococcus sobrinus 6715. The GTase-inhibitory substance in the OTE was purified successive adsorption chromatography on Diaion HP-21 and HP-20 columns; this was followed by further purification by Sephadex LH-20 column chromatography. A major fraction that inhibited GTase activity (fraction OTF10) was obtained, and the chemical analysis of OTF10 indicated that it was a novel polymeric polyphenol compound that had a molecular weight of approximately 2,000 and differed from other tea polyphenols. Catechins and all other low-molecular-weight polyphenols except theaflavin derived from balck tea did not show significant GTase-inhibitory activities. It was found that OTE amd PTF10 markedly inhibit GTase-I and yeast alpha-glucosidase, but not salivary alpha-amylase. Various GTases purified from S. sobrinus and Streptococcus mutans were examined for inhibition by OTE and OTF10. It was determined that S. sobrinus GTase-I and S. mutans cell-free GTase synthesizing water-soluble glucan were most susceptible to the inhibitory action of OTF10, while S. sobrinus GTase-Sa and S. mutans cell-associated GTase were moderately inhibited; no inhibition of S. sobrinus GTase-Sb was observed. Inhibition of a specific GTase or specific GTases of mutants streptococci resulted in decreased adherence of the growing cells of these organisms. The inhibitory effect of OTF10 on cellular adherence was significantly stronger than that of OTE.     

Electron microscopy of G-banded human mitotic chromosomes

Trypsin-treated human metaphase chromosomes stained with Giemsa and uranyl acetate showed clear, reproducible band structures under the transmission electron microscope (TEM). The banding pattern observed with TEM corresponded very closely to the G-band pattern visualized by light microscopy. The TEM images were used for karyotype analyses. Trypsin-treated chromosomes stained with uranyl acetate alone also showed clear G-bands under TEM. Shadow casting in addition to uranyl acetate staining revealed more structural detail of the chromosomes. Chromosome fibers, 200 Å–300 Å in diameter, were observed in the interband regions. Most chromosomes showed the major G-bands under the higher TEM magnification wit0out any trypsin treatment.     

PREFERENTIAL STAINING OF NUCLEIC ACID-CONTAINING STRUCTURES FOR ELECTRON MICROSCOPY

Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10^-2 M Ca⁺⁺, and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0°C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times (½ hour to 2 hours, at 0°C or 20°C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

Extraction of osmium-containing lipids by section staining for TEM

Postfixation with osmium-ferrocyanide or OSO4 renders lipid droplets in rat liver and kidney homogeneously electron dense without additional section staining. In sections of the same block that have been single stained by uranyl acetate or lead citrate, lipid droplets show a more electron translucent center surrounded by a dense rim. In sections double stained with uranyl acetate and lead citrate, lipid droplets frequently appear as empty vacuoles, from which the electron dense content has been completely extracted.     

A FINE STRUCTURAL ANALYSIS OF INTERCELLULAR JUNCTIONS IN THE MOUSE LIVER

Zonulae occludentes and gap junctions were examined both in the intact mouse liver and in a junction-rich membrane fraction from homogenized mouse liver. These preparations were visualized with the techniques of uranyl acetate staining en bloc, staining with colloidal lanthanum, negative staining with phosphotungstate, and freeze-cleaving. The zonula occludens is arranged as a meshwork of branching and anastomosing threadlike contacts sealing the lumen of the bile canaliculus from the liver intercellular space. The gap junction is characterized in section by a 20 A gap between the apposed junctional membrane outer leaflets, and permeation of this space with lanthanum or phosphotungstate reveals a polygonal lattice of subunits with a center-to-center spacing of 90–100 A. Freeze-cleaved gap junctions show a similar lattice. Extraction of junction-rich fractions with 60% aqueous acetone results in a disappearance of the 20 A gap in sectioned pellets and an inability to demonstrate the polygonal lattice with either the freeze-cleave or negative staining techniques. Extraction of the membranes with 50% acetone does not produce this effect. Thin-layer chromatography of the acetone extracts reveals a group of phospholipids in the 60% extract that are not detectable in the 50% extract. Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment. The effects of other reagents on the junctions are reported.     

人类微孢子虫检测方法研究进展

微孢子虫(microsporidia)是一类专性细胞内寄生的单细胞真核生物。是引起微孢子虫病的真菌类病原。在已知并被命名的1500多种微孢子虫中,共有9个属中的17个虫种可以感染人。人类微孢子虫可侵染包括肠道、肝、肺、脑等部位,引起慢性腹泻、肝炎、角膜炎、脑炎、血液系统性感染等,严重影响人类健康。研究开发快速高效的人类微孢子虫诊断方法成为当前病原微生物检测领域研究的热点。人类微孢子虫的发现历史实际上是伴随检测方法的不断进步而逐渐进行的。这些检测方法包括,透射电镜(transmission electron microscopy)、苏木精-伊红染色(hematoxylin-eosin stain,HE)、亚甲蓝染色(methylene blue)、吉姆萨染色(giemsa)、革兰氏染色(gram stain)、韦伯氏改良三色染色法(Weber’s chromotrope-based staining)、荧光增白剂染色法(calcofluor white staining)、抗原检测、抗体检测、实时荧光定量PCR (quantitative real-time PCR,q PCR)、环介导...     

Gram-Chromotrope: a New Technique that Enhances Detection of Microsporidial Spores in Clinical Samples

We have developed a new staining procedure that combines the traditional Gram staining for bacteria and the Weber's chromotrope staining method, the standard technique for the detection of microsporidia spores in clinical Specimens. This “Gram-chromotrope” staining technique enhances the staining characteristics of microsporidia spores and facilitates the easy detection and differentiation of spores from other microorganisms that are found in clinical specimens, especially stool samples. This new technique is fast, reliable, and simple to perform, and can be easily adapted for use in clinical laboratories.