Vacuolar localization of phosphorus in hyphae of Phialocephala fortinii, a dark septate fungal root endophyte

Phialocephala fortinii is a dark septate fungal endophyte that colonizes roots of many host species. Its effect on plant growth varies from being pathogenic to beneficial. The basic biology of this species has received little research, and thus the main objectives of this study were to determine cytological features of hyphae, including the nature of the vacuolar system, and whether polyphosphate was present in vacuoles. Both living hyphae and hyphae that had been rapidly frozen and freeze substituted before embedding were studied. A complex system of vacuoles, including a motile tubular vacuolar system, elongated vacuoles, and spherical vacuoles, was demonstrated in living hyphae by the fluorescent probe Oregon Green 488 carboxylic acid diacetate, using laser scanning confocal microscopy. The motile tubular vacuolar system was more prevalent at the hyphal tip than in more distal regions, whereas elongated vacuoles and spherical vacuoles were more abundant distal to the tip. All vacuoles contained polyphosphate as shown by labelling embedded samples with recombinant polyphosphate binding domain of Escherichia coli exopolyphosphatase, containing Xpress tag at the N-terminal end, followed by anti-Xpress antibody and a secondary antibody conjugated either to a fluorescent probe for laser scanning confocal microscopy or colloidal gold for transmission electron microscopy. The polyphosphate was dispersed in vacuoles. This was confirmed by staining embedded samples with 4',6-diamidino-2-phenylindole and viewing with UV light using epifluorescence microscopy. These cytological methods showed that the tubular vacuolar system had lower concentrations of polyphosphate than the spherical vacuoles. Lipid bodies were present around vacuoles.     

Immunocytochemical investigation of the causal agent of cassava mosaic disease in southern Africa

Cassava mosaic disease (CMD) exists throughout Africa, and cassava latent virus (CLV) has been implicated as the etiological agent in Kenya and West Africa. However, in Southern Africa, the causal agent of CMD was not until recently associated with CLV, and the possibility of a second flexuous virus particle has not been ignored. Attempts to isolate and visualize CLV antigen have been successful with Nicotiana benthamiana, an indicator host plant of CLV, but all efforts to isolate and visualize particles in infected cassava plants have failed. Immunocytochemical studies were undertaken in an attempt to localize virus antigen in infected cassava tissue.Cytochemical staining (light microscope) of infected cassava leaf material revealed the presence of inclusion bodies in epidermal and palaside mesophyll cells, and in epidermal collenchyma and outer parenchyma cells from the petiole and stem. However, transmission electron-microscopical (TEM) investigations revealed electron dense bodies in the cytoplasm, and no characteristic CLV nuclear inclusion bodies were evident. Transmission experiments to N. benthamiana and N. tabacum were attempted and leaves, exhibiting symptoms, examined microscopically. The nuclei appeared swollen (in comparison to uninfected leaves), a characteristic of CLV- infected N. benthamiana. However at the TEM level, no characteristic fibrillar-ring inclusion bodies or particles, could be visualized.Further immunocytochemical investigations were initiated, employing antisera raised against CLV isolated from N. benthamiana, and antisera for cassava common mosaic virus (CCMV), cassava brown streak virus (CBSV) and cassava X virus (CsXV). Goat anti-rabbit IgG-gold was used as a direct stain. No labelling occurred with CCMV and CBSV antisera. Intense gold labelling was located in the cytoplasm of phloem, mesophyll and epidermal cells of infected cassava and to a lesser extent in N. tabacum and N. benthamiana using affinity chromatography purified CLV antiserum. Little labelling was observed in nuclei of infected cells. Inconclusive results were obtained with CsXV antiserum.Immunogold labelling located CLV viral antigens in infected cassava leaf tissue. This observation, together with positive ELISA, transmission and DNA hybridization experiments, proves conclusively that CLV viral antigen is present in infected cassava in Southern Africa. However, most viral antigen in infected cassava, unlike N. benthamiana (fibrillar and granular nuclear inclusions) appears to be in the cytoplasm. This may tentatively suggest that the CLV protein is synthesized in the cytoplasm of its natural host, cassava, even though the virus may assemble in the nucleus at the appropriate time. However, as yet no virus inclusions have been observed in nuclei of infected cassava. Due to previous isolation of a flexuous rod and ambiguous staining results, the possibility of two viruses in cassava cannot be ruled out.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Summary Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tisues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.     

Polychromatic Stains for Thin Sections of Beta Embedded in Epoxy Resin

Thin sections of leaves and anthers of Beta vulgaris L., fixed in glutaraldehyde-OsO₄ and embedded in epoxy resin, were stained with different stains at pH ranges from 5 to 9 at 50 C to select those that provided polychromatic staining of suitable intensity. The thionin derivatives, Azure B, Toluidine Blue O, and polychrome Methylene Blue provided adequate staining, as did the commercially prepared stain Paragon PS 1301. Azure B stain was superior for sugar beet 0.5μ monitor sections: cytoplasm appeared grey; nuclei, blue-gray; nucleoli, blue; chloroplasts, blue-green; primary walls, blue; and secondary walls, light blue. Choice of one of the stains mentioned probably would depend upon the plant material under study.     

A puzzling feature of Colloidal Iron positive and Alcian Blue negative material

Summary A puzzling feature of Colloidal Iron positive and Alcian Blue negative substance is encountered in yolk sac of young larvae of a fish —Tilapia mossambica. This yolk material is PAS positive (proved to be due to neutral mucopolysaccharide) and negative to Toluidine Blue, Azure A (pH 2 to 4.5), Aldehyde Fuchsin and AB pH 1. More work is necessary to establish the exact chemical nature of the CI positive material.     

Influence of plastic embedding media on staining and morphology of lipid bodies

Summary The following light microscopy staining techniques were applied to plastic embedded tissues: Toluidine Blue, Sudan III, Sudan Black and Nile Blue sulphate. All these procedures stain the lipid bodies; however the Sudan III appears to be the most suitable.The ultramicroscopic appearance of lipid bodies of tissues embedded in Epon, Araldite and Vestopal W is presented. The effects of dehydration as well as of different electron microscopic staining techniques is also investigated.This work was supported by Grants of Consiglio Nazionale delle Ricerche of Italy: no. 115/1151/0-1247, no. 115/0815/0-1365.The excellent technical assistance of Miss Hermina Spiele is gratefully acknowledge.     

Changes in the plastid ultrastructure duringSedum rotundifolium leaf development

The ultrastructure of plastids was investigated in succulent leaves ofSedum rotundifolium to examine their changes during development. Leaves were categorized as etiolated, immature, young, and mature, based on their developmental stage and size. Of particular interest were the features of the tubular inclusion bodies (TIBs) and starch grains. These, along with vacuole size, showed remarkable changes over time. Etioplasts of unexposed leaves had prolamellar bodies, abundant starch grains, large TIB, few plastoglobuli, and thylakoid systems. Membranes of the thylakoids were still continuous with those of the prolamellar body. The plastids were often influenced by the presence and profile of inclusion bodies and starch grains throughout the early stages. Morphology was highly variable in the etioplasts but consistently hemispherical or ovoid in mature chloroplasts. TIB was most abundant in the etiolated leaves, but disappeared completely with development. Starch grains also became significantly reduced in size. Both young and mature mesophyll cells exhibited a normal chloroplast ultra-structure and huge central vacuoles, with an extremely thin peripheral cytoplasm. Grana were extensive and comprised a large portion of the chloroplasts. Traces of peripheral reticulum were also discovered in the chloroplasts of expanded leaves. The implications of these ultrastructural changes in the tubular inclusions and starch grains are discussed with relevance to Crassulacean acid metabolism (CAM).     

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

Chloroplast microtubules in young leaves ofSedum rotundifolium

Well-defined tubular inclusions were detected in mesophyll chloroplasts of youngSedum rotundifolium leaves. The size and distinctly uniform arrangement of tubular inclusions were the most noticeable features. The chloroplast usually contained a large inclusion, sometimes extending almost as long as the chloroplast. Such inclusions were built up from microtubules exhibiting aggregates of either large plate-like or paracrystalline structures depending on the section angles. These inclusions were often quite huge, measuring as much as 7.1 μm in length and 2.6 μm in width. The diameter of the microtubule was approximately 25 nm. The microtubule aggregates were non-membrane bounded structures enclosed partly by the thylakoids. The microtubules in the aggregate were all displaying the same definite orientation. Cross-sectional views clearly demonstrated the characteristic hexagonal arrangement within paracrystalline structures. Longitudinal sections of the chloroplast microtubules showed that they were in perpendicular orientation to the chloroplast envelope. They were not connected to these membranes in any case. In general, one microtubular aggregate was seen in each chloroplast section. The role of tubular inclusions in the chloroplasts related to the photosynthetic mode was discussed.     

Relationship between the axial periodicity and staining of collagen by the Masson trichrome procedure

Synopsis Although wide variations in the axial periodicity of collagen fibrils in tissue sections have been reported previously, the comparative study presented in this paper of the axial periodicity and the Masson trichrome staining of collagen fibres from different tensional situations demonstrates that marked variations in collagen axial periodicity can be directly correlated with the tensional state of the tissues before fixation and with their Masson trichrome staining reaction.Collagen fibres from tensional situations consistently exhibit a longer periodicity and retain the Acid Fuchsin component of the Masson trichrome procedure, whilst collagen fibres from non-tensional situations have a shorter periodicity, fail to retain the Acid Fuchsin component, and are coloured by the Light Green counterstain.     

番茄环纹斑点病毒与马铃薯Y病毒复合侵染烟草的细胞病理特征

在自然情况下, 番茄环纹斑点病毒(TZSV)和马铃薯Y病毒(PVY)通常复合侵染同一植株。该文首次报道了TZSV和PVY复合侵染烟草(Nicotiana sp.)植株的细胞病理特征, 并与单独侵染进行了比较分析。复合侵染的烟草植株细胞中, TZSV病毒颗粒明显增多, 并聚集于囊膜内形成包涵体, 与PVY的风轮状及片层状内含体和病毒颗粒聚集体交叉分布于细胞质(内含线粒体明显增多)中, 线粒体、叶绿体和细胞核结构较完整; 两种病毒的颗粒、包涵体和内含体数量均较单一侵染增多, 且对寄主亚细胞结构的破坏较单一侵染为轻, TZSV和PVY及其与寄主的互作可能存在协生作用。     

Electron Microscopy of Measles Virus Replication

Replication of measles virus in HeLa cells was examined by electron microscopy with ultrathin sectioning and phosphotungstic acid negative staining methods. The cytoplasmic inclusion bodies consisted of masses of helical nucleocapsid which was similar in structure to the nucleocapsid found in measles virions. The cytoplasmic helical nucleocapsid appeared to align near the HeLa cell membrane, and the membrane differentiated into the internal membrane of the viral envelope and the outer layer of the short projections. The viral particles were released by a budding process involving incorporation into the viral envelope of membrane which was contiguous to but morphologically altered from the membrane of the HeLa cells. The intranuclear inclusion bodies were composed of tubular structures similar to those found in the cytoplasmic inclusion bodies. These structures aggregated to crystalline arrangement. The relationship between nuclear inclusion body and replication of measles virus was not clear.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

Localization and compartmentation of Al in the leaves and roots of tea plants

Under acid soil conditions, solubility of aluminum (Al) increases leading to toxicity for plants. Al accumulator species such as tea, however, accumulate high levels of Al in tissues without toxicity symptoms. In this work, Al localization and compartmentation were studied in tea [Camellia sinensis (L.) O. Kuntze] grown hydroponically at 0 or 100 µM Al for eight weeks. Plant dry matter production was significantly higher in the presence of Al and accumulated up to 1.21 and 6.18 mg Al/g DW in the leaves and roots, respectively. About 40-50% of Al was partitioned into cell wall (CW)-bound fraction without any difference among leaves of different age and roots. A significant increase of the soluble phenolics fraction by Al was observed in both leaves and roots. Conventional and confocal laser scanning microscopy images of morin-stained roots indicated a high fluorescence signal in the caps and adjacent mersitematic cells. Towards basal parts, however, Al tended to accumulate mainly in the root hairs, rhizodermal and endodermal cell layers and slightly in the cortex while it was clearly excluded from the central cylinder. A high Al-morin signal was detected from the CW compared with other parts of the cells. Relatively high green fluorescence signal was emitted from the epidermal cell layer, trichomes, vascular bundle region and stomatal cells of particularly young leaves. Our study provides evidences for involvement of both avoidance and tolerance mechanisms for Al in tea plants.     

Fine Structure of Cellular Inclusions in Measles Virus Infections

Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell. The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mµ in diameter, the strands seen in the inclusions cannot be fully formed virus.     

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

NILE BLUE, ANILINE BLUE AND NEUTRAL RED AS INDICATORS OF LIPOLYSIS

SUMMARY: Dyes which have been used to detect lipolysis (Nile Blue, Aniline Blue and Neutral Red) and others (Methylene Blue, Toluidine Blue and Thionin) were critically examined for their inhibitory effects in both liquid and solid media. It was confirmed that Nile Blue would inhibit the Gram-positive bacteria tested, even at 2 × 10⁻⁵. Gram-negative bacteria were not inhibited at 1 × 10⁻⁴ and the colour change in fat media was good. Aniline Blue and Neutral Red did not inhibit Gram-positive organisms at 1 × 10⁻⁴ and the colour change was moderately good. The other three dyes were not toxic at 2·5 × 10⁻⁵, but there was no colour change. When butter fat saturated with the precipitated bases of Nile Blue, Basic Fuchsin, Crystal Violet or Malachite Green was incorporated in peptone-Yeastrel agar, the resulting media were toxic to Gram-positive bacteria. Fat saturated with the bases of Aniline Blue or Neutral Red permitted growth, but the colour change with lipolytic species was not striking, being one of intensity only. Satisfactory growth and colour changes were obtained using butter fat stained with the inert dye Waxolene Green in medium containing Neutral Red in the aqueous phase, and with butter fat stained with the oxazine base of Nile Blue in agar containing Aniline Blue.     

Further studies on ultrastructure of plants infected withPetunia ringspot virus

Summary New methods of fixation and embedding have revealed in plants infected withPetunia ringspot some structural features not described before. These include the X-bodies, 80 per cent of which are formed by tubular elements which are responsible for the positive staining specific for Golgi apparatus. The tubular elements are morphologically similar to agranular endoplasmic reticulum as described in some animal cells. The rest of the inclusion is formed by normal cytoplasmic elements in which are embedded long rod-shaped tubules 600 Å wide and more than 7,000 Å long, cross sections of which are formed by 10 subunits. These subunits are arranged in a helical way to form the large tubules. These subunits are probably the actual virus particles, which would be icosahedral and would form tubular crystals. Icosahedral virus particles would also form true crystalline inclusions.It is not known what the role of the agranular endoplasmic reticulum and of some dense osmiophilic bodies found in it may be in the multiplication of the virus. However, these components induced by the virus infection probably result in the manufacture of some proteins or other substances necessary for virus multiplication.     

Histochemical analysis of urea-unmasked glycosaminoglycans in the skin of the rat and mouse

Summary Histochemical analysis of urea-unmasked glycosaminoglycans has been performed in connective tissues of the rat and mouse skin by means of combined staining and enzyme digestion procedures. The staining procedures used were Alcian Blue pH 1.0, Alcian Blue pH 2.5, Aldehyde Fuchsin, periodic acid-Schiff (PAS), Alcian Blue pH 2.5-PAS, high iron diamine and low iron diamine methods. The digestive enzymes employed wereStreptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained indicated that the major components of the glycosaminoglycans in the connective tissues of the skin were hyaluronic acid, dermatan sulphate and chondroitin sulphate A and/or C, whereas the tissues were devoid of keratan sulphate.