Cytotoxicity of alkaline-tolerant dairy-associated Bacillus spp

The cytotoxicity of five Bacillus spp. isolated from alkaline cleaning solutions in South African dairies was evaluated against McCoy mouse cells using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)-based assay, confocal scanning laser microscopy and scanning electron microscopy. According to the MTT-based assay, two of the Bacillus isolates (Bacillus licheniformis 5 and B. pumilus 122) were cytotoxic to McCoy cells and the cytotoxic components were heat labile. Confocal scanning laser microscopy combined with fluorescent staining using propidium iodide and fluorescein diacetate indicated that cytotoxic effects occurred within 3 h, appeared to be membrane active and resulted in cell necrosis. Scanning electron microscopy showed that McCoy cells exposed to the cytotoxic components exhibited morphological damage.     

Peanut lectin binds to a subpopulation of mitochondria-rich cells in the rainbow trout gill epithelium

Fluorescently labeled peanut lectin agglutinin (PNA-FITC) was used to identify a subtype of mitochondria-rich (MR) cells in the gills of freshwater rainbow trout. In situ binding of PNA-FITC was visualized by inverted fluorescence microscopy and found to bind to cells on the trailing edge of the filament epithelium as demonstrated by differential interference contrast optics. The amount of PNA-FITC binding on the filament epithelium increased with cortisol pretreatment concomitant with an increased chloride cell fractional area as demonstrated by scanning electron microscopy. Dispersed gill cells were isolated by trypsinization and separated using a discontinuous Percoll density gradient. Cells migrating to the 1.06-1.09 g/ml interface were found to be MR as demonstrated by staining with the vital mitochondrial dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide and transmission electron microscopy (TEM). However, only approximately 40% of the MR cells were found to bind PNA-FITC. Cortisol pretreatment increased the relative numbers of MR cells isolated from the dispersed gill cell population, but the relative proportions of PNA binding cells remained unchanged. Ultrastructural analysis of isolated cells in the TEM demonstrated that the MR cell fraction was comprised of a mixed population of chloride cells and pavement cells.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Surface morphology of immunocompetent cells isolated from spleen of Bufo himalayanus (Günther)

Immunocompetent cells were isolated from spleen of B. himalayanus and studied surface morphology of the three different cell types--(i) plastic adherent; (ii) nylon wool adherent; and (iii) nylon wool non-adherent cells. As revealed by scanning electron microscopy, they resembled the macrophages, B and T cells, respectively. Presence of such cell types indicated that Bufo himalayanus possessed a well-organized immune system. Further work is needed to characterize the functional efficacy of these immunocompetent cells found in B. himalayanus.     

The neurite-promoting domain of human laminin promotes attachment and induces characteristic morphology in non-neuronal cells

The interaction of cells with laminin and laminin fragments was studied in short-term cell attachment assays. Neurite-promoting chymotrypsin fragments of laminin were isolated using a monoclonal antibody which blocks neurite outgrowth on laminin. The fragments were shown, by electron microscopy after rotary shadowing and by immunological reactivity with different monoclonal antibodies, to contain only the distal end of the long arm. These fragments promoted the attachment and spreading of glioma, sarcoma, carcinoma, muscle, and endodermal cells to the same extent as intact laminin. The attachment was unaffected by peptides containing the RGD sequence. The morphology of the cells on the chymotrypsin fragments was indistinguishable from that on intact laminin but different from the morphology of the same cells on fibronectin. Light microscopy and scanning electron microscopy showed extensive process formation on laminin but not on fibronectin suggestive of increased cell motility in response to laminin. We conclude that the neurite-promoting domain of laminin contains a major site of interaction for non-neuronal cells and that this site induces a cellular response in certain non-neuronal cells that is unique to laminin.     

The polypeptide composition and ultrastructure of nuclear ghosts isolated from mammalian cells

The polypeptide species of non-membranous nuclear ghosts from purified cell nuclei are conserved among a variety of human, hamster and mouse cell types studied, including HeLa, BHK, 3T6 and Hep-2 cell lines. The polypeptide species present in nuclear ghosts from HeLa cells synchronized in various stages of the cell cycle are largely the same with minor variations. The isolated nuclear ghosts are similar, in terms of polypeptide composition, to other residual nuclear structures isolated by independent techniques. The nuclear ghosts appear as flattened sac-like structures when viewed by scanning electron microscopy. Transmission electron microscopy of the nuclear ghosts reveals ring-like structures which may represent the nuclear pores. Also observed are novel rod-shaped structures approximately 260 nm in length and 50 nm in diameter. The latter images either arise by a rearrangement during isolation of the nuclear ghost macromolecules or are a heretofore undescribed structure of intact nuclei.     

Localization of α-galactomannan on the surface of Schizosaccharomyces pombe cells by scanning electron microscopy

Galactomannan was localized by scanning and transmission electron microscopy on the cells and cell walls of Schizosaccharomyces pombe. The markers were prepared from colloidal gold granules labelled with an -galactopyranosyl-binding lectin isolated from the seeds of Bandeiraea simplicifolia. Part or all of this -galactomannan was present in the outer layer of the cell wall and was uniformly distributed even on the fission scars.Non-Standard Abbreviations Au lectin-labelled colloid - SEM scanning electron microscopy - TEM transmission electron microscopy     

Brittle fracture of epidermal cells by liquid shear.

A suspension of epidermal cells obtained from pig tail skin by trypsinization was subjected to high liquid-shear forces in a French press. The material issuing from the press was examined by phase-contrast microscopy, transmission electron microscopy and scanning electron microscopy. The cytoskeleton of tonofibrils retained the shape of cell fragments, and subcellular organelles remained enmeshed in the network of tonofibrils. Examination of some cell fragments by scanning electron microscopy revealed the internal organization of the tonofibrils. The relevance of these findings to the problem of isolating subcellular fractions from epidermis is discussed.     

Electron Microscopic Identification and Morphologic Preservation of Enriched Populations of Lung Cells Isolated by Laser Flow Cytometry and Cell Sorting: A New Technique

There is increasing need to verify the identities of cell subpopulations enriched by laser flow cytometry and fluorescence-activated cell sorting (FACS). When cell subpopulations isolated from whole organs or tissues have similar characteristics (e.g., size, granularity, staining), light, phase contrast or fluorescence microscopy may not provide sufficient resolution to identify isolated cells accurately and many flow cytometric parameters (e.g., viability, fluorescence) require the cells to be live at the point of analysis where the cell transects the laser beam. In some studies, cells identified by fluorescence microscopy as a highly enriched subpopulation were found by electron microscopy to contain significant populations of other cell types. A technique, fixation-in-flow (FIF), has been developed to increase ability to correlate morphological and laser analyses of cell subpopulations. Sheath fluid is replaced by fixative, permitting fixation to be initiated immediately after laser beam analysis of live cells. This new procedure yields improved cytoarchitectural preservation of recovered cell subpopulation(s) for evaluation by transmission or scanning electron microscopy. This report presents results from applying the methodology to identify more accurately cell subpopulations of the distal lung, specifically type II pneumocytes, Clara cells and pulmonary macrophages. A modification of this procedure was employed to isolate fibroblast subpopulations from murine lung fibroblasts grown in vitro and the procedure is being used to determine the responses of cultured fibroblasts to other permutations (e.g., X-irradiation, cytokines).     

Ultrastructure of cells cultured on polycarbonate membranes.

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 micrometer or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Electron microscopic identification and morphologic preservation of enriched populations of lung cells isolated by laser flow cytometry and cell sorting: a new technique

There is increasing need to verify the identities of cell subpopulations enriched by laser flow cytometry and fluorescence-activated cell sorting (FACS). When cell subpopulations isolated from whole organs or tissues have similar characteristics (e.g., size, granularity, staining), light, phase contrast or fluorescence microscopy may not provide sufficient resolution to identify isolated cells accurately and many flow cytometric parameters (e.g., viability, fluorescence) require the cells to be live at the point of analysis where the cell transects the laser beam. In some studies, cells identified by fluorescence microscopy as a highly enriched subpopulation were found by electron microscopy to contain significant populations of other cell types. A technique, fixation-in-flow (FIF), has been developed to increase ability to correlate morphological and laser analyses of cell subpopulations. Sheath fluid is replaced by fixative, permitting fixation to be initiated immediately after laser beam analysis of live cells. This new procedure yields improved cytoarchitectural preservation of recovered cell subpopulation(s) for evaluation by transmission or scanning electron microscopy. This report presents results from applying the methodology to identify more accurately cell subpopulations of the distal lung, specifically type II pneumocytes, Clara cells and pulmonary macrophages. A modification of this procedure was employed to isolate fibroblast subpopulations from murine lung fibroblasts grown in vitro and the procedure is being used to determine the responses of cultured fibroblasts to other permutations (e.g., X-irradiation, cytokines).     

An ultrastructural investigation on the polyethylene glycol-induced adhesion of tobacco nuclei and uptake of micronuclei by soybean protoplasts

Nuclei isolated from tobacco protoplasts were induced to be taken up by soybean protoplasts using a protocol involving polyethylene glycol (PEG), osmotic shock and pH shift. Transmission electron microscopy revealed that PEG treatment condensed the chromatin of the isolated nuclei. Close adhesion of isolated nuclei to the plasma membrane of protoplasts following PEG treatment, was observed by both scanning and transmission electron microscopic methods. Ultrastructural observations were also made on the formation of micronuclei in tobacco cells following the treatment with amiprophosmethyl (APM). Nuclei and micronuclei isolated from APM-treated cells were induced to be taken up by soybean protoplasts. A single case of uptake of an isolated micronucleus was observed by transmission electron microscopy. The observations on the effects of PEG on the isolated nuclei, micronuclei and protoplasts are discussed in relation to the possible mechanism of uptake of nuclei by protoplasts using PEG.     

MOLECULAR,ENZYMATIC AND ULRASTRUCURE CHARACTERIZATION OF THE PYRENOID OF THE SCALY GREEN MONAD MICROMONAS SQUAMATA1,2

Pyrenoid material of micromonas squamata Manton & Parke was obtained free of cell and subcellular particle conamination by differential centrifugation of brei from osmoically lysed cells. The isolated pyrenoid particles were characterized by transmission and scanning electron microscopy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of pyrenoid extracts revealed a compled polypeptide composition with major components of 12, 54 and 66 kilodalton mol wt. Whole pyrenoids possessed the enzymatic properties of ribulose diphosphate carboxylase and fixed carbon dioxide with specific activity 10 times greaer than that of a pyrenoid-free high speed supenaant fracion of cell brei. Energy dispersive X-ray microanalysis revealed he presence of copper in masses of cryo-impacted pyrenoid material. Ultrastrucural cytochemistry was employed o determine he chemical nature of the reserve carbohydrate shell. Also, the pyrenoid of the intact cell was characterized by transmission electron microscopy.     

Attachment of Azospirillum to isolated plant cells

Abstract Azospirillum brasilense cells were shown to attach to isolated plant cells. A rapid semi-quantitative assay to measure plant cell attachment, as was described previously for Agrobacterium , was found to work especially well for Azospirillum . Attachment was measured in an indirect way and confirmed by direct observations through scanning electron microscopy.     

A Method for the Examination of the Same Cell Using Light, Scanning and Transmission Electron Microscopy

A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis.     

Isolated rat alveolar type II cells protrude intracellular lamellar bodies by forming bubble-like structures during surfactant secretion

Pulmonary surfactant is synthesized and secreted by pulmonary alveolar type II epithelial cells (type II cells). It passes through the alveolar lining fluid and adsorbs to the air-liquid interface. The process from secretion to adsorption is not yet entirely understood. To acquire a detailed understanding of this process, we used multiple observations of type II cells isolated from rat lungs under electron microscopy (EM) and confocal laser scanning microscopy (CLSM). Transmission EM observation demonstrated a loosening process of the intracellular lamellar bodies from the inside to the outside of the cell. Scanning EM observation revealed bubble-like protrusions from the cell surface, and differential interference contrast microscopy illustrated the protrusions expanding with time. CLSM observation with FM 1–43, a fluorescent membrane probe, revealed that the bubble-like protrusions were composed of phospholipids. Thus, we have demonstrated that isolated rat type II cells protrude intracellular lamellar bodies by forming bubble-like structures, possibly enabling them to adsorb to the air-liquid interface directly. These observations suggest a new mechanism for surfactant secretion from type II cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Encapsulation of coagulase-negative staphylococci of bovine origin

Capsule expression was assessed in six coagulase-negative staphylococcal strains in serum-soft agar and by india ink and electron microscopy. Classification of strains as encapsulated by serum-soft agar and india ink methods differed. Staphylococcus chromogenes, Staph. hyicus , and Staph. simulans grew as diffuse colonies in serum-soft agar and unstained halos were detected in india ink preparations. Staphylococcus hominis and Staph. simulans grew as diffuse colonies in serum-soft agar but no unstained halo was seen in india ink preparations. Staphylococcus hyicus was the only strain that gave negative results with serum-soft agar and india ink assays. Conventional electron microscopy revealed the presence of capsular polysaccharides on the cell surface of Staph. chromogenes, Staph. hominis and Staph. hyicus. Conventional electron microscopic technique used to examine the surface of cells was detrimental to capsule structure. During dehydration the capsule collapsed and appeared as electron dense aggregates at the surface of cells. To confirm results of conventional electron microscopy and to visualize clearly the cell surface, encapsulated Staph. hyicus and unencapsulated Staph. simulans were observed after freeze-fracture and etching by scanning electron microscopy. The fibrous nature of capsular polysaccharides surrounding cells of Staph. hyicus were distinct and confirmed observation by conventional electron microscopy. A rapid transmission electron microscopic technique is described also for observation of capsule. Results of the rapid TEM method agreed with conventional TEM and SEM. The finding that coagulase-negative staphylococci isolated from bovine milk are capable of capsule production may be important when investigating pathogenicity of these micro-organisms.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Non-granulated peripolar cells exist in the rat glomerulus

Summary The peripolar cell is a unique cell type in the mammalian glomerulus. Peripolar cells are said to be identifiable during light microscopy by their cytoplasmic granules and by their position at the vascular pole; and during scanning electron microscopy by their distinctive surface morphology. We used both techniques to count peripolar cells in 6 normal rat kidneys. Scanning microscopy revealed that 55(±5)% of glomeruli contained at least one peripolar cell whereas light microscopy revealed granulated peripolar cells in only 4(±2)% of glomeruli. Vascular poles which contained peripolar cells previously identified by scanning were then examined by light and by transmission electron microscopy. Serial sections through these peripolar cells demonstrated the absence of cytoplasmic granules. Our observations suggest that the majority of peripolar cells in the rat contain no granules.