The envelope of intracellular African swine fever virus is composed of a single lipid bilayer

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.     

Cell Envelope of an Iron-Oxidizing Bacterium: Studies of Lipopolysaccharide and Peptidoglycan

Further structural detail is presented of the cell envelope of the chemolithotroph Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans). Thin sections of purified lipopolysaccharide (LPS) and peptidoglycan show structures comparable to those seen in the envelope of intact cells, whereas negative stains of LPS appear as sheets, or ribbons, or both. The sugars common to LPS, namely, heptose, glucose, galactose, mannose, and 2-keto-3-deoxyoctulosonate, were identified. The cations, iron, calcium, and magnesium, were associated with LPS. The purified LPS had a density of 1.28 and an uncorrected sedimentation coefficient of 99.9S.     

FINE STRUCTURE IN FROZEN-ETCHED YEAST CELLS

The freeze-etching technique, which is a special kind of freeze-drying, allows electron microscopic investigation of cells and tissues in the frozen state. In regard to yeast cells (Saccharomyces cerevisiae) a freeze-fixation technique has been developed which does not kill the object. The electron micrographs therefore are considered to impart an image of high fidelity. The cutting of the frozen object, which actually consists of a fine splintering, produces not only cross-sectional views (cross-fractures) of the structures but also surface views of the membranes and organelles. Many surface structures are described which have not been shown by the usual sectioning techniques. The cytoplasmic membrane contains hexagonal arrangements of particles which are apparently involved in the production of the glucan fibrils of the cell wall. Alterations of the distribution of nuclear pores are shown in cells of different ages. Freeze-etching enables a clear distinction of endoplasmic reticulum and vacuoles in yeast cells. The membranes of the vesicular systems are covered by ribosomes arranged in circular patterns. The mitochondrial envelope shows small perforations which could allow the exchange of macromolecules. The storage granules consist of concentric layers of lipid, presumably phosphatide. A Golgi apparatus has been detected which may be involved in the storage of lipid. The structure of the unit membrane and the membrane structures of all organelles as revealed by chemical fixation are confirmed in principle. Glycogen agglomerations are identified in the ground plasm of older cells. Insight into artifacts introduced by common chemical fixation and embedding techniques is obtained and discussed.     

Superficial cell-wall layers on Spirillum "Ordal" and their in vitro reassembly.

The cell envelope of Sporillum sp. strain "Ordal" (possibly a variety of S. anulus) demonstrated multiple superficial wall layers which were diverse in their macromolecular arrays. Negative staining and freeze-etching techniques revealed an outer hexagonally packed layer and an inner tetragonally packed layer. However, both thin sections and freeze-etched cleavages of the wall showed that each of these regular structures rested upon a backing layer, and that there was a delicate amorphous layer overlying the outer hexagonal array. Rotary integration, optical deffraction, and reconstruction of image were used to clarify measurements of each array and to verify the validity of a diagrammatic model of the outer hexagonal system. The integrity of these layers required suitable cations (Ca2+ appeared essential) and pH (pH less than or equal to 4.6 dissociated most superficial layers). These observations aided in the development of a low-pH cationic-substitution technique, in which Na+ replaced essential Ca2+, for extraction of the layers from the cell surface. Dialysis to remove Na+ and restoration of Ca2+ initiated in vitro reassembly of the superficial layer components until regularly structured assembly products were formed.     

The fine structure of frozen-etched bacillus cereus spores

Summary The fine structure of Bacillus cereus spores was studied using the freeze-etching technique and compared with that already described in chemically fixed cells. Although the basic structures did not appear different, the freeze-etching technique permitted the examination of membrane surfaces and their involvement in sporulation. In this respect, some membranes were found to be covered with small 120 Å particles believed to represent multi-enzyme complexes, while other membranes appeared relatively smooth.One feature revealed by the freeze-etching technique and not demonstrated in chemically fixed cells, was the presence of relatively large (500 Å) vesicular structures. In many instances these vesicles were closely associated with different layers or membranes in the developing spore. While the role of these vesicular structures is as yet undetermined, their possible connection with the lytic process which liberates the spore from the sporangium, is discussed.     

Isolation and chemical characterization of outer envelope of Leptospira pomona.

Cells of Leptospira interrogans serotype pomona harvested from a chemically defined medium were resuspended in 0.01 M phosphate buffer of pH 7.3. Electron microscopy showed that 90 min of exposure effectively ruptured the outer envelope, freeing it from the cells as small flakes. Both zonal centrifugation in sucrose gradients and centrifugation in isopycnic KBr and CsCl gradients could be used to separate the outer envelope from the axial filaments and protoplasmic cylinders. The latter method resulting in higher yields of purified envelope with the particular protocols used. Thin sections of isolated outer envelope showed the same trilaminar structure seen in sections of intact cells. The outer layers were 1.5 nm thick and appeared as single layers of electron-dense particles. The central electron-transparent layer was 2.0-2.5 nm thick and appeared structureless. The gross chemical composition of the purified outer envelope was 47% protein, 27% carbohydrate, and 23% lipid. Colorimetric carbohydrate determinations revealed hexose, pentose, and 6-deoxyhexose; hexosamine was identified during amino acid analysis. Muramic acid, heptose, and 2-keto-3-deoxyoctonate were not detected. Thin-layer chromatography revealed only polar lipids, about 98% phosphatidylethanolamine and 2% lysophosphatidylethanolamine. Fatty acids identified by gas-liquid chromatography were octadecanoic, octadecenoic, hexadecanoic, and hexadecenoic. Amino acid analysis revealed 17 amino acids, histidine and glutamic acid being most abundant. The outer envelope was interpreted to be comparable with the outer double-track layer found in the cell covering of gram-negative eubacteria.     

Bacterial Oxidation of Pyritic Materials in Coal

Applicability of the manometric method for studying the oxidation of pyritic material in the presence of bacteria has been demonstrated. Resting cells of Ferrobacillus ferrooxidans accelerated the oxidation of coal pyrites and coarsely crystalline marcasite, but were inactive on coarsely crystalline pyrite. Resting cells of Thiobacillus thiooxidans were inactive on all pyrites tested. Oxidation rates in the presence of Ferrobacillus were increased by reducing the particle size of pyritic samples, and, in one case, by removing the CaCO(3) from a calcite-containing sample.     

Die Feinstruktur der Zellwand und Cytoplasmamembran von Clostridium nigrificans,dargestellt mit Hilfe der Gefrierätz- und Ultradünnschnittechnik

Zusammenfassung Der mit Hilfe der Gefrierätztechnik dargestellte Feinbau der Zellwand und Cytoplasmamembran von Clostridium nigrificans wurde mit den Ergebnissen der chemischen Fixation verglichen. Die Zellwand von chemisch fixierten Zellen zeigt einen Aufbau aus drei stark und zwei schwach elektronenstreuenden Schichten, wobei die innerste, der Cytoplasmamembran angrenzende Schicht nicht immer darstellbar ist. Die Cytoplasmamembran hat eine asymmetrische Elementarmembran-Struktur. Mit Hilfe der Gefrierätztechnik konnte die Zellwand in drei Schichten aufgespalten werden: Einer äußeren aus globulären, rechtwinkelig geordneten, etwa 9 nm messenden Partikeln aufgebauten Schicht und zwei darunter liegenden, 15 und 5 nm dicken Schichten. Die Cytoplasmamembran wird in jungen Kulturen vollkommen, in alten teilweise mit 5–15 nm großen Teilchen bedeckt. An Stellen wo die Teilchen abgespalten wurden oder entsprechend locker angeordnet waren, konnte die Oberfläche der Cytoplasmamembran sichtbar gemacht werden. Sie ist sowohl auf der der Zellwand als auch auf der dem Cytoplasma zugekehrten Seite mit systemlos verteilten, scheinbar verschieden tief eingebetteten Teilchen bedeckt.

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The fine structure of the cell wall and cytoplasmic membrane of Clostridium nigrificans demonstrated by means of freeze-etching and chemical fixation techniques

Summary A comparison was made between the results obtained using the freeze-etching technique in demonstrating the fine structure of the cell wall and the cytoplasmic membrane of Clostridium nigrificans with those using the chemical fixation technique. In chemically fixed cells, the cell wall appears to consist of three dense layers separated by two layers of low electron scattering power, whereby it is not always possible to observe that layer immediately bordering on the cytoplasmic membrane. The cytoplasmic membrane has an asymmetrical unit membrane structure. It was possible, to separate the cell wall in three distinct layers using the freeze-etching technique. The outermost is composed of globular, rectangularly arranged particles, approximataly 9 nm in size, the two inner layers are 15 and 5 nm wide respectively. The cytoplasmic membrane is covered with particles 5 to 15 nm in size, in younger cultures completely, in older cultures partially. On those places where the particles have been split off or where they are loosly arranged, it was possible to observe the surface of the cytoplasmic membrane. It appears to be covered with unsystematically scattered particles imbedded at different depths, not only on that side turned to the cell wall but also on that facing the cytoplasm.

     

Visualization of the nuclear lamina in mouse anterior pituitary cells and immunocytochemical detection of lamin A/C by quick-freeze freeze-substitution electron microscopy.

We examined the nuclear lamina in the quickly frozen anterior pituitary cells by electron microscopic techniques combined with freeze substitution, deep etching, and immunocytochemistry and compared it with that in the chemically fixed cells. By quick-freeze freeze-substitution electron microscopy, an electron-lucent layer, as thick as 20 nm, was revealed just inside the inner nuclear membrane, whereas in the conventionally glutaraldehyde-fixed cells the layer was not seen. By quick-freeze deep-etch electron microscopy, we could not distinguish definitively the layer corresponding to the nuclear lamina in either fresh unfixed or glutaraldehyde-fixed cells. Immunofluorescence microscopy showed that lamin A/C in the nucleus was detected in the acetone-fixed cells and briefly in paraformaldehyde-fixed cells but not in the cells with prolonged paraformaldehyde fixation. Nuclear localization of lamin A/C was revealed by immunogold electron microscopy also in the quickly frozen and freeze-substituted cells, but not in the paraformaldehyde-fixed cells. Lamin A/C was localized mainly in the peripheral nucleoplasm within 60 nm from the inner nuclear membrane, which corresponded to the nuclear lamina. These results suggest that the nuclear lamina can be preserved both ultrastructurally and immunocytochemically by quick-freezing fixation, rather than by conventional chemical fixation.     

Improved Staining of Extracellular Polymer for Electron Microscopy: Examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus

Phase contrast, ultraviolet microscopy, and freeze-etching were used to determine the amount of exocellular polymer surrounding unfixed cells of four genera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mesenteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectional electron microscopy was employed to measure the effectiveness of a modified ruthenium red staining method. The results obtained with this modification of ruthenium red staining technique were compared to results obtained when previously proposed ruthenium red methods of fixation were employed. The results of these relations were then compared to the amounts of exocellular material as determined with phase-contrast microscopy, ultraviolet microscopy, and freeze-etching. The data obtained indicate that improved fixation of exocellular polymer is achieved when cells are pretreated with ruthenium red as described herein. In addition, the modified methods also reveal cytological detail not apparent when other methods of ruthenium fixation are employed.     

Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.     

Transition of Chemolithotroph Ferrobacillus ferrooxidans to Obligate Organotrophy and Metabolic Capabilities of Glucose-Grown Cells

Transition of chemolithotrophic Ferrobacillus ferrooxidans to organotrophy occurred after 60 hr of incubation in an organic medium. Three distinct phases, based on metabolic activities of cells, were observed during the course of transition. Conversion of cellular nutrition to organotrophy resulted in a gradual loss of Fe(2+) oxidation and cessation of CO(2) fixation. These changes were concomitant with a rapid increase in uptake of glucose and phosphate during the latter part of transition period. The outcome of transition was governed by the pH of the medium, temperature of incubation, availability of oxygen, age of the chemolithotrophic cells, and the type of energy and carbon source available to the bacterium. Presence or absence of p-aminobenzoic acid and Fe(2+) ions did not influence transition of cells. A defined medium containing glucose, mineral salts, and p-aminobenzoic acid at pH 2.5 was found to be most suitable for transition and for culture of heterotrophic convertants. Maximum growth rate of the heterotrophic cells was attained with vigorous aeration at 35 C. The bacterium could be cultured on a variety of organic compounds, including complex organic media, provided they were used in low concentrations. Serological studies on autotrophic cells and the heterotrophic convertant have shown a definite antigenic relationship between the two cell types.     

Über die Feinstruktur des Zellkerns von Acetabularia nach Gefrierätzung

Summary The ultrastructure of the primary nucleus of the marine green algae Acetabularia (Polyphysa) cliftonii as revealed by the freeze-etching technique is described.The nuclear envelope is perforated by about 3×10⁶ pores which appear arranged nearly in rows. The nucleus is covered with a layer of cytoplasm which protects it in the isolated state and resists various manipulations. This cytoplasmic layer is protected by an elementary membrane. The latter appears rough on the cytoplasmic side, and smooth on the side facing the cell sap. The cytoplasmic layer and the surrounding perinuclear cytoplasm are connected by cytoplasmic ducts. The nucleus appears irregularly shaped owing to many invaginations in the nuclear envelope. Sometimes mitochondria are observed in these cavities.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

Ultrastructure of the Cell Envelope of Escherichia coli B After Freeze-Etching

The cell envelope of Escherichia coli B was investigated with the freeze-etching technique. A considerable gain in visible structural detail over more conventional electron microscopic techniques was obtained. The inner surface of the plasma membrane revealed a smooth surface sparsely studded with particles measuring from 5 to 10 nm in diameter, whereas the outer surface of the plasma membrane showed many more particles of corresponding diameter. The freeze-etched cell wall appeared to be a multilayered structure. The innermost layer could be observed as a profile studded with closely packed elements of about 10 nm in diameter. External to this layer was a smooth surface bordering the outermost cell wall layer. When frozen in the absence of glycerol the outermost surface observed in the cell wall was smooth, but when grown in the presence of glycerol it had a "wavy" appearance with small particles attached to it. The observations support current concepts on the ultrastructure of the enterobacterial cell envelope.     

Surface Structure of Yeast Protoplasts

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated.     

Pyruvate Inhibition of Ferrobacillus ferrooxidans Reversed by Rhodotorula Yeast

Ferrobacillus ferrooxidans was isolated in this laboratory from uranium ores mined at Jaduguda, Bihar. Investigations on a laboratory scale indicated that this organism could leach uranium from ores. Pyruvic acid (10^-4 M) was found to inhibit growth of F. ferrooxidans as well as leaching of uranium ore by F. ferrooxidans . However, in the presence of a red pigmented Rhodotorula yeast isolated from the same ores, the pyruvate inhibition was found to be reversed.     

Effect of Glucose on Carbon Dioxide Assimilation and Substrate Oxidation by Ferrobacillus ferrooxidans

Ferrobacillus ferrooxidans, grown on either elemental sulfur or ferrous sulfate, was able to use either substrate as an energy source for the assimilation of CO(2). In both cases, 0.01 mumole of carbon was incorporated per mumole of oxygen utilized. Glucose inhibited substrate oxidation and CO(2) fixation. Sulfur and iron oxidation were inhibited 5 to 15% and 40 to 50%, respectively, in the presence of 10% glucose. Under the same conditions, CO(2) assimilation was inhibited 50% with elemental sulfur as the energy source, and was almost totally inhibited when ferrous iron was used.     

On the morphology and ultrastructure of the cell wall of Spirulina platensis

The cell wall of the blue-green alga Spirulina platensis was studied with the electron microscope using ultra-thin sectioning, shadowing, carbon-replication or freeze-etching techniques for specimen preparation. The cell wall could be resolved into four layers, L-I through L-IV. The L-I and L-III layers contain fibrillar material. The septum is a three-layered wall: an L-II layer sandwiched between L-I layers. The shape in vitro of isolated septa might be an artifact due to the preparation technique used. Certain structural properties of the septum seem to allow tangential stretching; they might be reflected in the flexible gliding mobility of Spirulina species. The outer, L-IV layer contains material longitudinally arranged along the trichome axis.     

TISSUE PREPARATION AND FINE STRUCTURE OF THE RADICLE APEX FROM COTTON SEEDS

A comparative methodological study was made of the fine structure of apical cortical cells in excised radicles from cotton (Gossypium hirsutum L. var M-8) seeds. Radicles from dry seed had 12% moisture content and were prepared for electron microscopy using several different techniques. These included different methods of chemical fixation or freeze-fracture and etching of unfixed tissue for transmission electron microscopy (TEM) and cryofracturing of fixed and dehydrated radicles for scanning electron microscopy (SEM). Cortical cells had a similar appearance regardless of the method used in tissue preparation. Cell walls had a pronounced waviness which was particularly evident in SEM images of cells lining the elongated intercellular air spaces. The plasma membrane (PM) delimited the cytoplasm of each cell as an intact unit membrane. Single layers of tightly-packed lipid bodies (LB) were apposed to the PM and protein bodies (PB). Distension of cells, membranous organelles and LB was observed in radicles fixed by immersion in aqueous solutions, suggesting that a certain amount of hydration occurred during fixation. This interpretation was supported by the compact appearance of cells and organelles in tissue prepared by freeze-etch or vapor fixation. We conclude that freeze-fracture and etching of unfixed tissue provided the best information for cell morphology and structure of membranes and organelles in dry tissue. Complementary data on the fine details of nuclei and cytoplasmic organelles were best observed with TEM of fixed tissue. These data when viewed collectively indicate the advantage of using several techniques to obtain analogous and complementary information essential for establishing a baseline level of information on the fine structure of cells in dry tissue.