Thin-section Techniques Research Articles

Ultrastructure of a marine Thiobacillus.

SUMMARY: The ultrastructure of Thiobacillus sp. IV-85 was examined by thin section and freeze-etch techniques. The morphology of this marine isolate was similar to that of terrestrial species of Thiobacillus. Decline phase preparations showed a marked degeneration of internal organization. Membrane vesicles, characteristically bound by a trilaminar membrane, were observed only in preparations of decline phase cells. The examination of freeze-etched preparations of both first and second log phase organisms showed membrane-bound polyhedral inclusion bodies in intact cells and a characteristically rippled envelope.

Ultrastructure of the cell envelope and septation process in Caryophanon latum as revealed by thin section and freeze-etching techniques

With optimal conditions of thin-sectioning and freeze-etching, the cell wall of Caryophanon latum was composed of a thick peptidoglycan layer plus two external wall layers. The freeze-etched appearance of the external surface of the outer layer was smooth and lacked structural detail. The numerous cross septa within a trichome were formed by the symmetrical and concurrent ingrowth of the cytoplasmic membrane and the peptidoglycan layer. The site of septum initiation was identifiable by a dart-shaped ingrowth of the peptidoglycan layer rather than by the presence of a mesosome. However, small simple mesosomes were occasionally seen associated with the developing septum. The peptidoglycan in the septum had thickened to at least double the thickness of the wall peptidoglycan layer by the time of septum completion. The external wall layers did not participate in septum formation but did participate in trichome separation. The separation of the septal peptidoglycan was completed during the early ingrowth of the external wall layers. A unique cross-sectional view of a developing septum closing like an iris diaphragm as seen in a freeze-etched preparation was observed.

Surface properties of extracellular malaria parasites: morphological and cytochemical study.

Morphological and cytochemical surface characteristics of isolated malaria parasites (Plasmodium berghei) and host erythrocytes were compared by electron microscopy by using thin section and carbon replica techniques. Erythrocytes were uniform in shape and had fine, granular surfaces. In contrast, free parasites exhibited a variety of sizes, shapes, and surface textures. Fine surface stippling was a common topographical feature of isolated parasites. Small, infective forms often had patterned surfaces resulting from the protuberance of an underlying thick intermediate layer. Results of cytochemical analysis using a sialophilic colloidal iron stain indicated that the malaria parasite's surface lacked exposed sialic acid groups which would normally give rise to a net negative surface charge common to erythrocytes. Biochemical assay demonstrated that malaria parasites contained about one-half the amount of sialic acid per unit weight as did control red cell extracts. Similarly, external acidic mucopolysaccharide coats of free parasites, as revealed by ruthenium red staining were extremely thin as compared with the thick glycocalyx layer of red cells. Lipid plaques at the surface of parasites and red cells were localized by lipophilic iron colloid staining. Although the gross patchwork distribution of plaques was somewhat similar for the two cell types, the parasites were stained more intensely and had a closer-knit patchwork pattern than those exhibited by the erythrocytes. Such findings indicate that there are slight differences in the arrangement of phospholipids at the surfaces of limiting membranes of host cells and parasites. The significance of the above cytochemical surface properties of the malaria parasite (which are seemingly akin to those of intracellular organelles is discussed in relation to certain host-parasite interactions, such as parasite adhesion to target cells and enhanced clearance of extracellular parasites.

The structure of cytochrome oxidase membranes

Two preparations of oxidized membranous cytochrome oxidase were examined using both the negative stain and freeze fracture techniques of electron microscopy. The cholate preparation, containing 27% phospholipid by weight, appeared as membrane sheets. With the freeze fracture technique, cross fractures of the sheets showed particles abutted one against another. The Triton preparation, containing 42% phospholipid by weight, was decidedly vesicular and only particulate membrane fracture surfaces were usually seen. In the case of membranous cytochrome oxidase it is proposed that fractures follow the surface rather than a centre plane through the hydrophobic interior of the membrane. The observed particles appeared as prolate spheroids of 135 A X 68 A with the long axis extended through the membrane. From the size shape relationships, a molecular weight of 244 000 was calculated which is in agreement with values reported for dispersed cytochrome oxidase using other physical techniques. At least 50% of the protein is exposed to the aqueous environment. Cytochrome oxidase is a lipoprotein that spontaneously forms membranes under conditions of low ionic strength and the absence of detergents. Sun et al. [ 1 ] showed that these membranes were trilaminate structures 55 A thick when examined by conventional thin section techniques. Negatively stained preparations showed a membrane surface covered with 50 A particles. Seki and Oda [2] have shown

Diffusion of Ge in SiGe alloys

Germanium diffusion was measured in SiGe alloys of 100/0, 77.6/22.4, 69.2/30.8, 44.6/55.4, and 22.3/77.7 silicon to germanium atomic-percent ratios by the use of the radioisotope $^{71}\mathrm{Ge}$ and a thin-sectioning technique. As expected from the calculated, small, strain-energy contributions from the $^{71}\mathrm{Ge}$ impurities, the diffusion is similar to silicon self-diffusion for the silicon end member. Since the results fit an Arrhenius plot for the compositions and temperature ranges studied, activation energies and pre-exponentials were determined. These diffusion parameters indicate that the $^{71}\mathrm{Ge}$ diffusion is compatible with the monovacancy mechanism up to 70-at.% silicon in the SiGe alloys. For more silicon-rich material, the diffusion is quite analogous to the extended-defect mechanism previously suggested for high-temperature silicon self-diffusion.

High voltage electron microscopy on microgametogenesis of Haemoproteus columbae.

The microgametogenesis of Haemoproteus columbae was investigated by high voltage and conventional electron microscopy and the results were compared. The features of microgametogenesis which have become clear by high voltage electron microscopy include the distribution and arrangement of axonemes in the microgametocyte and the budding process and structure of the microgametes. High voltage electron microscopy shows that prior to exflagellation the axonemes occupy a major portion of the microgametocyte cytoplasm, and are arranged in an orderly fashion. Conventional electron microscopy suggests that the axonemes are randomly distributed in the microgametocyte cytoplasm. High voltage electron microscopy shows that a short portion of the axoneme protrudes outward in the early stage of microgamete budding, while the major portion remains within the cytoplasm. The microgamete of H. columbae as observed by the high voltage electron microscope, is composed of a slender nucleus and an axoneme which run along the same axis and spiral one another. The nucleus is slightly bulbous at the anterior end and at the midportion of the microgamete. These findings have not been obtained with certainty by conventional electron microscopy because the thin section technique could not provide a sufficient profile of the long wavy microgamete.

Morphology and development of dengue-2 virus employing the freeze-fracture and thin-section techniques

The freeze-fracturing of purified dengue-2 virus has shown at least three distinct fracture planes: a grazing fracture removing at most some surface projections; a fracture apparently splitting the membrane; and a cross fracture through the nucleocapsid of the virus. Morphogenesis in LLC-MK2 cell culture was studied by use of the freeze-fracture technique and also by thin sectioning. Freeze-fracturing has been helpful in determining the sites and extent of virus development before the virus appears in vacuoles, and in determining related phenomena of infection. There is no direct evidence from freeze-fracturing for the “budding” of virus into these vacuoles, and their release by reverse pinocytosis, both of which have been previously proposed. However, evidence from thin sections herein suggests that release of mature virus from the cell may yet be by a reverse pinocytosis from small vesicles involved with the normal mechanisms of intracellular transport.

Specialized intercellular junctions in human intracranial germinomas.

Three human intracranial germinomas were studied using thin section and freeze-fracture techniques. In thin sections, the spheroidal cells were similar to the seminoma cells of the testis. There were three types of specialized intercellular junctions; desmosomes, gap junctions, and tight junctions, were evident between the spheroidal cells. In freeze-fracture replicas, tight and gap junctions were easily differentiated by their characteristic deployment patterns of their subunits. The tight junction revealed a discontinuous, circular, serpentine, or ramifying complex of ridges and furrows, referred to as fascia occludens. The gap junction was also discontinuous and largely confined to the compartment formed by the ramifying ridges and furrows of tight junctions, and revealed aggregated particles and pits on fracture faces A and B, respectively.

Freeze-fracture images of capillaries in normal and neoplastic pituitaries

The capillaries in five human chromophobe adenomas were studied by thinsection and freeze-fracture techniques. Fenestrae were found in the attenuated portions of the endothelium, and plasmalemmal vesicles in the nonfenestrated portions. In freeze-fracture replicas, the fenestrae, 600–700 A in diameter, were revealed as circular holes in fracture face A and circular broken-off necks in face B, some 40–60 occurred per μm2. When the fracture process passed in a stepwise fashion from the luminal into the contraluminal plasma membrane, the fenestrae at the fracture edge involved both plasma membranes. Similar configuration of fenestrae was evident in the capillary endothelium of the rat pituitary. The fenestrae in the chromophobe adenoma were much less frequent than in the normal rat pituitary. The plasmalemmal vesicles occurring in replicas on one side of the cell did not appear to open to the other side of the cell. When the fracture followed the contours of the attached plasmalemmal vesicles, they appeared as smooth depressions in face A and domes in face B. When the fracture did not follow the contours of the vesicles, they were revealed as holes or depressed circular plaques in face A and broken-off necks or circular constellations of particles in face B. The plasmalemmal vesicles were more abundant and obvious in the tumour than in the normal rat pituitary.

Nuclear envelope isolation in peas

Nuclear envelopes were isolated from pea plumules by sonication of a highly purified nuclear fraction. The nuclear envelope fraction, examined in the electron microscope by both negative staining and thin sectioning techniques, was found to consists of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents frequently could be recognized. The chemical composition of the nuclear envelope fraction was determined and compared with that of the nuclear fraction. Significant amounts of DNA and RNA were found to remain associated with the nuclear envelope fraction. It was found that the specific activities of ATPase, 5′-nucleotidase and glucose-6-phosphatase were higher in the nuclear envelope fraction than in the total nuclei.

Characterization of an avian reovirus isolated in Queensland

An avian reovirus, RAM-1, produced cytopathic changes in chicken kidney, chick embryo fibroblast and duck embryo fibroblast cell cultures. The virus could also be titrated in chicken embryos, but this method was much less sensitive than cell culture. Following yolk sac inoculation, deaths occurred in two to four days. Inoculation onto the chorio-allantoic membrane resulted in necrotic plaques, with about 10 per cent. mortality. Surviving embryos had green discoloration of the liver, enlarged spleen and sometimes pericarditis. Features of the histopathological response were ectodermal hyperplasia and necrosis in the chorio-allantoic membrane, and accumulations of eosinophils in the liver, the spleen and the pericardium. Pericarditis developed in a few chickens inoculated at one day of age. The virus was not pathogenic for suckling mice. Virions typical of reoviruses could be demonstrated by electron microscopic examination of infected cell cultures by negative staining, or thin section techniques. The virus was stable on exposure to chloroform and pH 3·0, and was partially inactivated by exposure to 56 °C. for 30 min. Neutralizing antibody could be detected in cell cultures, or in embryonated eggs inoculated onto the chorio-allantoic membrane, but yolk sac inoculation gave unsatisfactory results. Precipitating antibody in hyperimmune rabbit serum was demonstrated with antigen derived from cell cultures and from chorio-allantoic membrane. Convalescent serum from chickens reacted only with the antigen derived from chorio-allantoic membrane.

The diffusion of germanium in silicon

The diffusion of germanium in single-crystal silicon has been measured using the radioactive tracer 71Ge and a thin sectioning technique. The activation energy of 4.7 eV determined from the data is analyzed by considering the expected contribution due to the wrong-sized impurity ions. It is concluded that the high activation energy observed is expected from analogy to silicon self-diffusion.

A structural comparison of cyst and germ tube wall inPhytophthora palmivora

An electron microscopic analysis of germinating cysts ofPhytophthora palmivora involving freeze-etching, thin sectioning, and replica techniques reveals that both cyst and hyphal wall comprise a two-phase system with a fibrillar and an amorphous component. The cyst wall is fibrillar throughout with the fibrils tightly interwoven and embedded in an amorphous matrix on the internal side of the wall. The hyphal wall consists of a fibrillar inner layer with the fibrils lightly covered by some amorphous material and an amorphous outer layer devoid of any fibrillar material. Both cyst and germ tube walls are wholly or partially covered by a fluffy coat of variable thickness. In the zone of germ tube emergence cyst wall and germ tube wall overlap and are tightly apposed. Thus, the germ tube wall is not a simple extension of the cyst wall but a new structural entity separated from the cyst wall by a thin line of demarcation.

Ultrastructure of Aspergillus fumigatus conidia development and maturation.

The development and maturation of conidia of the pathogenic fungus,Aspergillus fumigatus, were studied with the electron microscope. Thin sectioning and freeze-etching techniques were employed to examine young and mature phialides, as well as developing and mature conidia. The morphological changes observed during the process of conidia formation and their maturation centered primarily around the cell wall layers and the inclusions in the cytoplasm.

Structure and physico-chemical properties of bacteriophage G. II, The shape and symmetry of the capsid

The shape of the capsid of phage G, active against B. megatherium, has been studied by electron microscopy using a variety of approaches including negative staining of whole particles, examination of stereoscopic micrographs, shadowing, observations on capsids devoid of their DNA content (‘ghosts’), short and prolonged fixation and the use of single and serial ultra-thin sections. The results were conflicting. With negative staining the appearance of the capsid was not consistent with an icosahedral shape but could be related to that of an octahedron. The shape of the capsid in ‘ghost’ preparations was definitely octahedral. However, after fixing for 1h or longer and shadowing, the capsids were clearly icosahedral in shape with their sides equal to about half the length of those of the octahedron seen by negative staining. The same results were obtained by the thin sectioning technique. From a study of the two types of geometrical figures it was found that an octahedral container can be transformed into an icosahedral one with sides of half the original length if it is folded inwards on itself at 6 consecutive points. If this type of effect occurred during the preparative procedure it would account for the two apparently conflicting appearances of the capsid. Such an effect would involve a marked change or ‘jump’ through an intermediate unstable form. While it would reconcile the different results it is emphasized that such an interpretation is essentially speculative. To illustrate the complexities of the problem, an interesting geometrical exercise is presented in the Appendix. La forme de la capside du bactériophage G, actif par rapport au mégathérium de type B, a étéétudiée en microscopie électronique par différentes méthodes, parmi lesquelles: coloration négative de particules entières, inspections de micrographies stéréoscopiques, ombrage, observations de capsides démunies de leur contenu en ADN (en quelque sorte des ‘capsides fantômes’), fixation de courtes et longues durées et examens de sections ultra-minces uniques puis ultérieurement en séries. Les résultats furent pour le moins contradictoires. Dans le cas de la coloration négative l'apparence de la capside n'était pas compatible avec une forme icosaédrique, mais pouvait s'apparenter à celle d'un octaèdre. La forme observée dans la préparation ‘capside fantôme’ était définitivement celle d'un octaèdre. Toutefois il apparut, après fixation d'une durée égale ou supérieure à 1 heure et suivie d'ombrage, que les capsides avaient une forme icosaédrique bien définie, avec des côtés d'une longueur environ moitié plus petite que ceux obtenus par coloration négative. Les mêmes résultats furent obtenus par la technique des sections minces. Il résulte d'une étude des deux types de formes géométriques qu'un volume de forme octaédrique peut être transformé en un volume de forme icosaédrique avec des côtés de longeurs moitiés de celles des côtés du volume primitif si celui-ci est replié vers l'intérieur à 6 points consécutifs. Une explication serait alors possible pour les formes contradictoires observées sur la capside si ce genre de phénomène a lieu pendant la procédure préparative. Un tel effet serait le résultat d'un saut par l'intermédiaire d'une forme transitoire métastable. Une telle interprétation serait des plus spéculatives même si elle pouvait réconcilier les différents résultats. Un exercice géométrique illustrant la complexité du problème est présenté dans l'appendice. Mit verschiedenen Verfahren wurde versucht, die Form der Hülle des gegen B. megatherium aktiven Bakteriophages G zu ermitteln. Unter anderen verwendete man Negativfärbung intakter Körperchen, Stereomikroaufnahmen, Schrägbedämpfung, leere Hüllen ohne DNS-Gehalt, kurze und längere Fixierungszeiten und ultradünne Einzel- oder Serienschnitte. Die Ergebnisse waren widerspruchsvoll. Nach Negativfärbung ließ das Aussehen der Hülle nicht auf ein Ikosaeder sondern deutlich auf ein Oktaeder schließen. Auch leere Hüllen zeigten eine deutliche Oktaederform. Nach einstündiger oder längerer Fixierung und anschließender Schrägbedämpfung hingegen war die Hülle ikosaedrisch. Die Kanten waren ungefähr halb so lang wie die des nach Negativfärbung sichtbaren Oktaeders. Die gleichen Ergebnisse erhielt man mit dem Dünnschnittverfahren. Wenn man die Geometrie der beiden Formen untersucht, kann ein Oktaeder in ein Ikosaeder verwandelt werden, wenn das Oktaeder an sechs Stellen eingeknickt wird. Sollte dies beim Präparieren geschehen, wären damit die widerspruchsvollen Ergebnisse erklärt. In diesem Fall müßte eine markante oder möglicherweise sprunghafte Wandlung mit instabilem Zwischenstadium stattfinden. Obwohl durch diese Erklärung beide Resultate in Einklang gebracht werden, wird betont, daß eine solche Auslegung rein spekulativ ist. Um die Komplexität des Problems zu verdeutlichen, befindet sich im Anhang eine interssante geometrische Übung.

Protein bodies, lipid layers and amyloplasts in freeze-etched pea cotyledons

Freeze-etching and thin-sectioning techniques have been used to study the structure of storage parenchyma cells of pea cotyledons in mature, air-dry seeds and in seeds after various periods of imbibition. Protein bodies in air-dry tissue were coated with a complex of convoluted structure, apparently rich in lipid. After brief periods of hydration this complex appeared to have expanded and to consist of a system of tubules, and after 24 h imbibition it was no longer associated with the protein bodies. Sheets of lipid bodies were associated with amyloplast surfaces and the plasmalemma. In non-imbibed tissue, the cell walls were wavy, but in imbibed tissue they were smooth. The amyloplast-envelope membranes characteristically had areas with parallel folds, and membrane particles of protein-body envelopes were occasionally aligned in orderly rows.

Fine structure of interphase nuclei

The initiation and replication sites of DNA synthesis in the plasmodial nuclei of Physarum polycephalum were studied with electron microscopic autoradiography. By using both thin sectioning and whole mount techniques, it was shown that the dense chromatin masses in the nucleus consisted of predominantly elementary chromatin-like fibrils, approximately 300 A in diameter while the electron transparent region in the nucleus consisted of predominantly finer fibrils, less than 100 A in diameter. With electron microscopic autoradiography it was found that (1) the initiation sites of DNA synthesis were definitely in the boundary regions between the dense chromatin masses and the electron transparent region, (2) the initiation and replication sites of DNA synthesis were definitely not on the nuclear membrane, (3) within a few minutes, replication sites migrated from the initiation sites to the electron transparent region and (4) in this electron transparent region, almost all of the nuclear DNA was synthesized.

Das Gefrierätzbild von Lipidablagerungen in der Aortenintima cholesteringefütterter Kaninchen

In specimen preparation for freeze etching no lipid solvents are used. Therefore lipid deposits in cells and intercellular spaces and lipid droplets in cell organelles are perfectly preserved and can be differentiated far better by freeze etching than with conventional techniques of thin sectioning.

THE CELL WALL OFCOSMARIUM BOTRYTIS12

SUMMARYThe cell wall ofCosmarium botrytiswas studied through the use of the freeze‐etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls.The cell wall pore unit ofCosmarium botrytiswas studied through the use of scanning, freeze‐etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle‐like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net‐like fibril layers which in turn merge into a very electron dense region near the base of the pore.

Morphology and morphogenesis of Sindbis virus as seen with freeze-etching techniques.

Freeze-etch electron microscope studies of the morphogenesis and morphology of Sindbis virus confirmed results obtained by other workers employing thin-sectioning techniques. The 68-nm virion was found to have a nucleocapsid 36 nm in diameter surrounded by a double-layered, unit membrane. The membranous envelope is acquired as the capsid buds through the plasma membrane of the infected cell. The freeze-etch technique also provided the following new information. (i) At any one time, budding occurs in patches rather than evenly over the cell surface. (ii) The nucleocapsid is composed of capsomers 7 nm in diameter. (iii) The capsid interacts strongly with the membrane, both prior to budding and after maturation. (iv) The 7- to 10-nm particles characteristic of the internal faces of plasma membranes, which presumably represent host membrane proteins, are present in early stages of budding but disappear as morphogenesis progresses. (v) Fusion of the cell membrane at the base of the budding virion is a two-step process; the inner leaflet fuses into a sphere before the outer one. (vi) The outer surface of the viral envelope is covered with 4-nm subunits with a center-to-center spacing of 6 nm.