Direct embedding in epoxy resin of cells attached to cellophane

Macrophages were collected from mice or rats after subcutaneous implantation of cellophane or grown in Leighton tubes in which the flying coverslip was replaced by a strip of cellophane. The cellophane and attached cells were prepared for electron microscopy, embedded directly in epoxy resin and then sectioned. The cutting qualities of cellophane are adquate for ultrathin sectioning and permit the ultrastructural examination of the attached macrophage monolayer. The technique can be used for other cell types.     

Transmission and Scanning Electron Microscopy of Cell Monolayers Grown on Polymethylpentene Coverslips

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized. separated from the coverslip by liquid nitrogen immersion, and sectioned either “en face” or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.     

A Ralph Knife Holder Assembly for Use with the Sorvall “Porter-Blum” MT-1 Ultramicrotome

The superiority of plastic embedding for the production of high quality sections for light microscopy is well known, but the use of conventional glass knives with a cutting edge of approximately 4 mm has severely restricted the size of specimens in the past. Ralph knives provide a much longer cutting edge and adapters are available for certain models of microtomes and ultramicrotomes. A modified knife holder for use with the Sorvall “Porter Blum” MT-2 microtome was described by Gorycki and Sohm (1979); however, this is not suitable for the MT-1 model. We have therefore designed and made an adapter which enables Ralph knives to be used with this instrument. The design allows approximately 18 mm of cutting edge to be used on each knife, allowing larger specimens to be sectioned than with a conventional glass knife and reducing the frequency with which the knife needs to be changed when working with smaller blocks.     

Membrane disruption, fusion and resealing in the course of exocytosis in Paramecium cells

Ca²⁺-ionophore-mediated trichocyst exocytosis was followed by scanning electron microscopy, freeze-cleaving and ultrathin sectioning after surface labelling in vivo with negatively charged hemepeptides. The apical trichocyst membrane and the superposed cell membrane portion (encircled by ˜300 nm large “rings” of membrane-intercalated particles) undergo fragmentation, while both membranes involved fuse with each other within the “rings”. Subsequently cell membrane materials spread centropetally to the region within the “rings” allowing the cell membrane to become resealed and the trichocyst membrane to become detached. Exocytosis does not result in any remarkable integration of trichocyst membrane materials into the cell membrane.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.     

A re-description of the ciliate genus and type species, Balantidium entozoon

Members of the ciliate genus Balantidium possess a specialized “Villeneuve-Brachon's” field of somatic cilia to the right of the vestibule, or in a dextroral location. Specimens of the type species were collected in Italy and fixed for study by light microscopy, and scanning and transmission electron microscopy. The presence of the field in the type species and several other species of the genus indicates a need to re-describe the genus by including details of the ultrastructure of that field. Scanning electron microscopy shows that the field consists of one row of relatively short cilia of uniform length flanked on each side by 2–3 rows, or more, of very short cilia. Their kinetids have typical litostome structure in transmission electron micrographs. We speculate on a possible function for the Villeneuve-Brachon's field and also present morphometric data on the type species. The base sequence of the small subunit ribosomal RNA gene of Balantidium entozoon has been determined and found to differ by 5% from that of B. coli. Based on the location and ultrastructure, organelles found around the somatic kinetosomes and within inter-kinetal ridges of B. entozoon were identified as hydrogenosomes.     

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.     

Frequency and distribution of edge damage on Middle Stone Age lithic points, Pinnacle Point 13B, South Africa

Unretouched convergent flakes are frequently a well represented tool type in many Middle Stone Age (MSA) assemblages. Damage to the lateral margins of these points is frequent; however, analytical methods for dealing with the frequency and distribution of edge damage on points have not been developed and applied to a complete MSA lithic assemblage. A method for using GIS to quantify edge damage and statistically analyze the relative location and frequency of edge damage is presented here and applied to the complete assemblage of MSA points from Pinnacle Point Cave 13B (PP13B), South Africa. The results indicate a frequency of edge damage consistent with heavier utilization of the dorsal surface over the ventral surface, and the left side over the right, with the dorsal left lateral margin being most heavily damaged. Additionally, the distribution of edge damage and low frequency of impact damage to the points suggest that PP13B represents a location where points were used for cutting activities and discarded. Applying the recording procedures advocated here to controlled edge damage replication experiments will help provide the interpretive linkages to site assemblage edge damage distributions.     

Removal of Histological Sections from Glass for Electron Microscopy: Use of Quetol 651 Resin and Heat

A method is described for using the epoxy resin Quetol 651 and heat for convenient and rapid separation of conventional histological sections from glass slides for subsequent ultrathin sectioning for retrospective electron microscopy. The same method is useful when Epon-Araldite is substituted for the Quetol 651 resin.     

Removal of histological sections from glass for electron microscopy: use of Quetol 651 resin and heat

A method is described for using the epoxy resin Quetol 651 and heat for convenient and rapid separation of conventional histological sections from glass slides for subsequent ultrathin sectioning for retrospective electron microscopy. The same method is useful when Epon-Araldite is substituted for the Quetol 651 resin.     

Associations among components of the male germ unit followingin vivo pollination in barley

Summary The male gametophyte of barley has been studied during post-pollination stages from the time of pollen activation through pollen tube growth to the level of the ovule. Techniques included the use of serial thick sectioning, serial ultrathin sectioning of re-embedded thick sections, and computer generation as well as manual production of three-dimensional reconstructions. It was found that after pollination, but before the sperms exit the pollen grain, an intimate association is formed between the vegetative nucleus and the two sperms, and between the sperms themselves. The latter association is maintained through the duration of pollen tube growth observed in this study. These results indicate that the male germ unit of barley, a grass, is ultimately similar to that of other angiosperms studied thus far at the electron microscope level and that the concept of the male germ unit may have even greater significance and application within flowering plants than previously thought.     

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.     

Electron microscopic investigations of zymomonas mobilis cells grown in low and high glucose concentrations

Summary Zymomonas mobilis cells grown in the presence of low and high glucose concentrations were examined by electron microscopy. Using ultrathin sectioning and agar diffusion method, significant changes in morphology were observed. Although the fine structure resembles that of a typical gram-negative bacterium, changes in glucose concentration and phases of growth lead to large cell wall vesicle or blebs formation. The possible implications of this morphological change to glucose uptake and ethanol formation are discussed.     

Ultrastructure of the meningococcus in logarithmic and stationary development phases

The complex study of Neisseria meningitidis cultures A-208 in the time course of their development has disclosed that broth cultures in the logarithmic and stationary phases of their development are most valid on account of all their biological properties (the specific character of the reaction of agglutination, viability, the morphology of colonies and cells in light and electron microscopy). The use of scanning electron microscopy has made it possible to reveal bubbly endotoxin excretion in N. meningitidis and funnel-shaped depressions on their surface corresponding, probably, to nucleoid epicenters . In ultrathin sections some previously unknown features of the ultrastructure of N. meningitidis in the logarithmic and stationary phase of their development have been detected: (a) the morphological heterogeneity of N. meningitidis represented by cells of the "light" (L) and "dark" (D) types; (b) the surface structures of meningococcal cells from the cultures in the stationary phase of development show the tendency to smoothing out, which is accompanied by their sharper differentiation.     

Tubular spinae are long-distance connectors between bacteria

The marine pseudomonad D71 (NCMB 2018) ['Spinomonas maritima'] can be induced to produce long tubular surface appendages (spinae) in a growth medium of low osmolarity. In general, spina-carrying cells show these appendages with open distal ends. We examined cultured cells by scanning and transmission electron microscopy, using both critical-point drying and thin sectioning after embedding with agarose protection. By scanning electron microscopy, spinae were observed that connected cells over distances of several micrometers. Ultrathin sections often revealed an additional layer outside the outer membrane, resembling an S-layer. The inner and outer cell membranes were often joined at spina-insertion areas. Furthermore, evidence was found in ultrathin sections for uninterrupted tubes connecting two cells over a distance of up to 7 microns. We propose, therefore, that spinae form the framework for wide open cell clusters; we hypothesize that these spinae might also permit an exchange of cell-to-cell signals.     

Pressure Resin Infiltration of Aphids for Electron Microscopy

Fixed, dehydrated pea aphids, Acyrthosiphon pisum Harris, were partially infiltrated with epoxy resins by standard procedures, then placed in a pressure chamber at up to 1000 psi for varying lengths of time. Insects so treated were found to be more suitable than nonpressurized insects for ultrathin sectioning and electron microscopy.

It was necessary to remove one or more legs from the insects to obtain adequate infiltration even where high pressures were employed. Little damage was evident at light or electron microscope levels of examination.     

Vitreous Carbon: A New Material for Making Microtome Knives

The quality of sections obtained by microtomy depends to a large extent on the quality and characteristics of the microtome knife itself. Despite the need for improved microtomy techniques, there have been few significant developments since the introduction of glass and diamond knives in the 1950's. The manufacture of microtome knives from vitreous carbon provides new possibilities for developing both improved methods and improved equipment for specimen sectioning. Vitreous carbon has unique physical properties that lend themselves to the generation of precision cutting edges. Such an edge can be obtained either by breaking a piece of vitreous carbon or by using lapidary techniques. The resultant edge seems well adapted to both thick and thin sectioning. The introduction of vitreous carbon as a sectioning tool offers a significant alternative to metal, glass and diamond knives.     

Wafer embedding: Specimen selection in electron microscopic cytochemistry with osmiophilic polymers

Synopsis A new wafer embedding procedure is described that permits light microscopic screening of embedded tissue prior to ultrathin sectioning. It is particularly valuable when used on specimens obtained with an automatic sectioner and treated cytochemically to obtain visible intermediate or visible and electron opaque final reaction products. Aldehyde-fixed tissues are cut into sections with an automatic sectioner, incubated cytochemically including osmication if required, then embedded in epoxy resin between fluorocarbon-coated coverglasses which are supported by a platform specially designed for this purpose. The resultant wafer, less than 0.2 mm thick, is examined by light microscopy for optimal areas of cytochemical reaction and desirable structural features. Such areas are cut out and glued to blank blocks with fast curing cyanoacrylate cement for subsequent ultrathin sectioning. The usefulness of this technique is demonstrated by the location of: (1) esterase-positive lysosomes in kidney and trigeminal ganglia; (2) palatal sensory endings stained for acetylcholinesterase; and (3) phagosomes arising from the resorption of horseradish peroxidase tracer by the cuboidal parietal epithelial cells of Bowman's capsule in the male mouse.     

Cryo-ultramicrotomy after coating of the cut surface of the tissue block with carbon and metals before sectioning.

Working under improvised conditions it is shown that coating of the cut surface of a frozen tissue block with metal and carbon improves, when applied prior to each cutting, the sectioning properties and the handling of the subsequent ultrathin cryosection.