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Light and electron microscopic examination of isolated neurons, astrocytes and oligodendrocytes.

Astrocytes and neuronal and oligodendroglial perikarya isolated by the method of Norton and Poduslo (1970) were examined by transmission and scanning electron microscopy and inverted phase contrast microscopy. The viability of the cells, as determined by the eosin exclusion method, was also determined. The three cell fractions showed only slight cross-contamination, but the astrocyte fraction contained significant amount of small debris. The ultrastructural appearance of the cells indicated that much of the in situ properties were retained, with bundles of fibrils preserved in astrocytes with well-defined plasma membranes. Oligodendroglial perikarya were found to be the best preserved of the cell types. The viability studies indicated that about 90% of the cells excluded eosin. Scanning electron microscopy revealed the neuronal cell surface to be rough and studded with knob-like bodies. Oligodendrocytes tended to aggregate and demonstrated a much smoother surface than the neurons.

Animals↗

A model for demonstrating the adhesion of Actinobacillus seminis to epithelial cells.

The objective of this study was to demonstrate that a field isolate of Actinobacillus seminis (As8C) will adhere to epithelial cells and that this adhesion can be inhibited by pretreating the bacteria with mouse serum containing polyclonal antibodies (PoAbs) prepared against this isolate. An indirect fluorescent antibody test, transmission electron microscopy, and phase-contrast microscopy confirmed the adhesion of As8C to an established culture of bovine kidney epithelial cells (BKECs). In a bacterial adhesion assay, 40 As8C were estimated to adhere to each BKEC after 60 min. Using a bacterial inhibition assay, PoAbs diluted 10(-2) or 10(-3) inhibited the adhesion of As8C to BKECs by approximately 90%. Bacterial inhibition decreased to about 50% when the PoAbs were diluted to 10(-4). There was less than 10% inhibition of adhesion of As8C to BKECs when higher dilutions of PoAbs were used. The inhibition of As8C adhesion to BKECs was less than 20% following pretreatment of BKECs with 10(-2) to 10(-5) dilutions of PoAbs. Moreover, pretreatment of As8C with a 10(-2) dilution of PoAbs did not appear to adversely affect bacterial growth on agar. It is likely that the PoAbs interrupted the adhesion of As8C to BKECs by sterically interfering with a bacterial adhesin-epithelial cell receptor interaction.

Actinobacillus↗

Phase-contrast and electron microscopy of murine strains of Mycoplasma.

Nelson, John B. (The Rockefeller University, New York, N.Y.), and Michael J. Lyons. Phase-contrast and electron microscopy of murine strains of Mycoplasma. J. Bacteriol. 90:1750-1763. 1965.-Two strains of Mycoplasma pulmonis (associated with infectious catarrh) on examination in fluid culture (20% horse serum-bouillon) by phase microscopy were highly pleomorphic, with many bacilliform elements and fewer coccoid ones. Motility, characterized by gliding of rods and spinning of spherical forms, was observed through the 9th subculture of one strain and the 15th of the second. Motile elements were not seen in later transfers and pleomorphism was reduced. One strain of M. neurolyticum (associated with conjunctivitis and encephalitis) was much less pleomorphic and showed neither bacilliform elements nor motility at any time. When examined by negative-contrast electron microscopy, organisms of this strain were found to have an average diameter of 0.7 mu and to possess a concentrated peripheral layer of cytoplasm and a central mass which may represent the cells' nuclear equivalent. The latter feature was not prominent in spherical forms of M. pulmonis. These cells, when observed after 48 hr of culture, showed evidence of the generation of new progeny cells in their central area. The filamentous or bacilliform cells of M. pulmonis were frequently serpentine in appearance, 2.0 to 3.0 mu in length and 80 to 250 mmu in width. They appeared to generate new cells from terminal buds from which outpouchings initially developed. Older cells, in the stationary phase, showed evidence of undergoing multipolar germination. Microtubules, about 60 mmu wide, were found in association with most filamentous cells from 48-hr cultures; fragments of membrane, studded with closely packed ribosomelike particles, were also found. There was no evidence of flagella or any specialized structure that could account for the observed motility of the organisms.

Animals↗

Solid-phase immune electron microscopy (SPIEM) by use of protein A and its application for characterization of selected adenovirus serotypes.

Staphylococcal protein A was used for the anchoring of specific antibodies for a solid-phase immune electron microscope method (SPIEM). More IgG adsorbed to grids in the absence of protein A than in its presence. However, the virus trapping efficiency was markedly improved in the presence of protein A. The specificity of the test was evaluated by use of different adenovirus preparations and matching rabbit hyperimmune sera. The degree of serological reaction was evaluated by counting the number of particles attached to the grid. Type specific reactions and inter- and intrasubgroup reactions were identified.

Adenoviridae↗

UPTAKE OF MAMMALIAN CHROMOSOMES BY MAMMALIAN CELLS.

Chromosomes isolated from mouse leukemia L1210 cells were taken up by mouse macrophages, HeLa cells, and rat embryo fibroblasts following simple exposure in vitro. The process, which resembles pinocytosis or phagocytosis, was traced by autoradiography of chromosomes prelabeled with thymidine-H(3), and by staining techniques and phase contrast microscopy. During the first six hours, the uptake of chromosomes was restricted to the cytoplasm, but there was some evidence of penetration into the nucleus after 16 and 26 hours of exposure. Treatment of rat fibroblasts with glucose and insulin markedly enhanced the uptake of chromosomes, whereas iodoacetate inhibited their penetration.

Animals↗

Events in the movement of newt epidermal cells across implanted substrates.

Pieces of coverslip glass, polycarbonate filters, or coverslip plastic, coated with fibrinogen or type I collagen, were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Migratory events were then analyzed by phase contrast and electron microscopy. Phase-contrast microscopy revealed two types of lamellipodia on leading edge cells: one which was attached broadly to the cell body and one attached by a long, thin stalk. Stalkless forms were by far the most common type and we believe they provide the motive force for cell movement. Stalked-forms often moved at distinct angles to the direction of sheet movement, suggesting that they may be sensory appendages. Phase photographs of the leading edge of migrating sheet 4 hours and 8 hours after implantation showed that all cells that were on the leading edge at 4 hours continued to advance for the next 4 hours, demonstrating clearly that under these circumstances the distalmost cells do not become immobile upon contact with the substrate as others have suggested. TEM revealed that migrating sheets were modified monolayers and that regardless of proximodistal location in the sheet, and even in the intact skin adjoining a wound, each epidermal cell adjacent to the substrate puts forth a lamellipodium which underlaps the cell in front. This and the behavior of sheets as they were teased or pulled from the implant suggest strongly that all basal cells contribute to movement of the sheet by interacting with the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effect of artificial tears on cultured keratocytes in vitro.

We investigated the effects of artificial tears on cultured rabbit and human keratocytes in vitro. The cells were exposed to seven nonpreserved commercially available artificial tear formulations and examined under phase-contrast microscopy for 60 min. After 5-min exposures to the solutions, rabbit keratocytes were fixed for transmission electron microscopy (TEM). In rabbit keratocytes, phase-contrast microscopy and TEM demonstrated that Aqua Site (CIBA Vision Ophthalmics, Atlanta, GA, U.S.A.), Hypo Tears PF (Johnson & Johnson, Claremont, CA, U.S.A.), and Tears Naturale Free (Alcon, Humacao, Puerto Rico, U.S.A.) immediately induced intracytoplasmic vacuoles and cell swelling, and subsequent cell degeneration. Rabbit cells exposed to the other artificial tears, which contained Ca2+ and did not contain EDTA, maintained their normal shape and appearance for 60 min. Phase-contrast microscopy of human keratocytes showed that Aqua Site and Hypo Tears PF induced mild and delayed cellular swelling, but the other artificial tears tested did not affect the cell shape for the entire 60-min observation period. Electrolyte balance and osmolarity of artificial tears appear to be critical for keratocyte survival. Maintenance of keratocyte integrity may be an important factor to consider when selecting an artificial tear preparation to be used when corneal epithelium is not intact.

Animals↗

Characterization of testudine melanomacrophage linear, membrane extension processes--cablepodia--by phase and atomic force microscopy.

Melanomacrophages (MMs) are a component of an internal, pigmented cell system in liver and splenic tissues of some fishes, anurans, and reptiles. The cells have been found in centers or aggregates in sinusoids and are associated with cells capable of producing a peptide cytokine and immunoglobulins. A unique cell extension process has been observed in turtle MMs placed into cell culture, and this process has been studied by light and atomic force microscopy. These structures, referred to as cablepodia, are uniquely straight, narrow, and unbranching and appear to originate from growth cones opposite lamellipodia. Cablepodia were found to connect with other turtle MMs and fibroblasts forming cell networks. Dividing fibroblasts to which a cablepodium attached ceased cell division. The observations collectively suggest that a principal reason for aggregations of MMs in internal organs of lower vertebrates is their ability to form interconnected networks of cell processes for trapping and processing of particulate matter, cells, and infectious organisms and, possibly, for the communication of cell signals and transfer of intracellular materials.

Animals↗

Red blood cells, phase contrast, interference contrast microscopy and microspectrophotometry.

Following Teorell's (1) observation that the ghosts of hypotonically hemolysed erythrocytes reseal, it was shown that during the time they are permeable to hemoglobin, foreign macromolecules (dextran) can enter and that the hemolysed cell can achieve a final colloid-osmotic equilibrial state containing dextran and some residual Hb. In this way dextran reduces the hemoglobin loss in hypotonic hemolysis. Some hemoglobin loss is, however, inevitable, as it begins with a non-diffusive bulk outflow, sometimes observable as a jet, during which time a diffusive influx of the colloid-osmotic "balancer", dextran, is not possible. Finally, as expected from a process which is for the most part diffusive, transmembrane macromolecular transport is bidirectional; during hemolysis smaller molecules escape to a greater extent than larger ones.

Erythrocytes↗