Growth of mammalian cells on carbon-coated plastic substrata

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

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Biophysical Measurement of Molecular Weight of Foot-and-Mouth Disease RNA Fragments

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051323 ◽

1974 ◽

Vol 32 ◽

pp. 262-263

Author(s):

Sydney S. Breese ◽

Howard L. Bachrach

Keyword(s):

Chemical Properties ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Distilled Water ◽

Bovine Plasma ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Carbon Coated ◽

Rna Fragments ◽

Physical And Chemical

Continuing studies on the physical and chemical properties of foot-and-mouth disease virus (FMDV) have included electron microscopy of RNA strands released when highly purified virus (1) was dialyzed against demlneralized distilled water. The RNA strands were dried on formvar-carbon coated electron microscope screens pretreated with 0.1% bovine plasma albumin in distilled water. At this low salt concentration the RNA strands were extended and were stained with 1% phosphotungstic acid. Random dispersions of strands were recorded on electron micrographs, enlarged to 30,000 or 40,000 X and the lengths measured with a map-measuring wheel. Figure 1 is a typical micrograph and Fig. 2 shows the distributions of strand lengths for the three major types of FMDV (A119 of 6/9/72; C3-Rezende of 1/5/73; and O1-Brugge of 8/24/73.

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The Application of Ultracryotomy to Immunocytochemistry

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073969 ◽

1973 ◽

Vol 31 ◽

pp. 704-709

Author(s):

R. G. Painter ◽

K. T. Tokuyasu ◽

S. J. Singer

Keyword(s):

Frozen Sections ◽

Protein Antigens ◽

Carbon Coated ◽

Antibody Conjugates ◽

Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

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Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060696 ◽

1967 ◽

Vol 25 ◽

pp. 136-137

Author(s):

J. P. Petrali ◽

E. J. Donati ◽

L. A. Sternberger

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Mammalian Cells ◽

Specific Antiserum ◽

Electron Microscopic ◽

E Coli ◽

Cytoplasmic Membranes ◽

Viral Progeny ◽

Ultrathin Sections ◽

Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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Cell Fine Structure as Revealed in Dessicator-Dried Resinless Sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099532 ◽

1981 ◽

Vol 39 ◽

pp. 622-623

Author(s):

R. P. Becker ◽

J. J. Wolosewick ◽

J. Ross-Stanton

Keyword(s):

Fine Structure ◽

Tannic Acid ◽

Three Dimensional ◽

Albino Rats ◽

Cell Organelles ◽

Vascular Perfusion ◽

Thin Sections ◽

Carbon Coated ◽

Embedding Medium ◽

Polyethylene Glycol 6000

Methodology has been introduced recently which allows transmission and scanning electron microscopy of cell fine structure in semi-thin sections unencumbered by an embedding medium. Images obtained from these “resinless” sections show a three-dimensional lattice of microtrabeculfee contiguous with cytoskeletal structures and membrane-bounded cell organelles. Visualization of these structures, especially of the matiiDra-nous components, can be facilitated by employing tannic acid in the fixation step and dessicator drying, as reported here.Albino rats were fixed by vascular perfusion with 2% glutaraldehyde or 1.5% depolymerized paraformaldehyde plus 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4). Tissues were removed and minced in the fixative and stored overnight in fixative containing 4% tannic acid. The tissues were rinsed in buffer (0.2M cacodylate), exposed to 1% buffered osmium tetroxide, dehydrated in ethyl alcohol, and embedded in pure polyethylene glycol-6000 (PEG). Sections were cut on glass knives with a Sorvall MT-1 microtome and mounted onto poly-L-lysine, formvar-carbon coated grids while submerged in a solution of 95% ethanol containing 5% PEG.

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Low temperature x-ray microanalysis of frozen-hydrated tobacco leaves

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100111537 ◽

1984 ◽

Vol 42 ◽

pp. 284-285

Author(s):

S. Edith Taylor ◽

Patrick Echlin ◽

May McKoon ◽

Thomas L. Hayes

Keyword(s):

Low Temperature ◽

Lemna Minor ◽

Root Tips ◽

Tobacco Leaves ◽

X Ray ◽

Whole Cells ◽

Carbon Coated ◽

The Right ◽

Beam Currents ◽

The University

Low temperature x-ray microanalysis (LTXM) of solid biological materials has been documented for Lemna minor L. root tips. This discussion will be limited to a demonstration of LTXM for measuring relative elemental distributions of P,S,Cl and K species within whole cells of tobacco leaves.Mature Wisconsin-38 tobacco was grown in the greenhouse at the University of California, Berkeley and picked daily from the mid-stalk position (leaf #9). The tissue was excised from the right of the mid rib and rapidly frozen in liquid nitrogen slush. It was then placed into an Amray biochamber and maintained at 103K. Fracture faces of the tissue were prepared and carbon-coated in the biochamber. The prepared sample was transferred from the biochamber to the Amray 1000A SEM equipped with a cold stage to maintain low temperatures at 103K. Analyses were performed using a tungsten source with accelerating voltages of 17.5 to 20 KV and beam currents from 1-2nA.

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Examination of Rabbit Fc Crystals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100445 ◽

1968 ◽

Vol 26 ◽

pp. 140-141

Author(s):

J. P. Robinson ◽

P. G. Lenhert

Keyword(s):

Molecular Weight ◽

Flat Plate ◽

Phosphate Buffer ◽

Asymmetric Unit ◽

X Ray Diffraction ◽

X Ray ◽

Neutral Ph ◽

Small Crystals ◽

Carbon Coated ◽

Diffraction Patterns

Crystallographic studies of rabbit Fc using X-ray diffraction patterns were recently reported. The unit cell constants were reported to be a = 69. 2 A°, b = 73. 1 A°, c = 60. 6 A°, B = 104° 30', space group P21, monoclinic, volume of asymmetric unit V = 148, 000 A°3. The molecular weight of the fragment was determined to be 55, 000 ± 2000 which is in agreement with earlier determinations by other methods.Fc crystals were formed in water or dilute phosphate buffer at neutral pH. The resulting crystal was a flat plate as previously described. Preparations of small crystals were negatively stained by mixing the suspension with equal volumes of 2% silicotungstate at neutral pH. A drop of the mixture was placed on a carbon coated grid and allowed to stand for a few minutes. The excess liquid was removed and the grid was immediately put in the microscope.

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Microstructural Evaluation of Silicon Carbide Whisker Reinforced Alumina Fabricated with Carbon-Coated Whiskers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100088919 ◽

1991 ◽

Vol 49 ◽

pp. 920-921

Author(s):

K. B. Alexander ◽

P. F. Becher

Keyword(s):

Silicon Carbide ◽

High Resolution Electron Microscopy ◽

Ceramic Composites ◽

Interface Debonding ◽

Resolution Electron ◽

Microstructural Evaluation ◽

Carbon Coated ◽

Electron Energy Loss ◽

Interfacial Films ◽

Debonding Process

The presence of interfacial films at the whisker-matrix interface can significantly influence the fracture toughness of ceramic composites. The film may alter the interface debonding process though changes in either the interfacial fracture energy or the residual stress at the interface. In addition, the films may affect the whisker pullout process through the frictional sliding coefficients or the extent of mechanical interlocking of the interface due to the whisker surface topography.Composites containing ACMC silicon carbide whiskers (SiCw) which had been coated with 5-10 nm of carbon and Tokai whiskers coated with 2 nm of carbon have been examined. High resolution electron microscopy (HREM) images of the interface were obtained with a JEOL 4000EX electron microscope. The whisker geometry used for HREM imaging is described in Reference 2. High spatial resolution (< 2-nm-diameter probe) parallel-collection electron energy loss spectroscopy (PEELS) measurements were obtained with a Philips EM400T/FEG microscope equipped with a Gatan Model 666 spectrometer.

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The application of video-enhanced constrast/differential interference constrast microscopy in conjunction with whole mount electron microscopy to the study of organelle transport

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102419 ◽

1988 ◽

Vol 46 ◽

pp. 66-67

Author(s):

J. H. Hayden

Keyword(s):

Electron Microscopy ◽

Rana Pipiens ◽

Silver Film ◽

Immunofluorescence Microscopy ◽

Organelle Transport ◽

Linear Element ◽

Whole Mount ◽

Carbon Coated ◽

Linear Elements ◽

Dic Microscopy

In a previous study, Allen video-enhanced constrast/differential interference constrast (AVEC-DIC) microscopy was used in conjunction with immunofluorescence microscopy to demonstrate that organelles and vesicle move in either direction along linear elements composed of microtubules. However, this study was limited in that the number of microtubules making up a linear element could not be determined. To overcome this limitation, we have used AVEC-DIC microscopy in conjunction with whole mount electron microscopy.Keratocytes from Rana pipiens were grown on glass coverslips as described elsewhere. Gold London Finder grids were Formvar- and carbon coated, and sterilized by exposure to ultraviolet light. It is important to select a Formvar film that gives a grey reflection when it is floated on water. A silver film is too thick and will detract from the image in the light microscope.

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Cryomicroscopy of multiphase latices

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100089615 ◽

1991 ◽

Vol 49 ◽

pp. 1060-1061

Author(s):

O. L. Shaffer ◽

M.S. El-Aasser ◽

C. L. Zhao ◽

M. A. Winnik ◽

R. R. Shivers

Keyword(s):

Electron Microscopy ◽

Radiation Damage ◽

Freeze Fracture ◽

Particle Morphology ◽

Second Stage ◽

Transmission Electron ◽

Important Approach ◽

Carbon Coated ◽

Preparation Techniques

Transmission electron microscopy is an important approach to the characterization of the morphology of multiphase latices. Various sample preparation techniques have been applied to multiphase latices such as OsO4, RuO4 and CsOH stains to distinguish the polymer phases or domains. Radiation damage by an electron beam of latices imbedded in ice has also been used as a technique to study particle morphology. Further studies have been developed in the use of freeze-fracture and the effect of differential radiation damage at liquid nitrogen temperatures of the latex particles embedded in ice and not embedded.Two different series of two-stage latices were prepared with (1) a poly(methyl methacrylate) (PMMA) seed and poly(styrene) (PS) second stage; (2) a PS seed and PMMA second stage. Both series have varying amounts of second-stage monomer which was added to the seed latex semicontinuously. A drop of diluted latex was placed on a 200-mesh Formvar-carbon coated copper grid.

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