The negative staining of "difficult" bacteria like Arthrobacter globiformis for electron microscopy

1974 ◽  
Vol 20 (6) ◽  
pp. 901-903 ◽  
Author(s):  
E. C. S. Chan ◽  
M. Gomersall ◽  
J. Bernier
Keyword(s):  
Electron Microscopy ◽  
Cell Suspension ◽  
Phosphotungstic Acid ◽  
Negative Staining ◽  
Arthrobacter Globiformis ◽  
Glutaraldehyde Fixation ◽  
Plain Agar

A method is suggested for the negative staining of "difficult" bacteria like Arthrobacter globiformis for electron microscopy when the usual procedures do not give satisfactory results. It involves glutaraldehyde fixation, and thorough washing and drying of the cell suspension on a grid sitting on a bed of plain agar, followed by a rapid flushing with 2% phosphotungstic acid.

Some Morphologic Features of Axial Fibers and Body Fibrils of Treponemata

1970 ◽  
Vol 28 ◽  
pp. 110-111
Author(s):  
Ronald H. Jones ◽  
Zensaku Yoshii ◽  
Oscar J. Carver
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Phosphotungstic Acid ◽  
Defined Medium ◽  
Ammonium Molybdate ◽  
Negative Staining ◽  
Rabbit Serum ◽  
Cell Pole ◽  
Internal Cell ◽  
Cell Layers

Four strains (Reiter, Kazan 2,4,5) of treponemata were cultivated at 37° C for 3-7 days in standard media containing complement inactivated rabbit serum (Cannefax, Spirolate) and in a defined medium devised by Johnson and his associates (unpublished data). Electron microscope preparations included cells which were unwashed (harvested), washed up to seven times, autolysed, or ruptured by explosive decompression. Each preparation was fixed with gluteraldehyde or left unfixed. Negative staining was performed using phosphotungstic acid (PTA - pH 7.2) or ammonium molybdate (AM - pH 7.0-7.2). Electron microscopy was done with a JEM 6A model at 80kV using a magnification range of 10,000-50,000X.The autolytic and disruption techniques were most effective for visualizing the ultrastructure of the more internal cell layers or moieties. The number of axial fibers (AF) arising from each cell pole varied from 4-7, six being the most common.

Electron microscopy of keratinocytes cultured on a collagen substrate used as an allograft for the treatment of chronic wounds

1992 ◽  
Vol 50 (2) ◽  
pp. 1104-1105
Author(s):  
Debby A. Jennings ◽  
Michael J. Morykwas ◽  
Louis C. Argenta
Keyword(s):  
Electron Microscopy ◽  
Cell Suspension ◽  
Single Cell ◽  
Chronic Wounds ◽  
Mechanical Stability ◽  
Uv Light ◽  
Type I ◽  
Single Cell Suspension ◽  
Collagen Substrate ◽  
Dermal Collagen

Grafts of cultured allogenic or autogenic keratlnocytes have proven to be an effective treatment of chronic wounds and burns. This study utilized a collagen substrate for keratinocyte and fibroblast attachment. The substrate provided mechanical stability and augmented graft manipulation onto the wound bed. Graft integrity was confirmed by light and transmission electron microscopy.Bovine Type I dermal collagen sheets (100 μm thick) were crosslinked with 254 nm UV light (13.5 Joules/cm2) to improve mechanical properties and reduce degradation. A single cell suspension of third passage neonatal foreskin fibroblasts were plated onto the collagen. Five days later, a single cell suspension of first passage neonatal foreskin keratinocytes were plated on the opposite side of the collagen. The grafts were cultured for one month.The grafts were fixed in phosphate buffered 4% formaldehyde/1% glutaraldehyde for 24 hours. Graft pieces were then washed in 0.13 M phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Polybed 812.

Identification of the pulmonary syndrome hantavirus by direct and colloidal gold immune Electron Microscopy

1994 ◽  
Vol 52 ◽  
pp. 274-275
Author(s):  
C. D. Humphrey ◽  
C.S. Goldsmith ◽  
L. Elliott ◽  
S.R. Zaki
Keyword(s):  
Electron Microscopy ◽  
United States ◽  
Monoclonal Antibody ◽  
Colloidal Gold ◽  
Phosphotungstic Acid ◽  
Uranyl Acetate ◽  
Hantavirus Pulmonary Syndrome ◽  
Deer Mice ◽  
Immune Electron Microscopy ◽  
Negative Stain

An outbreak of unexplained acute pulmonary syndrome with high fatality was recognized in the spring of 1993 in the southwestern United States. The cause of the illness was quickly identified serologically and genetically as a hantavirus and the disease was named hantavirus pulmonary syndrome (HPS). Recently, the virus was isolated from deer mice which had been trapped near the homes of HPS patients, and cultivated in Vero E6 cells. We identified the cultivated virus by negative-stain direct and colloidal gold immune electron microscopy (EM).Virus was extracted, clarified, and concentrated from unfixed and 0.25% glutaraldehyde fixed supernatant fluids of infected Vero E6 cells by a procedure described previously. Concentrated virus suspensions tested by direct EM were applied to glow-discharge treated formvar-carbon filmed grids, blotted, and stained with 0.5% uranyl acetate (UA) or with 2% phosphotungstic acid (PTA) pH 6.5. Virus suspensions for immune colloidal gold identification were adsorbed similarly to filmed grids but incubated for 1 hr on drops of 1:50 diluted monoclonal antibody to Prospect Hill virus nucleoprotein or with 1:50 diluted sera from HPS virus infected deer mice.

scholarly journals SYNTHESIS, MIGRATION, AND RELEASE OF PRECURSOR COLLAGEN BY ODONTOBLASTS AS VISUALIZED BY RADIOAUTOGRAPHY AFTER [3H]PROLINE ADMINISTRATION

1974 ◽  
Vol 60 (1) ◽  
pp. 92-127 ◽  
Author(s):  
Melvyn Weinstock ◽  
C. P. Leblond
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Phosphotungstic Acid ◽  
Secretory Granules ◽  
Low Ph ◽  
Collagen Fibrils ◽  
Dentin Collagen ◽  
Odontoblast Process ◽  
High Radioactivity

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.

Postjunctional supersensitivity of the rat vas deferens and gap junctions

1976 ◽  
Vol 54 (3) ◽  
pp. 412-416 ◽  
Author(s):  
D. M. Paton ◽  
J. Buckland-Nicks ◽  
A. Johns
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Spontaneous Activity ◽  
Vas Deferens ◽  
Rat Vas Deferens ◽  
Sprague Dawley ◽  
Glutaraldehyde Fixation ◽  
Sprague Dawley Rats

Tissues from the duodenum and vas deferens of Sprague–Dawley rats were examined of the rat vas deferens and gap junctions. Can. J. Physiol. Pharmacol. 54, 412–416. by electron microscopy after glutaraldehyde fixation and postosmication. Gap junctions (nexuses) were readily demonstrated in the duodenum in both control and reserpine treated animals (1.0 mg/kg per day for 7 days). However, gap junctions could not be demonstrated in vas deferens. It is concluded that the postjunctional supersensitivity and spontaneous activity induced by reserpine in vas deferens, does not result from the formation of gap junctions.

The fine structure and the protein composition of γ phage of Bacillus anthracis

1975 ◽  
Vol 21 (11) ◽  
pp. 1889-1892 ◽  
Author(s):  
Takashi Watanabe ◽  
Akinori Morimoto ◽  
Toshiro Shiomi
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Bacillus Anthracis ◽  
Protein Composition ◽  
Staining Technique ◽  
Negative Staining ◽  
Polyacrylamide Gels ◽  
Long Tail

The fine structure of γ phage of Bacillus anthracis was studied by electron microscopy with a negative-staining technique. The phage has a hexagonal head and a long tail without a sheath. By electrophoresis on polyacrylamide gels, the proteins of the phage particles are separate into 10 polypeptides with moleclar weights ranging from 140 000 to 12 000.

Electron microscopy of interphase chromosomes in situ; binding of permanganate to chicken erythrocytes

1976 ◽  
Vol 20 (2) ◽  
pp. 289-307
Author(s):  
H.G. Davies
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Chemical Composition ◽  
Protein Interaction ◽  
Phosphotungstic Acid ◽  
Intact Cell ◽  
Anomalous Behaviour ◽  
Protein Protein Interaction ◽  
Chicken Erythrocytes

From quantitative electron-microscope observations on the binding of permanganate to regions of erythrocytes and reticulocytes of known chemical composition, it is concluded that KMnO4, like phosphotungstic acid (PTA), binds preferentially to sites on proteins. Compared with PTA, KMnO4 binding exhibits less anomalous behaviour. The data support the hypothesis previously put forward that the 2 regions, or phases, in condensed chromatin differ in both molecular composition and concentration. The increase in binding to protein which occurs during nuclear haemolysis is interpreted in terms of protein-protein interaction in the chromatin of the intact cell.

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