Fixation of Yeast Protoplasts for Electron Microscopy

Nature ◽  
1961 ◽  
Vol 191 (4792) ◽  
pp. 1022-1023 ◽  
Author(s):  
P. F. ELBERS
Keyword(s):  
Electron Microscopy ◽  
Yeast Protoplasts

Enlarged Crystalline Patches on the Plasma Membrane of Yeast Protoplasts

1980 ◽  
Vol 38 ◽  
pp. 686-687
Author(s):  
G. Sosinsky ◽  
R. Schekman ◽  
R. Glaeser
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Plasma Membrane ◽  
High Resolution Electron Microscopy ◽  
Yeast Cells ◽  
Physiological Conditions ◽  
Resolution Electron ◽  
Yeast Protoplasts ◽  
Intramembraneous Particles ◽  
Crystalline Array

The crystalline patches of intramembraneous particles that form in the yeast plasma membrane, under stationary state physiological conditions, represent a potentially interesting specimen for high resolution electron microscopy. Isolation of these crystalline membrane patches first requires removal of the cell wall and the formation of osmotically fragile yeast protoplasts. In developing a procedure for the isolation of these crystalline membrane patches, we have found that the intramembraneous particles form much larger crystalline patches in protoplasts than in intact yeast cells. We have performed deep etch experiments and have found that the crystalline array of particles is not expressed on the extracellular surface of the plasma membrane.

scholarly journals A Method of Isolating Anucleated Yeast Protoplasts Unable to Synthesize the Glucan Fibrillar Component of the Wall

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Marie Kopecká ◽  
M. Gabriel ◽  
O. Nečas
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Plasma Membrane ◽  
Light And Electron Microscopy ◽  
Sharp Contrast ◽  
Yeast Protoplasts ◽  
Inhibition Of Protein Synthesis ◽  
Reproducible Method ◽  
Fibrillar Component

A mixture of nucleated and anucleated protoplasts was produced from log-phase Saccharomyces cerevisiae by the use of snail enzymes. The mixture was separated by centrifugation, and anucleated protoplasts were studied by means of light and electron microscopy. Anucleated protoplasts did not synthesize glucan fibrils even though they seemed to contain all other basic structures in their cytoplasm, and the structure of the plasma membrane was unchanged. This was in sharp contrast to ordinary nucleated protoplasts which synthesized glucan fibrils even after inhibition of protein synthesis by cycloheximide. The reason for this behaviour of anucleated protoplasts is not clear. Such anucleated yeast protoplasts represent the first example of uniform anucleated fungi produced by a reproducible method.

Selective contrast enhancement with ruthenium tetroxide for observation of reverting yeast protoplasts

1990 ◽  
Vol 48 (3) ◽  
pp. 608-609
Author(s):  
Nobuko Naito ◽  
Naoko Yamada ◽  
Mamiko Sato ◽  
Hiromi Kobori ◽  
Masako Osumi
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Golgi Apparatus ◽  
Osmium Tetroxide ◽  
Low Voltage ◽  
Secretory Vesicles ◽  
Oxidizing Agent ◽  
Yeast Protoplasts ◽  
High Resolution Images ◽  
Very High

Rutenium tetroxide (RuO4) is a strong oxidizing agent like osmium tetroxide (OsO4). Therefore its possible use as a fixative and staining agent for electron microscopy is promissing. It also has the advantage of reacting with polar lipids which do not react with OsO4. Despite these advantages, however, RuO4 was not put into practical use because it is easily decomposed. Recently we used RuO4 fixation to observe the glucan fibrils on reverting protoplasts by low-voltage SEM, and obtained very high resolution images of glucan fibrils (Fig. 1). The purpose of this study was to evaluate the effectiveness of RuO4 as a stain for TEM. The results showed that RuO4 treatment is very effective for observing the u1trastructure of reverting yeast protoplasts. The treatment provided clear images of regenerating glucan fibrils, Golgi apparatus, secretory vesicles and filasomes in the cytoplasm.

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Immune Electron Microscopy (IEM) of a Canine Adeno-Associated Virus

1974 ◽  
Vol 32 ◽  
pp. 256-257
Author(s):  
Gunter F. Thomas ◽  
M. David Hoggan
Keyword(s):  
Electron Microscopy ◽  
Cell Cultures ◽  
Complement Fixation ◽  
Hepatitis Virus ◽  
Wide Distribution ◽  
Immune Electron Microscopy ◽  
Adeno Associated Virus ◽  
Virus Like Particle ◽  
Dog Kidney ◽  
Green Monkeys

In 1968, Sugimura and Yanagawa described a small 25 nm virus like particle in association with the Matsuda strain of infectious canine hepatitis virus (ICHV). Domoto and Yanagawa showed that this particle was dependent on ICHV for its replication in primary dog kidney cell cultures (PDK) and was resistant to heating at 70°C for 10 min, and concluded that it was a canine adeno-associated virus (CAAV). Later studies by Onuma and Yanagawa compared CAAV with the known human serotypes (AAV 1, 2, 3) and AAV-4, known to be associated with African Green Monkeys. Using the complement fixation (CF) test, they found that CAAV was serologically related to AAV-3 and had wide distribution in the dog population of Japan.

High Voltage Electron Microscopy of Ion-Milled Dental Enamel

1974 ◽  
Vol 32 ◽  
pp. 60-61
Author(s):  
L. D. Ackerman ◽  
S. H. Y. Wei
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Third Molar ◽  
Polarized Light ◽  
Dental Enamel ◽  
Longitudinal Section ◽  
Ion Milling ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron

Mature human dental enamel has presented investigators with several difficulties in ultramicrotomy of specimens for electron microscopy due to its high degree of mineralization. This study explores the possibility of combining ion-milling and high voltage electron microscopy as a means of circumventing the problems of ultramicrotomy.A longitudinal section of an extracted human third molar was ground to a thickness of about 30 um and polarized light micrographs were taken. The specimen was attached to a single hole grid and thinned by argon-ion bombardment at 15° incidence while rotating at 15 rpm. The beam current in each of two guns was 50 μA with an accelerating voltage of 4 kV. A 20 nm carbon coating was evaporated onto the specimen to prevent an electron charge from building up during electron microscopy.

Pathogenesis of Human Hair Defects

1974 ◽  
Vol 32 ◽  
pp. 12-13
Author(s):  
P.S. Porter ◽  
T. Aoyagi ◽  
R. Matta
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Human Hair ◽  
Standard Techniques ◽  
Scanning Electron

Using standard techniques of scanning electron microscopy (SEM), over 1000 human hair defects have been studied. In several of the defects, the pathogenesis of the abnormality has been clarified using these techniques. It is the purpose of this paper to present several distinct morphologic abnormalities of hair and to discuss their pathogenesis as elucidated through techniques of scanning electron microscopy.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

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