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Results for “Negative Staining”

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PubMed1980

Fidelity of structure representation in electron micrographs of negatively stained protein molecules.

We have investigated the fidelity of structure representation in electron micrographs of negatively stained proteins by conducting a systematic evaluation of such micrographs in terms of a known molecular structure, solved by x-ray crystallography. Microcrystals of immunoglobulin G Dob were used as specimens in this comparison between micrograph images, optimized by computer image processing, and reference images derived computationally from the crystal structure. To an effective resolution of 2 nm, we observed a remarkably good correlation between the experimental images and their idealized counterparts, which are unaffected by those factors--electron irradiation and dehydration--that are thought to be primarily responsible for perturbation of protein structure during electron microscopy. Separate structural features resolved in these micrographs do not, in general, correspond to specific components of individual molecules but arise instead from complex superpositions involving several overlapping molecules.

Crystallography↗
PubMed1992

Has negative staining still a place in biomacromolecular electron microscopy?

Transmission electron microscopy of proteins has provided molecular- and in a few cases near-atomic-resolution structural information. In this review, we critically evaluate the potential and the limitations in obtaining molecular resolution, particularly with negatively stained specimens, and put these into perspective with cryomicroscopy of unstained frozen-hydrated and sugar-embedded preparations.

Histocytological Preparation Techniques↗
PubMed1992

Three-dimensional reconstruction of single particles negatively stained or in vitreous ice.

The random-conical reconstruction method has been highly successful in three-dimensional imaging of macromolecules under low-dose conditions. This article summarizes the different steps of this technique as applied to molecules prepared with negative staining or vitreous ice, and sketches out the current directions of development. We anticipate that by using new instrumental developments, transfer function correction and computational refinement techniques, a resolution in the range of 7-10 A could ultimately be achieved.

Calcium Channels↗
PubMed1977

Negative staining of a non-haemadsorbing strain of African swine fever virus.

Since the application of negative staining, preceded by fixation, prevents the disruption and distortion of the capsid of the African swine fever virus, improved contrast and evaluation of the appearance and size of virus particles in the electron microscope is possible and, in addition, the icosahedral shape of the virus is demonstrable. The mature virus particle contains at least 2 capsid layers and an outer envelope.

African Swine Fever Virus↗
PubMed2002

Noncytopathic hepatitis A virus induces surface alterations in LLC-MK2 cells revealed by thin sections, negative staining, and scanning electron microscopy.

Previous electron microscope studies of ultrastructural events during hepatitis A virus replication in experimentally infected cells have used only ultrathin section techniques. Nevertheless, no important differences were observed between infected and uninfected cells. This study was carried out using scanning electron microscopy and negative staining of whole LLC-MK2 cells grown directly on grids covered with support membranes, and then infected with an hepatitis A virus strain. Thin sections of infected and uninfected controls were also analyzed. An intricate web of projections forming a net between cell interfaces was observed only in infected cells. Some of these projections were more than 700 nm long and had ballooning tips. Nevertheless, HAV particles were not visualized in the infected cells.

Animals↗
PubMed1985

Distinction between Bunyaviridae genera by surface structure and comparison with Hantaan virus using negative stain electron microscopy.

Ultrastructural studies of glutaraldehyde-fixed viruses of the Bunyaviridae were performed by negative-stain electron microscopy. The surface structure of viruses of each genus was compared with that of the other genera and with Hantaan virus, the prototype of a proposed new genus of Bunyaviridae. Viruses of each genus had a surface structure distinct for that genus. In addition, Hantaan virus had a surface structure composed of a grid-like pattern of morphologic subunits not previously described for animal viruses. Careful morphologic studies of suspected Bunyaviridae may be used in considering preliminary generic assignment. This study also supports the assignment of Hantaan-related viruses to a separate generic status within the Bunyaviridae.

Bunyaviridae↗
PubMed1978

Pseudo-exfoliation fibrils examined by negative staining.

Pseudo-exfoliation (PE) material, collected from lenses extracted because of cataract, has been examined by negative staining. The material was fragmented by ultrasound into single fibrils and small collections of fibrils. The fibrils were composed of a small number of filamentous subunits associated with or surrounded by a fuzzy material which had side excrescences at regular intervals of 50--55 nm or 25--30 nm. The side excrescences correspond to the cross-bands of the PE fibrils. It is proposed that the PE fibrils are composed of a few subunits of a fibrillar protein, which form a core to which glycosaminoglycan side chains are attached. The visible periodicity of the PE fibrils, appearing as cross-bands, is thought to be caused by clusters of glycosaminoglycan distributed at regular intervals along the fibrils.

Cytoskeleton↗
PubMed1983

Molecular sieve in bovine descemets membrane as revealed by negative staining.

Descemet's membrane was isolated from the corneas of cows and observed by electron microscopy after negative staining with 1% phosphotungstic acid solution, pH 7.2. Ultrastructurally, bovine Descement's membrane had a very regular hexagonal pattern. Nodes were connected to the six others around each of them by thin filaments to form a hexagon. The distance between the nodes was approximately 120 nm, the diameter of the nodes approximately 30 nm, and the width of the connecting filaments approximately 10 nm. Bovine Descement's membrane was a molecular sieve composed of nodes and filaments substantiating our molecular sieve theory of basement membranes.

Animals↗
PubMed1975

Infectious bursal disease agent: morphology by negative stain electron microscopy.

The virus of infectious bursal disease of chickens was studied by immune electron microscopy. Negatively-stained preparations revealed morphological similarities with both the bluetongue virus group, and the virus of infectious pancreatic necrosis of trout. Results indicated that the small particle found in such preparations is a degradation product of the large particle.

Centrifugation, Density Gradient↗
PubMed1987

Tubulofilaments in negatively stained scrapie-infected brains: relationship to scrapie-associated fibrils.

A simple method was devised for negative-stain transmission electron microscopy of brain infected with the agent of scrapie. Brains of infected hamsters contained large masses of tubulofilamentous structures with irregular fuzzy surfaces. Brains of mice infected with Creutzfeldt-Jakob disease agent contained similar tubulofilaments in smaller numbers. The abnormal tubulofilaments resembled but were distinguished from normal microtubules. On grids soaked in sodium dodecyl sulfate the abnormal tubulofilaments were found in stages of fragmentation, an outer coat appearing t0 strip from the surface to reveal thinner fibrillary structures resembling scrapie-associated fibrils (SAF). The unmasked fibrils were identified as SAF by immunogold labeling, while the larger tubulofilaments were not labeled. The findings indicate that in infected brain tissue SAF may occur as an internal part of a larger structure that is disrupted by detergent and are not likely to be an artifact formed during extraction procedures.

Actin Cytoskeleton↗
PubMed2005

High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy.

Helicobacter pylori secretes a vacuolating toxin (VacA) that can assemble into water-soluble oligomeric complexes and insert into membranes to form anion-selective channels. Previous studies have described multiple types of oligomeric VacA structures, including single-layered astral arrays, bilayered forms, and two-dimensional crystalline arrays. In the current study, vitrified VacA complexes were examined by cryo-negative staining electron microscopy, views of the different oligomeric structures in multiple orientations were classified and analyzed, and three-dimensional models of the bilayered forms of VacA were constructed with a resolution of about 19 angstroms. These bilayered forms of VacA have a "flower"-like structure, consisting of a central ring surrounded by symmetrically arranged peripheral "petals." Further structural insights were obtained by analyzing a mutant form of VacA (VacADelta6-27), which lacks a unique amino-terminal hydrophobic segment and is defective in the capacity to form membrane channels. Bilayered oligomeric complexes formed by wild-type VacA contained a visible density within the central ring, whereas bilayered complexes formed by VacADelta6-27 lacked this density. These results indicate that deletion of the VacA amino-terminal hydrophobic region causes a structural alteration in the central ring within VacA oligomers, and suggest that the central ring plays an important role in the process by which VacA forms membrane channels.

Bacterial Proteins↗
PubMed1978

Negative staining of whole cells: transmission electron microscopy of peripheral organelles in rat venous endothelial cells.

The study of whole negatively stained cells has revealed details of cellular organelles in rat venous endothelial cells. In particular, details of surface membrane organelles and small tubular structures were demonstrated. The surface membrane organelles which appeared "vesicular-like" were found to be connected with small tubular attachments. These findings were correlated with those described by other techniques. It is significant that this simple technique appears to permit the demonstration of fine details of three-dimensional cytoplasmic structures.

Animals↗
PubMed1985

The use of polylysine during negative staining of viral suspensions.

The use of 0.1% aqueous solution of polylysine (poly-L-lysine) is proposed as a prior step to negative staining of viral or particle suspensions. Particles spread better on films precoated with polylysine than with other substances used for the same purpose. This applies particularly to samples from sucrose or CsCl gradients. The use of negative contrast substances is discussed.

Adenoviridae↗
PubMed1978

Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining.

The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.

Animals↗
PubMed1977

Electron microscopy of negatively stained jackbean urease at three levels of quaternary structure, and comparison with hydrodynamic studies.

Electron microscopy, with sodium phosphotungstate as negative stain, has been carried out on purified jackbean urease prepared at three levels of quaternary structure: (a) A1 urease, Mr = 240 000, S20,W = 11.5 S (b) alpha urease, Mr = 480 000, S20,w = 18.3 S (c) polymers of alpha urease above the tetramer stage. The compatibility of the images from level to level leaves no doubt that the enzyme itself is being visualized, and the following geometry is suggested by electron microscopy: A1 molecules are cyclic trimers, which pair up in eclipsed position across a 1-nm cleft to form the hexameric alpha, which displays D3 (or 32) symmetry of a trigonal prism. Polymers consist of alpha molecules aligned with their clefts coplanar and an angle of 120 degrees between each triplet of 3-fold axes. These features correspond reasonably well with sedimentation and electrophoretic studies of the solvated enzyme, which have indicated a hemispherical A1, a spherical alpha, and string-of-beads polymers. Sedimentation constants of the urease polymers up through the pentamer level were found to be compatible with the rosette, straight-chain, and zig-zag forms seen in the electron microscope, and with the suggested protomer arrangement in A1 and alpha urease.

Macromolecular Substances↗