Electron microscopy in diagnostic virology

1984 ◽  
Vol 42 ◽  
pp. 70-73
Author(s):  
S. E. Miller
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Molecular Identification ◽  
Point Of View ◽  
Negative Stain ◽  
Viral Diagnosis ◽  
Electron Microscopist ◽  
Diagnostic Virology

The techniques for detecting viruses are many and varied including FAT, ELISA, SPIRA, RPHA, SRH, TIA, ID, IEOP, GC (1); CF, CIE (2); Tzanck (3); EM, IEM (4); and molecular identification (5). This paper will deal with viral diagnosis by electron microscopy and will be organized from the point of view of the electron microscopist who is asked to look for an unknown agent--a consideration of the specimen and possible agents rather than from a virologist's view of comparing all the different viruses. The first step is to ascertain the specimen source and select the method of preparation, e. g. negative stain or embedment, and whether the sample should be precleared by centrifugation, concentrated, or inoculated into tissue culture. Also, knowing the type of specimen and patient symptoms will lend suggestions of possible agents and eliminate some viruses, e. g. Rotavirus will not be seen in brain, nor Rabies in stool, but preconceived notions should not prejudice the observer into missing an unlikely pathogen.

scholarly journals Virus Identification by Electron Microscopy

2000 ◽  
Vol 8 (5) ◽  
pp. 28-29
Author(s):  
Sara E. Miller
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Aqueous Extract ◽  
Western Blots ◽  
Virus Identification ◽  
Negative Stain ◽  
Biochemical Identification ◽  
The Right ◽  
Specific Reagent ◽  
Do So

Electron microscopy is clearly the best way to look at enteric viruses, many of which do not grow in tissue culture, and those that can be so coaxed, do so under special conditions that are not routinely found in the culture lab. Biochemical identification (e.g., immunological kits, PCR, Western Blots) require a specific reagent to recognize the virus, and if the right reagent is not used, the viruses will be missed (e.g., if you run a test for rotavirus, you will miss adenovirus, etc.). Furthermore, there are not biochemical reagents for all viruses. With electron microscopy, one can see a wide variety of viruses by doing a negative stain of an aqueous extract of stool.

Optimization of diagnostic EM for enteric viruses

1992 ◽  
Vol 50 (1) ◽  
pp. 678-679
Author(s):  
C. D. Humphrey ◽  
H.H. Huang ◽  
H. Tanaka
Keyword(s):  
Electron Microscopy ◽  
Enteric Viruses ◽  
Virus Identification ◽  
Negative Stain ◽  
Enteric Adenovirus ◽  
Negative Stain Electron Microscopy ◽  
Viral Diagnosis

Virus identification by negative stain electron microscopy (EM) is one of the most versatile and sensitive assays for rapid viral diagnosis. Electron microscopy is particularly important for the study of enteric viruses because they are difficult to cultivate and characterize. The gastroenteritis viruses most frequently identified include rotavirus (Fig. 1), enteric adenovirus (Fig. 2), enteric coronavirus (Fig. 3), calicivirus (Fig. 4), Norwalk agent, and astrovirus (Fig. 6). In addition, there are several unclassified viruses categorized as either small round structured viruses (Fig. 5) or small round viruses (Fig. 7).

Tissue section lifting for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 86-87
Author(s):  
Adrian F. van Dellen
Keyword(s):  
Infectious Disease ◽  
Electron Microscopy ◽  
Tissue Culture ◽  
Organ System ◽  
Surrounding Tissue ◽  
Paraffin Sections ◽  
Surface Areas ◽  
Specific Determination ◽  
High Degree ◽  
Accuracy And Repeatability

The morphologic pathologist may require information on the ultrastructure of a non-specific lesion seen under the light microscope before he can make a specific determination. Such lesions, when caused by infectious disease agents, may be sparsely distributed in any organ system. Tissue culture systems, too, may only have widely dispersed foci suitable for ultrastructural study. In these situations, when only a few, small foci in large tissue areas are useful for electron microscopy, it is advantageous to employ a methodology which rapidly selects a single tissue focus that is expected to yield beneficial ultrastructural data from amongst the surrounding tissue. This is in essence what "LIFTING" accomplishes. We have developed LIFTING to a high degree of accuracy and repeatability utilizing the Microlift (Fig 1), and have successfully applied it to tissue culture monolayers, histologic paraffin sections, and tissue blocks with large surface areas that had been initially fixed for either light or electron microscopy.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

A Method for Electron Microscopic Study of Tissue Culture Cell Monolayers Grown in Tubes

1967 ◽  
Vol 25 ◽  
pp. 132-133
Author(s):  
R. Stephens ◽  
G. Schidlovsky ◽  
S. Kuzmic ◽  
P. Gaudreau
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Light Microscopy ◽  
Cell Sheet ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Electron Microscopic ◽  
Cell Sheets ◽  
Morphological Alterations ◽  
Culture Cells

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.

Ultrastructure of two neoplasms in culture compared with original biopsies

1984 ◽  
Vol 42 ◽  
pp. 50-51
Author(s):  
Joseph M. Harb ◽  
James T. Casper ◽  
Vlcki Piaskowski
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Biopsy Specimen ◽  
Cultured Cells ◽  
Light And Electron Microscopy ◽  
Human Tumor ◽  
Soft Agar ◽  
Human Tumor Cells ◽  
Morphological Distinction ◽  
Tumor Types

The application of tissue culture and the newer methodologies of direct cloning and colony formation of human tumor cells in soft agar hold promise as valuable modalities for a variety of diagnostic studies, which include morphological distinction between tumor types by electron microscopy (EM). We present here two cases in which cells in culture expressed distinct morphological features not apparent in the original biopsy specimen. Evaluation of the original biopsies by light and electron microscopy indicated both neoplasms to be undifferentiated sarcomas. Colonies of cells propagated in soft agar displayed features of rhabdomyoblasts in one case, and cultured cells of the second biopsy expressed features of Ewing's sarcoma.

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 A Cellular Reaction to Antibody in Tissue Culture Studied with Electron Microscopy

1959 ◽  
Vol 5 (3) ◽  
pp. 405-410 ◽  
Author(s):  
Harrison Latta
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Normal Serum ◽  
Butyl Methacrylate ◽  
Heart Cells ◽  
Plasma Clot ◽  
Outer Nuclear Membrane ◽  
Cell Components

The reaction of embryonic chick heart cells grown in tissue culture to specific guinea pig antiserum has been studied with electron microscopy. Heart fragments from chick embryos were cultured with a plasma clot. After being tested with antiserum or normal serum, they were fixed with buffered osmium tetroxide and embedded in butyl methacrylate before removal from the glass culture chamber. Thin cells found by phase microscopy to have reacted were sectioned in a plane parallel to the glass surface on which they had grown. The results confirm and extend observations made previously while the reactions were occurring. The plasma membrane, like that of the red cell, becomes disrupted or less resistant to trauma following the action of antiserum. The membranes of mitochondria and endoplasmic reticulum vesiculate and swell. Before nuclear shrinkage becomes prominent, the outer nuclear membrane separates over a large portion of the nuclear envelope and forms one or more large swollen blebs. Thus, the outer nuclear membrane shows a reactivity similar to endoplasmic reticulum. It is suggested that the various physical and chemical changes observed to follow the action of antibody and complement on fibroblasts may be explained by osmotic pressure differences between various cell components. Some basic similarities to the action of hemolytic agents on red cells are noted.

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