Osmium tetroxide vapor fixation for preserving fungal structures for SEM

1990 ◽  
Vol 48 (3) ◽  
pp. 748-749
Author(s):  
L.R. Tiedt
Keyword(s):  
Room Temperature ◽  
Osmium Tetroxide ◽  
Petri Dish ◽  
Usual Method ◽  
Water Soluble ◽  
Fungal Morphology ◽  
Drying Method ◽  
Critical Point Drying ◽  
Inner Surface ◽  
Scanning Electron

Scanning electron microscopy has provided valuable information in studies of fungal morphology and ontogeny, but undisturbed preservation of sporulating structures is often difficult.In this study of sporodochia development in Fusarium spp. in the section Liseola, the usual method of aqueous fixation, dehydration and the subsequent critical-point drying method, could not be used. Macroconidia forms sporodochia by means of water soluble slime and when aqueous solutions come in contact with the sporodochia, the conidia become scattered. To preserve these morphological structures, the samples had to be vapour fixed with osmium tetroxide. Sample blocks were excised from the sporulating cultures and carefully transferred to the inner surface of a Petri dish cover. The cover with several adherent agar blocks were inverted over a Petri dish bottom containing several drops of 2% osmium tetroxide. The Petri dish was left at room temperature for 24h. The Petri dish cover with the fixed samples was transferred to a clean Petri dish bottom and left to air dry.

Observations of thick and thin sections in a field-emission SEM

1994 ◽  
Vol 52 ◽  
pp. 1018-1019
Author(s):  
W. P. Wergin ◽  
S. Roy ◽  
E. F. Erbe ◽  
C. A. Murphy ◽  
C. D. Pooley
Keyword(s):  
Electron Microscope ◽  
Field Emission ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope ◽  
Scanning Electron

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.

scholarly journals Vapor Coating: A Simple, Economical Procedure for Preparing Difficult Specimens for Scanning Electron Microscopy

2007 ◽  
Vol 15 (3) ◽  
pp. 44-45 ◽  
Author(s):  
E. Ann Ellis ◽  
Michael W. Pendleton
Keyword(s):  
Osmium Tetroxide ◽  
Petri Dish ◽  
Specimen Preparation ◽  
Fume Hood ◽  
Imaging Center ◽  
User Facility ◽  
Small Container ◽  
Set Up ◽  
Preparation Techniques ◽  
Scanning Electron

The Microscopy and Imaging Center at Texas A&M University is a multi-user facility involved with preparation and analysis of many different biological and materials sciences projects. Vapor stabilization and coating is an important part of our specimen preparation methodology for difficult biological and materials, especially polymer, samples. The procedure for all our vapor preparation techniques is done in a simple, economical apparatus set up in a properly functioning fume hood with a flow rate of at least 100 ft/min (Fig. 1). The apparatus is made from a glass petri dish or a glass petri dish for the bottom and an appropriate size beaker for the top. Specimens, mounted on stubs, are placed inside the chamber and the fixative (osmium tetroxide, ruthenium tetroxide or acrolein) is placed in a small container (plastic bottle cap) near the specimens.

Preparation of Helminths For Scanning Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 438-439
Author(s):  
Robert W. Weise
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Room Temperature ◽  
Parasitic Nematodes ◽  
Live Animal ◽  
Transmission Electron ◽  
Critical Point Drying ◽  
Scanning Electron

The role that scanning electron microscopy (SEM) is playing in descriptive helminthology is becoming more apparent in the literature. However, the majority of papers on the SEM of helminths have used conventional or modified light microscope techniques of fixation and dehydration, and not established SEM techniques in which freeze- and critical point-drying are routinely used. The present investigation was undertaken to examine the applicability of modified scanning and transmission electron microscope techniques for the preparation of certain helminths for SEM.Method I.– Live animal-parasitic nematodes were fixed in 6% phosphate buffered glutaraldehyde for 24 hr at room temperature.

A technique for preparing Beauveria spp. for scanning electron microscopy

1980 ◽  
Vol 58 (15) ◽  
pp. 1700-1703 ◽  
Author(s):  
E. C. Quattlebaum ◽  
G. R. Carner
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Freeze Drying ◽  
Osmium Tetroxide ◽  
Air Drying ◽  
Preparation Methods ◽  
Critical Point Drying ◽  
Scanning Electron

Vapor fixation for 96 h with 1% osmium tetroxide (OsO4) and 3–4 days air drying produced distortion-free specimens of Beauveria spp. for examination with the scanning electron microscope. A combination of 4 h OsO4 vapor fixation and freeze-drying also reduced disruption satisfactorily but specimens were not as well preserved as with the first method. Preparation methods that were ineffective in preventing collapse of hydrophilic structures were Cling Free® sprayed on specimens prior to examination, freeze-drying, critical-point drying (of unfixed material), and vapor fixation with glutaraldehyde.

scholarly journals Methods of preparing flower stem samples for scanning electron microscopy

2015 ◽  
Vol 21 (1) ◽  
pp. 17 ◽  
Author(s):  
Poliana Cristina Spricigo ◽  
Jéssica Prada Trento ◽  
Joana Dias Bresolin ◽  
Viviane Faria Soares ◽  
Viviane Faria Soares ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Stainless Steel ◽  
Critical Point ◽  
Freeze Drying ◽  
Osmium Tetroxide ◽  
Cut Flowers ◽  
Critical Point Drying ◽  
Flower Stem ◽  
Scanning Electron

Brazil has great capacity for expansion in the floriculture sector. Studies on postharvest cut flowers contribute to development of the sector, helping to maintain the quality of domestic production. Scanning electron microscopy (SEM) is a powerful tool that allows viewing of flower structures and also microorganisms. The aim of this study was to evaluate methods of preparing flower stem samples for viewing in SEM as a support for studies on postharvest cut flowers. Ways of cutting, fixing, and drying samples were tested. Cutting with a stainless steel blade and through freeze-fracture were tested; fixation was carried out without the use of osmium tetroxide (OsO4); and drying of the samples was performed through freeze-drying and through critical point dryingwithCO2. Cutting with a stainless steel blade proved to be a satisfactory method for stem samples, with low cost and simple application compared to freeze-fracturing. Good fixation and high image contrast were obtained without the use of osmium tetroxide, thus avoiding the use of this toxic compound. Freeze-drying allowed the structure and morphological composition to be viewed, while critical point drying withCO2 preserved the microorganisms present in the samples.

Scanning Electron Microscopy of Blaberus Giganteus (L.) Maxillary Palp Sensilla

1976 ◽  
Vol 34 ◽  
pp. 194-195
Author(s):  
P. Dailey
Keyword(s):  
Osmium Tetroxide ◽  
Ventral Surface ◽  
Maxillary Palp ◽  
Sodium Cacodylate Buffer ◽  
Sodium Cacodylate ◽  
Sensilla Basiconica ◽  
Critical Point Drying ◽  
Cacodylate Buffer ◽  
Maxillary Palps ◽  
Scanning Electron

The maxillary palps of B. giganteus are located on the maxillae which lie between the labrum and labium. They are modified appendages used in feeding. Each appendage bears 5 segments, the most distal of which contains the palp organ or sensory pad (Fig. 1). The maxillary palps were fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.1) containing .15 M sucrose and post-fixed in 1% osmium tetroxide in buffer. This was followed by dehydration and critical point drying. Cryofractography was performed on some of the palp organs prior to drying (1).The sensory pad is composed of an exocuticular membrane upon which lie numerous sensilla basiconica in an elliptical pattern on the ventral surface. The membrane enables the sensory pad to expand and, in living specimens it appears dome-like due to the pressure exerted on it by the hemolymph.

Hexamethyldisilazane: A modified procedure

1989 ◽  
Vol 47 ◽  
pp. 1006-1007
Author(s):  
Verdon Laliberté ◽  
L.L. Hayes ◽  
B.M. Stanulis-Praeger
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Uranyl Acetate ◽  
Cell Contact ◽  
Critical Importance ◽  
Drying Procedures ◽  
Critical Point Drying ◽  
Scanning Electron ◽  
Better Than

Scanning electron microscopy is a powerful tool in the study of cell contact in vitro, and has shown, specifically, that fibroblasts under conditions of growth restriction increase cell contact by filopodia. Accurate quantitation of surface features like filopodia, however, depends particularly upon artifact-free drying procedures. Critical point drying (CPD) of fibroblast monolayers all too often causes cell flattening, shrinkage, loss of surface detail and cracking (Fig. 1). Newer methods utilizing hexamethyldisilazane (HMDS) and uranyl acetate (UA) preserve fibroblast contour and surface architecture better than CPD, but solvent choice in the preparation of UA is of critical importance. UA in 70% ethanol results in the formation of precipitate (Fig. 2) whether the specimens are in the cold (4°C) or at room temperature, overnight or for periods less than 1 hour regardless of filtering. It occurs when incubation is carried out in the dark and when it is followed by copious rinses in 70% ethanol.

The Critical Point Drying Method Applied to Scanning Electron Microscopy of L-929 Cells

1970 ◽  
Vol 28 ◽  
pp. 278-279
Author(s):  
Charles TurnbiLL ◽  
Delbert E. Philpott
Keyword(s):  
Electron Microscopy ◽  
Carbon Dioxide ◽  
Surface Tension ◽  
Critical Point ◽  
Freeze Drying ◽  
Attractive Alternative ◽  
Crystal Formation ◽  
Critical Point Drying ◽  
Time Required ◽  
Scanning Electron

The advent of the scanning electron microscope (SCEM) has renewed interest in preparing specimens by avoiding the forces of surface tension. The present method of freeze drying by Boyde and Barger (1969) and Small and Marszalek (1969) does prevent surface tension but ice crystal formation and time required for pumping out the specimen to dryness has discouraged us. We believe an attractive alternative to freeze drying is the critical point method originated by Anderson (1951; for electron microscopy. He avoided surface tension effects during drying by first exchanging the specimen water with alcohol, amy L acetate and then with carbon dioxide. He then selected a specific temperature (36.5°C) and pressure (72 Atm.) at which carbon dioxide would pass from the liquid to the gaseous phase without the effect of surface tension This combination of temperature and, pressure is known as the "critical point" of the Liquid.

Sign in / Sign up

Export Citation Format

Share Document