Freeze substitution fixation of fungal reproductive structures and spores

1989 ◽  
Vol 47 ◽  
pp. 990-991
Author(s):  
C. W. Mims ◽  
E. A. Richardson
Keyword(s):  
Plant Pathogens ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Dialysis Membrane ◽  
Razor Blade ◽  
Thin Sections ◽  
Reproductive Structures ◽  
Dry Down ◽  
Diamond Knife

The advantages of freeze substitution fixation over conventional chemical fixation for preservation of ultrastructural details in fungi have been discussed by various authors. As most ascomycetes, basidiomycetes and deuteromycetes do not fix well using conventional chemical fixation protocols, freeze substitution has attracted the attention of many individuals interested in fungal ultrastructure. Thus far most workers using this technique on fungi have concentrated on thin walled somatic hyphae. However, in our laboratory we have experimented with the use of freeze substitution on a variety of fungal reproductive structures and spores with promising results.Here we present data on freeze substituted samples of sporangia of the zygomycete Umbellopsis vinacea, basidia of Exobasidium camelliae var. gracilis, developing teliospores of the smut Sporisorium sorghi, germinating teliospores of the rust Gymnosporangium clavipes, germinating conidia of the deuteromycete Cercosporidium personatum, and developing ascospores of Ascodesmis nigricans.Spores of G. clavipes and C. personatum were deposited on moist pieces of sterile dialysis membrane where they hydrated and germinated. Asci of A. nigricans developed on pieces of dialysis membrane lying on nutrient agar plates. U. vinacea was cultured on small pieces of agar-coated wire. In the plant pathogens E. camelliae var. gracilis and S. sorghi, a razor blade was used to remove smal1 pieces of infected host issue. All samples were plunged directly into liquid propane and processed for study according to Hoch.l Samples on dialysis membrane were flat embedded. Serial thin sections were cut using a diamond knife, collected on slot grids, and allowed to dry down onto Formvar coated aluminum racks. Sections were post stained with uranyl acetate and lead citrate.

Ultrastructure of the Infection of Poinsettia by Oidium SP. using High Pressure Freezing and Freeze Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 690-691
Author(s):  
G. J. Celio ◽  
E. A. Richardson ◽  
C. W. Mims
Keyword(s):  
High Pressure ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Euphorbia Pulcherrima ◽  
High Pressure Freezing ◽  
Thin Sections ◽  
Transmission Electron ◽  
Dextran Solution ◽  
Leaf Disks ◽  
Diamond Knife

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.

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.

Secretory PITS in Backswimmers (Heteroptera: Notonectidae).

1996 ◽  
Vol 54 ◽  
pp. 310-311
Author(s):  
R. J. Williams ◽  
N. R. Dollahon ◽  
E. Larsen ◽  
S. O’Neill ◽  
R. Chapman
Keyword(s):  
Dorsal Surface ◽  
Uranyl Acetate ◽  
Absolute Ethanol ◽  
Razor Blade ◽  
Glass Coverslip ◽  
Aluminum Specimen ◽  
Cacodylate Buffer ◽  
Lateral Margins ◽  
Ethanol Series ◽  
Diamond Knife

In studies of several families of aquatic heteropterans we have found exoskeletal pits not described in the literature. These structures are associated with the lateral margins of the pronotum and/or the dorsal surface, posterior to the scutellum in notonectids, nepids, and corixids. The function of these pits is unknown, but we presumed that they might be either sensory or secretory in nature. We undertook this study of the microscopy of pits in the notonectid, Buenoa margaritacea to learn if either of these functions is consistent with the fine structure.For TEM, adult insects were submerged in 1.0% paraformaldeyde and 2.0% glutaraldehyde in 0.02M sodium cacodylate buffer with 0.01% calcium chloride at a pH of 7.2, then dissected with a razor blade cleaned with acetone. Tissues were fixed overnight at 4°C in paraformaldehyde and glutaraldehyde, then fixed in 1% osmium tetroxide in cacodylate buffer, dehydrated in a graded series of acetone, embedded in Spurr resin and polymerized at 70°C for 21 hours. Blocks were thin sectioned with a diamond knife, and sections were stained with uranyl acetate and lead citrate. Whole specimens for SEM were similarly fixed, dehydrated in an ethanol series and critical point dried. SEM micrographs of internal pit anatomy were produced by adhering 500nm sections to a glass coverslip, then removing the embedding resin by incubation for 5 minutes in a saturated solution of potassium hydroxide in absolute ethanol, followed by three 20 minute rinses in absolute ethanol and air drying. Cover slips were attached to an aluminum specimen stub and sputter coated for 60 seconds with gold/palladium as were whole insects .

Freeze substitution studies on cultured human fibroblasts

1989 ◽  
Vol 47 ◽  
pp. 1008-1009
Author(s):  
Julian P. Heath ◽  
Donna Turner
Keyword(s):  
Osmium Tetroxide ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Lung Fibroblasts ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Cultured Human Fibroblasts

We are using rapid freezing and freeze substitution to study the three dimensional organisation of membrane systems and cytoskeletal filaments in motile fibroblasts. This study has two objectives: first, to provide material for structural and immunocytochemical analysis of membrane-cytoskeletal interactions in cells that have been preserved with minimum artefact (1,2,3) and second, to refine and develop existing rapid freezing and freeze substitution techniques to allow for the study of single cells that have been experimentally manipulated and observed by digital video microscopy before fixation.The cells used were human lung fibroblasts (IMR90) either growing on Lux Thermanox coverslips or as pelleted suspensions. The cells were slam frozen on a Med-Vac Cryo Press against a liquid nitrogen cooled copper block. Coverslips were trimmed to 2 x 2 mm in size, excess fluid was drained off, and they were placed on top of a 1mm thick gelatin cushion on an aluminium planchette. For cell suspensions, 3 ul was placed on top of the gelatin cushion. Frozen samples were placed in acetone containing 1% osmium tetroxide for 72 hours at 192 K, wanned to 253 K for 4 hours, and then brought to room temperature. The samples were rinsed in acetone and embedded in Spurr’s resin. Thin sections were cut on a RMC6000 ultramicrotome, stained in uranyl acetate and Reynolds' lead citrate and photographed on a Philips EM410 electron microscope at 60 keV.

Ultrastructural Study of Trophozoites and Cysts of Acanthamoeba-Hartmannella and Naegleria Species

1974 ◽  
Vol 32 ◽  
pp. 192-193
Author(s):  
A. Julio Martinez ◽  
Richard J. Duma ◽  
Doris G. Fultz ◽  
Ruth B. Finley
Keyword(s):  
Present Report ◽  
Uranyl Acetate ◽  
Osmic Acid ◽  
Acetate Solution ◽  
Lead Citrate ◽  
Thin Sections ◽  
Cacodylate Buffer ◽  
In Situ Fixation ◽  
Diamond Knife

Ultrastructural components of trophozoites and cysts of Acanthamoeba-Hartmannella (A-H) and Naegleria amebas have been investigated. The present report describes a technique to study the morphological features of such protozoa.The A-H strains selected were cultured for 7 days in axenic amebic media, and the Naegleria strains were cultured for 7 days in axenic media with blood. In situ fixation was accomplished by adding an equal volume of 4% Osmic Acid in Cacodylate buffer at 4¶C for one hour. Following frequent agitation, the tubes were then spun at 2500 RPM for 10 minutes to obtain a pellet. The pellets were dehydrated in graded series of alcohols (from 50% to absolute) and propylene oxide and then embedded in EPON. One micron sections were stained with Paragon (PS 1301) for thirty seconds. Thin sections were cut with a diamond knife, double stained with 5% alcoholic uranyl acetate solution and lead citrate and observed in an Hitachi-HS-8F2 electron microscope.

Three Dimensional Immuno-Localization of Green Fluorescent Protein Chimeras Using Rapid Freezing and Freeze-Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 298-299
Author(s):  
Mary Morphew ◽  
David Mastronarde ◽  
Eileen O'Toole ◽  
Mark Ladinsky ◽  
Brad Marsh ◽  
...  
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Green Fluorescent ◽  
Structural Preservation

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

A New Smaller Sized Virus-Like Particle in Drosophila Melanogaster

2001 ◽  
Vol 7 (S2) ◽  
pp. 742-743
Author(s):  
Jeffrey G. Ault ◽  
Ellen Shimakawa
Keyword(s):  
Drosophila Melanogaster ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
High Pressure Freezing ◽  
Chromosome Behavior ◽  
Thin Sections ◽  
Behavior Study ◽  
Virus Like Particle

During a chromosome behavior study involving high-pressure freezing (HPF)/freeze substitution (FS) of Drosophila melanogaster testes, we discovered quasi-crystalline inclusions in the nuclei of adjacent gut epithelial cells (Fig. 1). The HPF and FS protocols were standard. The viscera of adult flies were packed in yeast paste for HPF. The tissue was fixed by FS with 1% osmium tetroxide in acetone for 72 hours at -90° C then 48 hours at -60° C. Afterwards, it was washed several times at room temperature in 100% acetone and embedded in Epon/Araldite. Thin sections were cut and stained with uranyl acetate and lead. As expected with HPF/FS, the material was well-preserved with straight microtubules, smooth membranes, dense mitochondria, and abundant ribosomes both on the rough endoplasmic reticulum and in the full cytoplasm (Fig. 1).The inclusions consisted of virus-like particles packed loosely together in orderly arrays. Particles were usually hexagonally packed with spaces disrupting the periodicity (Figs. 2 and 3).

scholarly journals How Long Should A Diamond Knife Stay Sharp?

1998 ◽  
Vol 6 (2) ◽  
pp. 6-7
Author(s):  
Charles A. Garber ◽  
Bernard Mesa
Keyword(s):  
Cutting Speed ◽  
Metal Particles ◽  
List Type ◽  
Distilled Water ◽  
Knife Edge ◽  
Razor Blade ◽  
Common Causes ◽  
Dry Down ◽  
Block Face ◽  
Diamond Knife

The answer to this question is about as elusive as predicting which way the Dow Jones average will close tomorrow! But seriously, there are the “ten commandments” for a diamond knife to enjoy a long life, the most important ones being as fallows:1)Because of the extreme sharpness of a diamond knife edge, it should not be touched with any solid object, even for cleaning, This is controversial since some manufacturers actually recommend that the edges be cleaned with sticks of varying types. We ourselves believe such treatment accelerates the wear of a diamond knife.2)Don't let sections or the remains of sections or other debris dry down onto the knife edge. Keep the knife edge wet until it is ready for cleaning before being put to bed for the night.3)Use a diamond knife cleaner sold by several firms (including ours) specifically for this purpose, Some typical laboratory ultrasonic cleaners can have enough power to be damaging to a knife.4)Wash the knife edge one last time with distilled water, and then dry with some kind of "blast", such as from a clean duster,5)Avoid conditions of chatter at all times. Reduce chatter by varying the clearance angle or slowing the cutting speed. Other common causes of chatter are insufficient tightening of the boat in the microtome, an insufficiently tightened block, or an incompletely cured block.6)Final block trimming with a razor blade can ieave metal particles from the blade, which are of course damaging to the knife edge, This can be minimized by using a fresh razor blade each time, Then wash the end of the freshly cut block face with distilled water, followed by drying with a duster blast is the final step before the first cut with the knife, This is a final chance to wash away metal particles that could damage the knife edge.

Cellular and structural diversity of Campylobacter pylori

1989 ◽  
Vol 47 ◽  
pp. 1058-1059
Author(s):  
A. R. Crooker ◽  
M. C. Myers ◽  
W. G. Kraft
Keyword(s):  
Structural Diversity ◽  
Uranyl Acetate ◽  
Campylobacter Pylori ◽  
Chemical Fixation ◽  
Liquid Media ◽  
En Bloc ◽  
Thin Sections ◽  
Strain Nctc ◽  
Upper Gastrointestinal

Campylobacter pylori is a recently recognized microaerophilic, gram-negative bacterium found in the upper gastrointestinal tract of humans. The role of this organism in the pathogenesis of gastritis and ulceration is currently under intense investigation. The rapidly growing literature on C. pylori describes the bacterium as spiral shaped; however, oval and coccal forms are occasionally noted. We report here on the cellular and structural diversity of this organism, including a new, pleomorphic form.Two Peruvian clinical isolates of C. pylori and the type strain NCTC 11638 were grown for up to 120 h in liquid media. Thin sections and negatively-stained whole mounts were prepared for electron microscopy. Chemical fixation was performed in suspension with 2% cacodylate-buffered glutaraldehyde followed by 2% OsO4 In the same buffer, then uranyl acetate en bloc. Specimens were dehydrated in acetone and embedded in Spurr's resin. Ultrathin silver sections were stained with uranyl acetate and Reynolds’ lead citrate. Briefly prefixed bacteria were negatively-stained with 1% PTA on SiO/Forravar-coated 300 mesh grids.

An ultrastructural study of ascospore delimitation and maturation in Ascodesmis nigricana using freeze substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 630-631
Author(s):  
C. W. Mints ◽  
E. A. Richardson ◽  
J. Kimbrough
Keyword(s):  
Ultrastructural Study ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Dialysis Membrane ◽  
Liquid Propane

Fungi belonging to the class Ascomycetes are characterized by the production of sexually derived spores termed ascospores inside a microscopic, sac-like structure termed an ascus. In the Euascomycetidae, typically uninucleate spore initials are delimited within the ascus as the result of the invagination of membranes that arise from the so-called "ascus vesicle", a discontinuous cylinder of two closely spaced unit membranes that develops around the extreme periphery of the ascus. To date, there are conflicting data regarding the origin of the ascus vesicle. We therefore decided to address the problem using freeze substitution fixation. The advantages of this procedure over conventional chemical fixation protocols for preservation of ultrastructural details in fungi have been well-documented. In this study small pieces of dialysis membrane bearing developing ascocarps of Ascodesmis nigricans were plunged into liquid propane and processed for TEM according to the procedures of Hoch and Mints et al.

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