Application of ruthenium red/osmium tetroxide to bacteria, plant and animal tissue

1986 ◽  
Vol 44 ◽  
pp. 54-55
Author(s):  
R. L. Grayson ◽  
N. A. Rechcigl
Keyword(s):  
Electron Microscopy ◽  
Electron Density ◽  
Osmium Tetroxide ◽  
Biological Materials ◽  
Ruthenium Red ◽  
Animal Tissue

Ruthenium red (RR), an inorganic dye was found to be useful in electron microscopy where it can combine with osmium tetroxide (OsO4) to form a complex with attraction toward anionic substances. Although Martinez-Palomo et al. (1969) were one of the first investigators to use RR together with OsO4, our computor search has shown few applications of this combination in the intervening years. The purpose of this paper is to report the results of our investigations utilizing the RR/OsO4 combination to add electron density to various biological materials. The possible mechanisms by which this may come about has been well reviewed by previous investigators (1,3a,3b,4).

Ruthenium red-osmium bridging with TCH: new technique to stain biological specimens for light and TEM and to coat them for SEM

1988 ◽  
Vol 46 ◽  
pp. 20-21
Author(s):  
B. Giammara ◽  
J. Hanker
Keyword(s):  
Electron Microscopy ◽  
Microscopic Study ◽  
Osmium Tetroxide ◽  
Light And Electron Microscopy ◽  
Ruthenium Red ◽  
Surface Coat ◽  
Repeated Application ◽  
Electron Microscopic ◽  
Sputter Coating ◽  
Selection Of

The demonstration and coating of glycomacromolecular surface coat components of biological specimens (1) with ruthenium red (RR, Fig. 1) is improved by treating with osmium tetroxide (2) probably due to its attachment to glycolipids. Since 1966 studies have shown how bridging osmium to osmium with thiocarbohydrazide (TCH, Fig. 2) can result in improvement in contrast of biological specimens (3,4) for light and electron microscopy. Since 1973 this bridging procedure has widely been applied (5,6) to obtain a conductive coating for biological specimens for SEM eliminating the need for sputter coating. Improvement in conductance of uncoated specimens for EM has also been obtained (6) by bridging osmium with p-phenylenediamine hydrochloride (PPD). The improvement in conductance of RR coated biological specimens for SEM by OsO4 treatment without TCH (2) required repeated application of the reagent solutions and did not result in sufficient staining of the glycomacromolecules and glycolipids for the light microscopic selection of areas for electron microscopic study.

High Voltage Electron Microscopy for Thick Sections of Biological Materials Combined with Molecular Tracers

1972 ◽  
Vol 30 ◽  
pp. 480-481
Author(s):  
Eichi Yamada ◽  
Harunori Ishikawa
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Biological Materials ◽  
Ruthenium Red ◽  
Mouse Kidney ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Structural Details ◽  
Molecular Tracers

One of the advantages of high voltage electron microscopy (HVEM) is the great penetration of electron beam, which permits the examination of thicker sections of resin-embedded biological materials. However, when stained thick sections are examined under the HVEM, all structural details within the whole thickness are brought into focus as superimposed image. Hence, certain structures are not easily observable on such an image, even on the stereo view obtained by specimen tilting technique. To overcome this difficulty, molecular tracers are introduced to the HVEM in an attempt to visualize certain structures selectively.Mouse kidney and cardiac muscle were examined using ruthenium red and peroxidase as molecular tracers. Thick Epon sections ranging from 0.5 to 3 μ were observed in a JOEL JEM-1000 or a Hitachi HU-1000 HVEM, with an accelerating voltage of 1000 KV. Pictures were taken routinely as stereo pairs using a tilting stage device with ±10° or ±8°.

Electron microscopy of an endoparasitic fungus, Gonimochaete pyriforme, infecting nematodes

1985 ◽  
Vol 63 (12) ◽  
pp. 2326-2331 ◽  
Author(s):  
Masatoshi Saikawa ◽  
Takashi Anazawa
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Electron Density ◽  
Germ Tube ◽  
Adhesive Layer ◽  
Ruthenium Red ◽  
Ruthenium Red Staining ◽  
Dense Vesicles

Gonimochaete pyriforme Barron was studied using the electron microscope. Protoplasm in the pyriform-shaped aplanospore is filled with electron-dense vesicles (0.1–0.3 μm) except at the base where it is vacuolated. The globose knob at the apical end of the spore is covered with a very thin adhesive layer (ca. 0.1 μm) whose electron density is slightly enhanced by ruthenium red staining but which does not show a fibrillose appearance. After attachment to the nematode's cuticle, a narrow germ tube (0.15 μm) arises from the globose knob and penetrates through the adhesive layer and the host's cuticle into the nematode body. The adhesive knob of the aplanospore in G. pyriforme is very similar in ultrastructure to the encysted zoospore of Myzocytium humicola Barron and Percy in the Lagenidiales after the cyst has germinated.

Spontaneous Cataract in Nude Athymic Mice

1977 ◽  
Vol 35 ◽  
pp. 512-513
Author(s):  
Ronald H. Bradley ◽  
R. S. Berk ◽  
L. D. Hazlett
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Nude Mouse ◽  
Cellular Immunity ◽  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Athymic Mice ◽  
Transmission Microscopy ◽  
Mouse Lens ◽  
Scanning Electron

The nude mouse is a hairless mutant (homozygous for the mutation nude, nu/nu), which is born lacking a thymus and possesses a severe defect in cellular immunity. Spontaneous unilateral cataractous lesions were noted (during ocular examination using a stereomicroscope at 40X) in 14 of a series of 60 animals (20%). This transmission and scanning microscopic study characterizes the morphology of this cataract and contrasts these data with normal nude mouse lens.All animals were sacrificed by an ether overdose. Eyes were enucleated and immersed in a mixed fixative (1% osmium tetroxide and 6% glutaraldehyde in Sorenson's phosphate buffer pH 7.4 at 0-4°C) for 3 hours, dehydrated in graded ethanols and embedded in Epon-Araldite for transmission microscopy. Specimens for scanning electron microscopy were fixed similarly, dehydrated in graded ethanols, then to graded changes of Freon 113 and ethanol to 100% Freon 113 and critically point dried in a Bomar critical point dryer using Freon 13 as the transition fluid.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

New Design for a Differentially Pumped Hydration Chamber for a Siemens IA

1971 ◽  
Vol 29 ◽  
pp. 44-45
Author(s):  
R. C. Moretz ◽  
G. G. Hausner ◽  
D. F. Parsons
Keyword(s):  
Electron Microscopy ◽  
Water Vapor ◽  
Electron Diffraction ◽  
Water Vapor Pressure ◽  
Biological Materials ◽  
Thin Membrane ◽  
Reliable System ◽  
System A ◽  
Water Films ◽  
Aperture Opening

Electron microscopy and diffraction of biological materials in the hydrated state requires the construction of a chamber in which the water vapor pressure can be maintained at saturation for a given specimen temperature, while minimally affecting the normal vacuum of the remainder of the microscope column. Initial studies with chambers closed by thin membrane windows showed that at the film thicknesses required for electron diffraction at 100 KV the window failure rate was too high to give a reliable system. A single stage, differentially pumped specimen hydration chamber was constructed, consisting of two apertures (70-100μ), which eliminated the necessity of thin membrane windows. This system was used to obtain electron diffraction and electron microscopy of water droplets and thin water films. However, a period of dehydration occurred during initial pumping of the microscope column. Although rehydration occurred within five minutes, biological materials were irreversibly damaged. Another limitation of this system was that the specimen grid was clamped between the apertures, thus limiting the yield of view to the aperture opening.

A Resorcinol-Formaldehyde Resin as an Embedding Material for Electron Microscopy of Membranes

1969 ◽  
Vol 27 ◽  
pp. 328-329 ◽  
Author(s):  
J. G. Robertson ◽  
D. F. Parsons
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Neutral Lipids ◽  
Lipid Extraction ◽  
Water Soluble ◽  
Formaldehyde Resin ◽  
Electron Microscope Autoradiography ◽  
Resorcinol Formaldehyde ◽  
Major Disadvantage ◽  
Cell Organization

The extraction of lipids from tissues during fixation and embedding for electron microscopy is widely recognized as a source of possible artifact, especially at the membrane level of cell organization. Lipid extraction is also a major disadvantage in electron microscope autoradiography of radioactive lipids, as in studies of the uptake of radioactive fatty acids by intestinal slices. Retention of lipids by fixation with osmium tetroxide is generally limited to glycolipids, phospholipids and highly unsaturated neutral lipids. Saturated neutral lipids and sterols tend to be easily extracted by organic dehydrating reagents prior to embedding. Retention of the more saturated lipids in embedded tissue might be achieved by developing new cross-linking reagents, by the use of highly water soluble embedding materials or by working at very low temperatures.

Selective Staining of Acid Mucopolysaccharides By Ruthenium Red

1968 ◽  
Vol 26 ◽  
pp. 38-39
Author(s):  
J. H. Luft
Keyword(s):  
Electron Microscope ◽  
Light Microscopy ◽  
Light Microscope ◽  
Osmium Tetroxide ◽  
Single Cells ◽  
Ruthenium Red ◽  
Collagen Fibers ◽  
Selective Staining ◽  
Pectic Substances ◽  
Solid Tissues

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).

Effect of Acetone and Kerosene on Skin Ultrastructure

1972 ◽  
Vol 30 ◽  
pp. 92-93
Author(s):  
A. P. Lupulescu ◽  
H. Pinkus ◽  
D. J. Birmingham
Keyword(s):  
Electron Microscopy ◽  
Human Skin ◽  
Osmium Tetroxide ◽  
Human Epidermis ◽  
Ultrastructural Changes ◽  
Chemical Agents ◽  
Skin Ultrastructure ◽  
Volar Surface

Our laboratory is engaged in the study of the effect of different chemical agents on human skin, using electron microscopy. Previous investigations revealed that topical use of a strong alkali (NaOH 1N) or acid (HCl 1N), induces ultrastructural changes in the upper layers of human epidermis. In the current experiments, acetone and kerosene, which are primarily lipid solvents, were topically used on the volar surface of the forearm of Caucasian and Negro volunteers. Skin specimens were bioptically removed after 90 min. exposure and 72. hours later, fixed in 3% buffered glutaraldehyde, postfixed in 1% phosphate osmium tetroxide, then flat embedded in Epon.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

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