Experiments on Fixation for Electron Microscopy

1963 ◽  
Vol s3-104 (65) ◽  
pp. 123-127
Author(s):  
S. K. MALHOTRA
Keyword(s):  
Electron Microscopy ◽  
Acetic Acid ◽  
Osmium Tetroxide ◽  
Proximal Convoluted Tubule ◽  
Mitochondrial Matrix ◽  
Zymogen Granules ◽  
Exocrine Cells ◽  
Acid Ph ◽  
Pancreatic Exocrine ◽  
Tubule Cells

The effect of fixation with acidified solutions of osmium tetroxide (pH 1.5 to 3.5 has been studied on the first (proximal) convoluted tubule cells of the kidney and the pancreatic exocrine cells of the mouse, by electron microscopy. Partially prepolymerized methacrylate was used for embedding. The various membranous structures and the ribosomes retain their individuality even after prolonged fixation in solutions containing 5% acetic acid (pH 1.5). However, the mitochondrial matrix and the ground cytoplasm are not preserved; the zymogen granules are also partially washed out.

scholarly journals The fine structure produced in cells by primary fixatives 2. Potassium dichromate

1965 ◽  
Vol s3-106 (73) ◽  
pp. 15-21
Author(s):  
JOHN R. BAKER
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Fine Structure ◽  
Golgi Apparatus ◽  
Nuclear Membrane ◽  
Osmium Tetroxide ◽  
Potassium Dichromate ◽  
Potassium Dichromate Solution ◽  
Zymogen Granules ◽  
Mouse Pancreas

The exocrine cells of the mouse pancreas were fixed in potassium dichromate solution, embedded in araldite or other suitable medium, and examined by electron microscopy. Almost every part of these cells is seriously distorted or destroyed by this fixative. The ergastoplasm is generally unrecognizable, the mitochondria and zymogen granules are seldom visible, and no sign of the plasma membrane, microvilli, or Golgi apparatus is seen. The contents of the nucleus are profoundly rearranged. It is seen to contain a large, dark, irregularly shaped, finely granular object; the evidence suggests that this consists of coagulated histone. The sole constituent of the cell that is well fixed is the inner nuclear membrane. The destructive properties of potassium dichromate are much mitigated when it is mixed in suitable proportions with osmium tetroxide or formaldehyde.

scholarly journals STUDIES ON THE GOLGI APPARATUS BY ELECTRON MICROSCOPY WITH PARTICULAR REFERENCE TO AOYAMA'S TECHNIQUE

1956 ◽  
Vol 2 (4) ◽  
pp. 395-402 ◽  
Author(s):  
Dennis Lacy ◽  
Cyril E. Challice
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Osmium Tetroxide ◽  
Silver Impregnation ◽  
Impregnation Method ◽  
Exocrine Cells ◽  
Golgi Membranes ◽  
Osmium Tetroxide Solution

1. Aoyama's silver impregnation method for the Golgi apparatus has been used on exocrine cells of the pancreas of the mouse and studied by electron microscopy in order to determine as precisely as possible where the silver is deposited. Similar cells have also been fixed in buffered osmium tetroxide solution and compared with cells treated by the silver technique. 2. Examination of the Aoyama preparations usually revealed a light deposition of silver in the cytoplasm (hyaloplasm or matrix) and a heavy deposition of silver around a series of closely apposed vacuoles. The heavy deposition of silver was regarded as revealing the chromophilic region of the Golgi apparatus while the vacuoles were identified as the chromophobic component. 3. Comparison of the silver preparations with those fixed in buffered osmium tetroxide solution showed that the silver was primarily deposited in the region of the Golgi membranes.

Experiments on Fixation for Electron Microscopy 2. Alkalized Osmium Tetroxide

1962 ◽  
Vol s3-103 (63) ◽  
pp. 287-296
Author(s):  
S. K. MALHOTRA
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Potassium Acetate ◽  
Exocrine Cells ◽  
Osmium Tetroxide Solution ◽  
Convoluted Tubules ◽  
Test Objects ◽  
Proximal Convoluted Tubules

The effect of fixation with osmium tetroxide solution, made alkaline by the addition of potassium acetate, was studied by electron microscopy. Exocrine cells of the pancreas and the cells of the first (‘proximal’) convoluted tubules of the kidney of the mouse were used as test-objects. Partially prepolymerized methacrylate was used for embedding. The preservation of the various cell inclusions was similar to what is generally produced after fixation with Palade's buffered osmium tetroxide.

The Fine Structure Produced in Cells by Primary Fixatives

1963 ◽  
Vol s3-104 (65) ◽  
pp. 101-106
Author(s):  
JOHN R. BAKER ◽  
BARBARA M. LUKE
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Golgi Apparatus ◽  
Mercuric Chloride ◽  
Chloride Solution ◽  
Zymogen Granules ◽  
Electron Dense Material ◽  
Mouse Pancreas ◽  
Exocrine Cells ◽  
Mercuric Chloride Solution

The exocrine cells of the mouse pancreas were fixed in mercuric chloride solution, embedded in plexigum, and examined by electron microscopy. The cytoplasm was found to be coagulated as a continuous substance containing innumerable subspherical cavities, mostly between 40 and 200 mµ in diameter and separate from one another. The zymogen granules were preserved, but no trace remained of mitochondria or Golgi apparatus. The nuclear sap was coagulated as a coarse network with thickenings at the nodes. Lumps of electron-dense material (? DNA) were present at the periphery of the nucleus and round the nucleolus. The proteins of the cell appear to have been fixed by mercuric chloride, but the membranous constituents, which rely for their form on a phospholipid component, are not clearly recognizable. The lipids have presumably been lost during dehydration and embedding.

Experiments on Fixation for Electron Microscopy

1963 ◽  
Vol s3-104 (66) ◽  
pp. 155-167
Author(s):  
S. K. MALHOTRA
Keyword(s):  
Electron Microscopy ◽  
Sodium Acetate ◽  
Butyl Methacrylate ◽  
Distilled Water ◽  
Exocrine Cells ◽  
Alkaline Side ◽  
Veronal Buffer ◽  
Tubule Cells ◽  
Test Objects

Michaelis's sodium acetate / sodium veronal buffer is generally used for holding the pH of fixing solutions for electron microscopy at about pH 7.3 to 7.5. The acetate, however, has no buffering action on the alkaline side of neutrality. Experiments were therefore made to study the effect on preservation of cellular constituents when sodium acetate is omitted from Palade's (or its variants) and Luft's fluids. Exocrine cells of the pancreas, convoluted tubule cells of the kidney, and the testes of the mouse were used as test-objects, n-butyl methacrylate (generally partially prepolymerized) and epikote 812 were used for embedding. The preservation visualized in the micrographs did not seem to suggest any marked differences in the quality of fixation from that produced by fixation in Palade's and Luft's fluids. However, there is some evidence that comparable micrographs could be produced by fixation in simple solutions of OsO4 in distilled water and subsequent embedding in suitable media.

Experiments on Fixation for Electron Microscopy

1962 ◽  
Vol s3-103 (61) ◽  
pp. 5-15
Author(s):  
S. K. MALHOTRA
Keyword(s):  
Electron Microscopy ◽  
Proximal Tubule ◽  
Electron Micrographs ◽  
Osmium Tetroxide ◽  
Simple Solution ◽  
Distilled Water ◽  
Exocrine Cells ◽  
Osmium Tetroxide Solution ◽  
Test Objects

The effect of fixation with a simple solution of osmium tetroxide in distilled water was studied by electron microscopy. Exocrine cells of the pancreas and cells of the proximal tubule of the kidney of the mouse were used as test-objects. Partially prepolymerized methacrylate was used for embedding. There did not appear to be any marked disorganization of the cell inclusions. The appearance of the inclusions in the electron micrographs was similar to what is generally seen after fixation with the buffered osmium tetroxide solution of Palade.

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).

Ectodesmata as Revealed By Freeze-Etch Preparations of Plant Epidermal Cell Walls

1973 ◽  
Vol 31 ◽  
pp. 468-469
Author(s):  
N.C. Lyon ◽  
W. C. Mueller
Keyword(s):  
Electron Microscopy ◽  
Acetic Acid ◽  
Nitric Acid ◽  
Oxalic Acid ◽  
Light Microscopy ◽  
Epidermal Cell ◽  
Cell Walls ◽  
Plant Viruses ◽  
Plant Leaves ◽  
Thin Sections

Schumacher and Halbsguth first demonstrated ectodesmata as pores or channels in the epidermal cell walls in haustoria of Cuscuta odorata L. by light microscopy in tissues fixed in a sublimate fixative (30% ethyl alcohol, 30 ml:glacial acetic acid, 10 ml: 65% nitric acid, 1 ml: 40% formaldehyde, 5 ml: oxalic acid, 2 g: mecuric chloride to saturation 2-3 g). Other workers have published electron micrographs of structures transversing the outer epidermal cell in thin sections of plant leaves that have been interpreted as ectodesmata. Such structures are evident following treatment with Hg++ or Ag+ salts and are only rarely observed by electron microscopy. If ectodesmata exist without such treatment, and are not artefacts, they would afford natural pathways of entry for applied foliar solutions and plant viruses.

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.

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