The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

Lipid fixation during preparation of chloroplasts for electron microscopy

Reaction of osmium tetroxide with isolated spinach chloroplasts fixed completely the glycolipids, phosphatidyl glycerol, and phosphatidyl choline. Under the same reaction conditions only 30% of the chlorophyll was fixed. Reaction of potassium permanganate with isolated spinach chloroplasts fixed more than 90% of the glycolipids, phosphatidyl glycerol, and phosphatidyl choline, provided the reaction period was long enough. Potassium permanganate also fixed the chlorophyll. Reaction of osmium tetroxide and potassium permanganate with isolated (14)C-lipids from Chlorella pyrenoidosa fixed 59% and 66% of the radioactivity, respectively. The lipids that were not fixed included sterols and pigments. Electron micrographs show that chloroplasts extracted with chloroform-methanol after fixation in osmium tetroxide or potassium permanganate differ from those dehydrated with acetone mainly in that in the former, osmiophilic globules have been removed and there seems to be some fusion of the boundary membranes and grana membranes. These effects may be due to the extraction of unfixed, neutral lipids such as sterols and quinones.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : I. Fixation and Electron Staining Reactions

Chick embryo mitochondria, studied with the electron microscope, show crista-free areas of low electron opacity. These areas are observable after fixation with osmium tetroxide, calcium permanganate, potassium permanganate, formaldehyde, acrolein, acrolein followed by osmium tetroxide, uranyl acetate followed by calcium permanganate, and acetic acid-alcohol. Staining of sections with lead hydroxide or uranyl acetate, or with both, resulted in an increased density of a fibrous material within these areas. The appearance of the fibrous structures varied with the fixative employed; after fixation with osmium tetroxide the material was clumped and bar-like (up to 400 A in diameter), whereas after treatment of osmium tetroxide-fixed tissues with uranyl acetate before dehydration the fibrous structures could be visualized as 15 to 30 A fibrils. Treatment with ethylenediaminetetraacetate (EDTA) in place of uranyl acetate coarsened the mitochondrial fibrils. After fixation with calcium permanganate or potassium permanganate, or a double fixation by uranyl acetate followed by calcium permanganate, the fibers appeared to have a pattern and ultrastructure similar to that observed after the osmium tetroxide-uranyl acetate technique, except that some of them had a slightly greater diameter (up to 50 A). Other fixatives did not preserve the fibers so well. The fibers appeared strongly clumped by formaldehyde fixation, and were difficult to identify after fixation with acrolein or acetic acid-alcohol. The staining of nucleic acid-containing structures by uranyl acetate and lead hydroxide was improved by treatment of osmium tetroxide-fixed sections with hydrogen peroxide, and the mitochondrial fibers also had an increased density in the electron beam after this procedure. The staining characteristics suggest the fibrous material of chick embryo mitochondria to be a nucleic acid-containing structure, and its variable appearance after different fixations parallels that previously reported, or described in this paper, for the nucleoplasm of bacteria and blue-green algae. The results, in addition to those described in the accompanying communication, indicate that these mitochondria contain DNA.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Does lateral specialization apply to the frog's brain? An electron microscope observation.

The ultrastructure of polymorphic crystal-like inclusions occurring in the cytoplasm of neurons of only the medial portion of the left habenula has been studied in the adult frog Rana esculenta and in the tadpole with the use of three types of fixatives: osmium tetroxide, a mixture of aldehydes and potassium permanganate.     

Fixation of proteins by osmium tetroxide potassium dichromate and potassium permanganate

Summary The crosslinking abilities of osmium tetroxide, potassium dichromate and potassium permanganate towards bovine serum albumin and bovine -globulin were investigated by chromatography with Sephadex G-200. Osmium tetroxide had a moderate crosslinking ability towards these proteins, the others had little or none. Chromatography with Sephadex G-50 permitted the oxidative cleavage of the proteins by these oxidative fixation agents to be studied. Potassium permanganate caused much fragmentation of the proteins and destruction of the tyrosine and tryptophan residues. Osmium tetroxide and potassium dichromate caused only a small amount of protein cleavage. These results were corroborated by polyacrylamide gel electrophoresis and viscosimetric studies. The significance of the results for tissue fixation is discussed.     

The Ultrastructure of Chlorobium tepidum Chlorosomes Revealed by Electron Microscopy

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.     

Triple fixation of Bacillus subtilis dormant spores.

A triple-fixation method with a sequential application of 5% glutaraldehyde, 1% osmium tetroxide, and 2% potassium permanganate gave superior preservation of the ultrastructure of Bacillus subtilis dormant spores with a thick spore coat.