INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : II. Enzymatic and Other Hydrolytic Treatments

The effects of proteolytic enzymes, ribonuclease, and deoxyribonuclease upon a fibrous component of chick embryo mitochondria, which was previously shown to have many fixation and staining properties characteristic of the bacterial nucleoplasm, are reported. Pepsin digestion of formaldehyde-fixed tissues removed the membranes and matrices of mitochondria, but a pepsin-resistant fibrous material remained which was heavily stained by uranyl and lead ions. Experiments on a DNA "model system" showed that DNA treated with osmium tetroxide can be depolymerized by deoxyribonuclease. Zinc ions strongly inhibited the depolymerization of DNA. Digestion of osmium tetroxide-fixed tissues (fixed only briefly) with deoxyribonuclease for 1 hour greatly reduced the Feulgen staining of the nuclei, and after 4 hours the Feulgen reaction was completely abolished. The reduction and the disappearance of the Feulgen reaction in nuclei was paralleled by partial to complete digestion of the mitochondrial fibers in the regions studied (after 1 and 4 hours, respectively), without any other obvious changes in cellular structures. When deoxyribonuclease was inhibited by the addition of zinc ions, the nuclear Feulgen reaction was not diminished, nor were the mitochondrial fibers removed. Buffer control incubations for deoxyribonuclease and ribonuclease did not alter the structure or staining properties of the mitochondrial fibers, nor did incubation with ribonuclease. The latter reaction digested the cytoplasmic and nucleolar ribosomes after a 4-hour incubation period, in parallel with the abolishment of toluidine blue staining. The results contribute further evidence that these mitochondria contain deoxyribonucleic acid.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

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.     

The effect of chemical fixatives on cell walls of Bacillus subtilis.

Cell walls of Bacillus subtilis were treated with several chemical fixatives which are commonly used preparatory to electron microscopy; i.e., osmium tetroxide, formaldehyde, acrolein, crotonaldehyde, and glutaraldehyde. Dimensional analysis was performed on thin sections of fixed walls from plastic embeddings and, by means of the statistical technique of multiple comparisons, significant differences were found between wall thicknesses from the various fixations. These differences varied with the fixation time and the type of fixative used in the reaction. When compared to embedded walls which had been stained before fixation, the overall effect was a reduction in wall thickness which was attributed to fixative action and not to the embedding or staining processes. The reduction of wall thickness was even more apparent when dimensions of fixed walls were compared to published dimensions of both frozen sections and freeze-etch profiles. Since these fixatives bind to reactive sites within the wall fabric, a change in electrochemical charge density is effected which can be monitored in terms of heavy-metal-binding capacity. Most monoaldehyde fixatives and osmium tetroxide render the wall as reactive, or less reactive, to uranyl acetate as unfixed walls, whereas glutaraldehyde can significantly increase the binding capacity.     

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.     

PLANT MICROTECHNIQUE: SOME PRINCIPLES AND NEW METHODS

Some easily seen structural features of living plant cells are destroyed or badly distorted by most of the common fixatives and embedding media used in plant histology. In stained sections of plant tissues fixed in FAA (formalin-acetic acid-alcohol mixtures) and embedded in paraffin wax, for example, mitochondria and fine transvacuolar strands of cytoplasm are usually not visible. Many structural features such as these can be preserved, however, with suitable fixatives and embedding media. Specifically we recommend fixation in non-coagulant fixatives (e.g., osmium tetroxide, acrolein, glutaraldehyde, formaldehyde) and the use of plastics as embedding media, and we describe in detail a method of fixation in acrolein and embedding in glycol methacrylate polymer. In a wide range of plant specimens prepared in this way, stained sections 1–3 microns thick showed excellent preservation of tissue and cell structures.     

Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution.

Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the pond snail, Cipangopaludina malleata Reeve, were fixed in 1% osmium tetroxide, 3% permanganate, or 4% formaldehyde followed by 1% osmium tetroxide, each being buffered to pH 7.2 with Veronal-acetate or Sörensen's phosphate buffer. On the other hand, testes fixed with 4% formaldehyde adjusted to pH 7.2 with 0.075 M Na-cacodylate were incubated in Novikoff-Goldfischer medium for demonstrating thiamine pyrophosphatase, uridine or inosine diphosphatase, uridine monophosphatase or adenosine triphosphatase. The specimens incubated were postfixed in 1% osmium tetroxide buffered to pH 7.2 with Veronal-acetate buffer. Thin sections of the epoxy Epon resin-embedded tissue were stained either singly with saturated aqueous uranyl acetate or doubly with saturated aqueous uranyl acetate followed by lead citrate.In a concentric lamellar structure consisting of the granular endoplasmic reticulum in the cytoplasm of early atypical spermatids, disappearance of ribosomes attached to the outer surface of cisternae seems to have initiated at the central part of the structure, and the cisterna-attached ribosomes seem to participate in the formation of dense granules appearing in the vesicles representing the endoplasmic reticulum of atypical spermatids.The Golgi apparatus of the atypical spermatids in the advanced stages of development is composed of at least three different layers, the central part consisting of an amorphous material, the following lamellar and vesicular elements, and the peripheral fine vesicles.It has been assumed that the mechanism by which the nucleic acid, especially DNA is converted into the polysaccharide might be attributed to the function of the Golgi apparatus, because the transformation of dense granules into less dense granules as well as diphosphatase activities have been detected within the Golgi apparatus.This study was supported by Grant GM-8327-06 from the United States Public Health Service.     

A note on some non-membranous structures in plant mitochondria

Summary Electron-opaque inclusions, seen in the mitochondrial matrix of several plant tissues after glutaraldehyde and osmium tetroxide fixation, with uranyl acetate and lead citrate poststaining, are described. Some tentative proposals about their composition and function are put forward.     

Electron microscope studies of the human epidermis: the clear cell of Masson (dendritic cell or melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

Electron Microscope Studies of the Human Epidermis The Clear Cell of Masson (Dendritic Cell or Melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

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.     

Mitochondrial matrix granules in soft tissues : I. Elemental composition by X-ray microanalysis

The elemental composition of individual matrix granules in mitochondria of rat brown fat, mouse gall bladder and guinea pig kidney has been examined by X-ray microanalysis. The matrix granules showed a similar elemental composition that was strongly dependent upon the method of sample preparation. Low-temperature oxygen plasma microincineration or wavelength-dispersive X-ray spectrometers were used to demonstrate the presence of phosphorus in matrix granules of osmium tetroxide-fixed specimens. Matrix granule osmiophilia was retained in glutaraldehyde-fixed brown fat only if exposure to polar organic solvents was avoided during subsequent steps, e.g. by cryosectioning. As normally prepared, matrix granules lack detectable calcium but had bound this at detectable levels after fixation in osmium tetroxide, but not glutaraldehyde, supplemented with 5 mM Ca²⁺. The results demonstrate that mitochondrial matrix granules of normal soft tissues are not calcium phosphate deposits and contain phospholipids, apparently as a major constituent. Thus they provide evidence against the hypothesis that matrix granules are primarily involved in mitochondrial calcium sequestration and, indirectly, for the hypothesis that the granules may be related to inner membrane assembly.     

THE FINE STRUCTURE OF THE ELECTRIC ORGAN OF TORPEDO MARMORATA

The fine structure of the electric organ of the fish Torpedo marmorata has been examined after osmium tetroxide or potassium permanganate fixation, acetone dehydration, and Araldite embedment. This organ consists of stacks of electroplaques which possess a dorsal noninnervated and a ventral richly innervated surface. Both surfaces are covered with a thin basement membrane. A tubular membranous network whose lumen is continuous with the extracellular space occupies the dorsal third of the electroplaque. Nerve endings, separated from the ventral surface of the electroplaque by a thin basement membrane, contain synaptic vesicles (diameter 300 to 1200 A), mitochondria, and electron-opaque granules (diameter 300 A). Projections from the nerve endings occupy the lumina of the finger-like invaginations of the ventral surface. The cytoplasm of the electroplaques contains the usual organelles. A "cellular cuff" surrounds most of the nerve fibers in the intercellular space, and is separated from the nerve fibre and its Schwann cell by a space containing connective tissue fibrils. The connective tissue fibrils and fibroblasts in the intercellular space are primarily associated with the dorsal surface of the electroplaque.     

Investigations into the structure and fixation properties of cytoplasmic lamellae in the hamster oocyte

Summary Lamellar structures have been revealed in the cytoplasm of rapidly growing hamster oocytes by glutaraldehyde fixation and by fixation in 30% ethanol followed by osmication. The structures are not preserved after osmium tetroxide either used alone or followed by glutaraldehyde; nor are they preserved by absolute ethanol, formaldehyde, glyceraldehyde, glyoxal, 2-hydroxy-adipaldehyde or potassium permanganate. Immersion in 30% ethanol followed by extraction in distilled water and fixation in glutaraldehyde and osmium tetroxide exposes the lattice-like skeletal structure of the lamellae. The lamellae are present but slightly altered after short digestion in pepsin. Longer digestion results in complete dissolution of the structures.Supported by U.S.P.H.S. Post-doctoral Fellowship 5 F2 HD-25, 190–02.I wish to thank Prof. R. E. Coupland for his continued interest in this work and for his helpful criticisms.     

PREPARATION OF ISOLATED RAT LIVER MITOCHONDRIA FOR ELECTRON MICROSCOPY

A comparative study of the fixation of isolated rat liver mitochondria was undertaken. If the criterion is adopted that after processing, the mitochondria should resemble as closely as possible rat liver mitochondria in situ, the procedure found to produce such preservation was that of fixation in suspension in veronal-buffered 2% potassium permanganate. Fixation in osmium tetroxide produced variable results, while mitochondria fixed in glutaraldehyde were contracted. We suggest that in cases where fixation procedures modify the morphological appearance of mitochondria, the significance of such changes must be treated with caution.     

Ultrastructural localization of calcium in prothoracic gland cells of Galleria mellonella L. (Insecta,Lepidoptera) in relation to secretory activity

Summary Localization of intracellular calcium was demonstrated by precipitation with potassium hexahydroxoantimonate in the fixation medium containing osmium tetroxide or osmium tetroxide and glutaraldehyde. The presence of calcium in the precipitates was confirmed by X-ray microanalysis. Cells from active prothoracic glands contain more calcium deposits than inactive glands. The calcium precipitates are mainly localized in the nucleus, in the smooth endoplasmic reticulum, in the hyaloplasm and to a lesser degree in the mitochondria. These findings are consistent with the proposed role of calcium in the stimulation of steroidogenesis.     

Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate

Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method.     

Preservation of alveolar type II pneumocyte lamellar bodies for electron microscopic studies.

Simultaneous fixation with glutaraldehyde and osmium tetroxide, followed by an uranyl acetate (UA) treatment before dehydration and embedding (Hirsch and Fedorko 1968) ensures a very good preservation of lamellar bodies (LB's) as well as of the cellular membranes in type II pneumocyte. The uranyl acetate treatment appeared to be the most efficient step of the procedure. The morphological aspect of lamellar bodies after such a preparation was similar to that observed after freeze-etching of lipid retaining methods. Moreover, the Hirsch-Fedorko procedure is very simple and can easily be used for routine ultrastructural and radioautographic studies. On the other hand, it appeared that the uranyl acetate phospholipid "complex" is very sensitive to the pH of chemical solutions used after sectioning. The "complex" is variously dissolved by alkaline solutions, photographic developers or stains. The best preservation of ultrastructure was obtained with neutral or acidic developers and acidic stains.     

Size changes in single muscle fibers during fixation and embedding.

During fixation of single muscles fibers with glutaraldehyde, the volume of the fiber shrinks 20%, recovers in rinse and osmium tetroxide to near normal volume and shrinks 20% again when staining with uranyl acetate. This suggest that osmotic properties of membranes may not have been completely lost during fixation, post-fixation and en bloc staining. Dehydration in ethanol and propylene oxide produces a further 10% shrinkage in volume. Infiltration and embedding with Epon causes an additional 15% change in volume. This gives a total shrinkage in volume of 45% which is nearly twice that of the apparent shrinkage in the volume of the myosin lattice as determined by electron microscopy.