Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

The intracytoplasmic membrane system of the bacteroids of subterraneum clover nodules (Trifolium subterraneum L.)

Summary A vesicular intracytoplasmic membrane system is demonstrated in bacteroids from the leghaemoglobin filled zone of effective Trifolium subterraneum nodules after KMnO₄ and OsO₄ fixation. The system appears to be present in all mature bacteroids from this zone, and is derived from tubular invaginations of the plasma membrane of the bacteriod. A granular substance similar to the bacteroid cytoplasm is found in the vesicles which are bounded by a tripartite membrane approximately 80 Å wide, while the interspace between the vesicles is filled with a material of similar appearance to that in the interspace between bacteroid plasma membrane and cell wall.     

PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

Observations on the fine structure of KMnO₄-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO₄. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO₄-fixed testes in view of what has already been reported for mammalian testes fixed in OsO₄.     

Development of the bacteroid in the root nodule of barrel medic (Medicago tribuloides Desr.) and subterraneum clover (Trifolium subterraneum L.)

Summary The development of the bacteriod is traced from thin sections of slices of nodules fixed in KMnO₄ and OsO₄. While in the infection thread the Rhizobium cell has the ultrastructure characteristic of gram-negative bacteria, with two unit membranes bounding a granular cytoplasm containing dense bodies, a nucleoid area and inclusion granules. A 10–12 fold increase in size, a loss of inclusion granules and the formation of a membrane envelope around each Rhizobium cell follows the dispersal of the rhizobia through the host cytoplasm. As the bacteriods develop there is a loss of fibrillar material from the nucleoid region and changes occur in the distribution of ribosome-like particles in both host and bacterial cells. When fully differentiated and presumably fixing nitrogen the bacteroids from the red zone of subterraneum clover nodules but not barrel medic have a well developed intra-cytoplasmic membrane system.     

Fine structure changes in the development of the nodules of Trifolium subterraneum L. and Medicago tribuloides Desr.

Summary Electron microscope observations of thin sections of nodules of subterranean clover and barrel medic, after fixation in KMnO₄ or OsO₄, show that following infection there is a marked increase in the amount of endoplasmic reticulum, in the number of ribosomes, Golgi bodies, mitochondria and proplastids in the host cells.As the infection thread approaches the nucleus, large gaps appear in the nuclear membrane. During the formation of the membrane envelopes around the rhizobia, after their release from the infection thread, the reticulum changes from a predominantly plate-like to a vesicular form. As the bacteroids develop the plastids of the host cells become filled with starch, and become aligned, with the mitochondria, against the cell walls of the host cells. Plastids in noninvaded cells also become starch-filled. Bacteroids and host cells enlarge further and finally the bacterioids occupy most of the cytoplasm of the host cell, except for the nuclear region and vacuole. With OsO₄ fixation the nucleoplasm, predominantly fibrillar before infection, with a dense staining nucleolus, becomes packed with dense ribosome-like (150 A° diameter) granules. No such changes occur in the nuclei of non-infected cells. In the proplastids and plastids many small, electron dense particles (60 A° diameter) (phytoferritin?) are observed.     

The Spermatid Nucleus in Two Species of Grasshopper

The nuclear changes accompanying spermatid elongation have been studied in two species of grasshopper, Dissosteira carolina and Melanoplus femur-rubrum. Testes were fixed in 1 per cent buffered OsO₄, imbedded in butyl methacrylate, and examined as thin sections in the electron microscope. In both species nuclear changes during spermatid development involve (1) an early period, during which the nuclear contents are predominately fibrous; (2) a middle period, characterized by the lateral association of the nuclear fibers to form plates or lamellae which are oriented longitudinally in the major axis of the elongated nucleus; and (3) a late period, involving coalescence of the lamellae into a crystalline body which eventually becomes so dense that all resolvable detail is lost. The fibers seen in the early spermatid nucleus are about 150 A in diameter and so are similar to fibers described from other types of nuclei. The thickness of the lamellae varies from about 150 A when first formed to 70 A during the later stages. The lack of evident chromosomal boundaries in the spermatid nucleus makes it difficult to relate either the fibers or lamellae to more familiar aspects of chromosome structure. We see no apparent reason to consider that the fiber alignment described here is related to conventional chromosome pairing.     

Cytological Studies of the Nematocysts of Hydra : I. Desmonemes, Isorhizas, Cnidocils, and Supporting Structures

Entire hydras or tentacles were fixed in OsO₄ or in KMnO₄ and thereafter washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on an experimental model, thermal expansion type ultramicrotome or on a Porter-Blume microtome. The sections were examined in an RCA electron microscope. Type EMU-2 D. "Squash preparations" for light microscopy, were made from the hydra mouth region and the attached tentacles. These were observed with an AO Baker interference microscope. In the mature organism, three of the four types of nematocysts normally found in hydra could be positively identified with the electron microscope. The desmonemes, the smallest type, have a dense matrix and a thin capsule. The two different types of mature isorhizas could not be distinguished with certainty. They are intermediate in size between the desmonemes and stenoteles and have a capsule with a dense matrix. The cnidocil, or triggering hair, which is composed of a dense core and a fibrillar sheath has nine supporting elements arranged in a semi-circle near its base. Twenty "supporting structures" are arranged around the nematocyst capsule and interconnections between the supporting elements and these latter structures have been observed. Development of the nematocysts involves an increase in density of the matrix. Spines can be seen in the interior of tubular structures within the capsules of the holotrichous isorhizas.     

Electron Microscopic Observations on the Taste Buds of the Rabbit

An examination of the fine structure of the taste buds in the rabbit was undertaken. Gustatory epithelium was fixed in OsO₄ or 1 per cent KMnO₄ solution, containing polyvinylpyrrolidone (PVP). Thick sections were examined in the phase microscope and contiguous sections prepared for the electron microscope. The bud contains two types of cells, gustatory receptors and sustentacular cells. The receptors are characterized by a dark nucleus and densely granular cytoplasm. The apical processes bear numerous microvilli which extend into the taste pore. Imbedded between the microvilli there is a dense substance, which is also present in the apical cytoplasm of the receptors. The sustentacular cells contain a large pale nucleus and less dense cytoplasm. Their basal surfaces rest upon a basement membrane. The subepithelial nerve plexuses comprise the fibers which innervate the gustatory receptors. The nerve fibers vary in diameter from 500 A to 0.3 µ, and are ensheathed by Schwann cells. The intragemmal fibers enter the taste bud between adjacent cells, and are ensheathed by the plasma membranes of the supporting cell until they synapse upon the gustatory cell. The synaptic terminals contain synaptic vesicles, which at this junction reside in the postsynaptic element. This observation is discussed with reference to synapses described elsewhere in the nervous system.     

ALTERATION OF THE CONFORMATION OF PROTEINS IN RED BLOOD CELL MEMBRANES AND IN SOLUTION BY FIXATIVES USED IN ELECTRON MICROSCOPY

The effects of several commonly employed fixatives on the three-dimensional conformations of two soluble proteins and the protein of intact red blood cell membranes have been studied by means of circular dichroism measurements in the spectral region of the peptide absorption bands. The fixatives used produced significant and parallel conformational changes in all of the proteins, in the increasing order: glutaraldehyde; OsO₄; glutaraldehyde followed by OsO₄; and KMnO₄. The last two treatments obliterated most of the helical character of the proteins. The significance of these observations to the preparation of specimens for electron microscopy is discussed.     

Permanganate Fixation of Plant Cells

In an evaluation of procedures explored to circumvent some of the problems of osmium tetroxide-fixation and methacrylate embedding of plant materials, excised segments of root tips of Zea mays were fixed for electron microscopy in potassium permanganate in the following treatment variations: unbuffered and veronal-acetate buffered solutions of 0.6, 2.0, and 5.0 per cent KMnO₄ at pH 5.0, 6.0, 6.7, and 7.5, and temperatures of 2–4°C. and 22°C. After fixation the segments were dehydrated, embedded in epoxy resin, sectioned, and observed or photographed. The cells of the central region of the rootcap are described. The fixation procedures employing unbuffered solutions containing 2.0 to 5.0 per cent KMnO₄ at a temperature of 22°C. gave particularly good preservation of cell structure and all membrane systems. Similar results were obtained using a solution containing 2.0 per cent KMnO₄, buffered with veronal-acetate to pH 6.0, and a fixation time of 2 hours at 22°C. The fixation procedure utilizing veronal-acetate buffered, 0.6 per cent KMnO₄ at 2–4°C. and pH 6.7 also gave relatively good preservation of most cellular constituents. However, preservation of the plasma membrane was not so good, nor was the intensity of staining so great, as that with the group of fixatives containing greater concentrations of KMnO₄. The other fixation procedures did not give satisfactory preservation of fine structure. A comparison is made of cell structures as fixed in KMnO₄ or OsO₄.     

ULTRASTRUCTURE OF THE SPOON TYPE SYNAPTIC ENDINGS IN THE NUCLEUS VESTIBULARIS TANGENTIALIS OF THE CHICK

The fine structure of the "spoon" type synaptic endings of the chick tangential nucleus was studied with the electron microscope. These endings often measure ~18 µ in length by ~3–4 µ in width. The axoplasm of the endings contains very few synaptic vesicles, a large number of neurofilaments oriented parallel to the long axis of the nerve fiber, and microtubules and numerous mitochondria. The synaptic membrane complex shows areas of localized occlusion of the synaptic cleft with the formation of an external compound membrane. It has not been decided whether these areas have a disc shape; their length measures between 0.04 and 0.47 µ. The five-layer pattern characteristic of an external compound membrane is shown in specimens fixed with formalin—OsO₄, glutaraldehyde—acrolein—OsO₄, and acrolein KMnO₄ but it does not appear in the glutaraldehyde-OsO₄-fixed specimens. The over-all thickness of the external compound membrane varies depending upon the fixative used. The synaptic clefts in the regions between the external compound membrane discs are widened and measure ~300 A. A condensation of dense material occurs in pre- and postsynaptic cytoplasms all along the synaptic membrane complex. The morphological relationships described in the spoon endings are suggestive of electrical transmission.     

SPERMATOGENESIS IN ANIMALS AS REVEALED BY ELECTRON MICROSCOPY : II. SUBMICROSCOPIC STRUCTURE OF DEVELOPING SPERMATID NUCLEI OF GRASSHOPPER

The submicroscopic structure of the maturing spermatid nucleus of the grasshopper, Gelastorrhinus bicoler de Haan, has been studied in thin tissue sections by electron microscopy. In the early spermatid the nucleus appears dense with no clearly resolvable fine structure. In the advanced spermatid with a conical-shaped nucleus, the karyoplasm begins to show a fibrillar structure. At subsequent stages, the elongated spermatid nucleus displays in cross-sections a hexagonal honeycomb pattern and in longitudinal sections an array of parallel lines, 70 A in diameter and spaced 100 to 220 A apart. As differentiation of the spermatid proceeds further, the space between the lines becomes narrower and narrower until it can no longer be resolved.     

An Electron Microscope Study of Myelin Figures

In the electron microscope, thin sections of OsO₄-fixed myelin figures from the phospholipide fraction of human brain show a pattern of parallel dark lines with a repeating period of about 40 A. It is shown that the dark lines probably represent the reaction product of OsO₄ with double bonds in the fatty acid chains, thereby marking the central portion of one bimolecular lamella. The addition of globin results in dense lines 25 to 50 A wide that cover the surface of the myelin figures. When such a figure consists of only two bimolecular leaflets of lipide covered with globin, the structure shows striking similarity to the image of cell membranes in fixed tissue sections. A hypothetical schema is given of the molecular structure of the figure, and the distribution of OsO₄ in it.     

Fine structural relation between the Sertoli cell and the differentiating spermatid in the human testis

Summary The structural relationship between the Sertoli cell and the developing spermatid was studied with the electron microscope. In the contact area of the Sertoli cell with the anterior part of the developing spermatid, a filamentous structure is observed. This structure consists of fine tubular filaments about 100 Å in diameter associated with dense material. The functional significance of the structure is discussed.Supported by Grant HD-00593-05 of National Institutes of Health, United States Public Health Service. The author is indebted to Dr. T. Katayama, Department of Urology, Chiba University, for supplying the materials.     

ELECTRON MICROSCOPY OF SPORES OF BACILLUS MEGATERIUM WITH SPECIAL REFERENCE TO THE EFFECTS OF FIXATION AND THIN SECTIONING

Resting spores of Bacillus megaterium appear uniformly opaque and undifferentiated under the electron microscope. Germinated spores and spores which have lost their dipicolinic acid underwent characteristic changes in structure. Spores fixed with KMnO₄ lose their dipicolinic acid. Spores fixed with OsO₄ under certain conditions retain their dipicolinic acid. When conventional sectioning procedures are used with either method of fixation, abnormal spore structure is produced as a result of the solution of cellular constitutents. Dry sections of unfixed spores embedded in methacrylate reveal the spore structure in a more normal state. Indirect evidence has been obtained for the existence of a penetration barrier at or near the outer edge of the cortex.     

The electron microscopic observation of haustoria of Erysiphe graminis hordei

The fine structure of haustoria ofErysiphe graminis hordei was studied using samples fixed with 3 per cent KMnO₄ or 2 per cent OsO₄. The cell wall of the infected leaves of barley seedlings was extremely electron-dense around the penetration point of this fungus. This may be due to chemical change of components in cell wall by the enzymatic action of the fungus. The cell wall was invaginated towards the cytoplasm at the point of penetration and formed a sheath around the infection hypha. Unknown electron-dense substances were accumulated around the infection hypha outside the sheath. The haustorial cell wall was surrounded with encapsulation and distinguished clearly from the cytoplasm of epidermal cell. The cell wall of the haustorium was very thin and electron-transparent, when compared with that of conidia and hyphae. A septum with a septal pore was observed between the infection hypha and the haustorium. Besides the nucleus, mitochondria, endoplasmic reticula and the like, many kinds of vesicles and specific coiled membraneous structure were found in the haustorium. The origin and the function of the encapsulation remain obscure.

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Zusammenfassung Die Feinstruktur von Haustorien vonErysiphe graminis hordei war untersucht, indem Stücke mit 3 % KMnO₄ oder 2 % OsO₄ fixiert worden sind. Die Zellwand der infizierten Blätter der Gerstenkeimlinge war elektron-dicht um die Eindringungsstelle des Pilzes. Dies mag die Folge der chemischen Veränderung der Komponenten in der Zellwand durch die enzymatische Wirkung des Pilzes sein. Die Zellwand war an der Eindringungsstelle zum Zytoplasma gebogen und hat eine Hülle um die Infektionshyphe gebildet. Unbekannte, elektron-dichte Substanzen waren um die Infektionshyphe außerhalb der Hülle angesammelt. Die Haustorialzellwand war abgegrenzt und war vom Zytoplasma der Epidermalzelle unterscheidbar. Die Zellwand des Haustoriums war dünn und elektron-durchsichtig im Vergleich mit denen der Konidien und Hyphen. Ein Septum mit einer Septalpore war zwischen der Infektionshyphe und dem Haustorium beobachtet. Neben dem Nucleus, Mitochondrien, endoplasmatischen Netz sind mancherlei Blister, spezifische, eingerollte Membranstruktur im Haustorium gefunden worden. Die Herkunft und die Funktion dieser Strukturen blieb ungeklärt.

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Contribution No. 232     

Spermatogenesis in animals as revealed by electron microscopy

Summary The fine structure of the spermatids in late stages of the differentiation, which appeared in the testis of early pupa of the silkworm Bombyx mori Linné, was studied in the electron imcroscope, being fixed in buffered (pH 8.2) 2.5% osmium tetroxide or 3% potassium permanganate.The clear band differentiates into elaborated elements consisting of an array of at least 12 membranes which run loosely winding along the major axis of the spermatid. The elaborated clear band, i. e., clear band derivatives, may be an apparatus to facilitate the activity of spermatozoa, since they are present along the full length of the remarkably elongated premature spermatozoa.It has been revealed that the clear band derivatives possess a highly ordered, fine structure which is seen to be of a paracrystalline nature. The periodic pattern has first occurred along the long axis of the clear band derivatives. After such structure is decomposed into an apparently homogeneous material, a characteristic periodic pattern occurs again crossed the major axis of the clear band derivatives, the significance of such ultrastructural changes remaining obscure.The tubular structure appears through the head part of the developing spermatids, revealing even in an apical region where the nucleus is not visible, and it appears enlarged at the base of the nucleus, but no more visible in the tail piece. In the stages when the clear band becomes progressively specialized, the tubular structure appears attached to the nucleus, although it situated at the peripheral part of the cell in a more early stage of the differentiation. The tubule is incompletely separated into two layers by a dense septum projected from the tubular wall, suggesting that such structure provides the tubule with a relatively large interior surface for metabolic reactions.This study was supported by Grant GM-8327-04 from the United States Public Health Service.     

ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about -180°C., dried, at -55 to -60°C., embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade's granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO₄. The structural organization in the nucleus is distorted by vacuolization. The mitochondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are encountered. After staining the sections with OsO₄ or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OsO₄-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO₄ or Giemsa solution also renders more distinct the membrane of the ER and Palade's granules but not the Golgi apparatus and cellular membrane. Treatment with ribonuclease results in the disappearance of Palade's granules. On the strength of these observations the authors conclude that OsO₄ fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade's granules, though it may induce slight swelling of these cell components.     

ELECTRON MICROSCOPE AND LOW-ANGLE X-RAY DIFFRACTION STUDIES OF THE NERVE MYELIN SHEATH

1. A close correlation has been obtained between high resolution electron microscopy and low-angle x-ray diffraction studies of the myelin sheath of frog and rat peripheral and central nerves. Extensive studies were performed by application of both techniques to the same specimens, prepared for examination by OsO₄ or KMnO₄ fixation, and embedding either in methacrylate or in gelatin employing a new procedure. Controlled physical and chemical modifications of the myelin sheath prior to fixation were also investigated. 2. A correspondence was established between the layer spacings observed in electron micrographs and the fundamental radial repeating unit indicated by the low-angle x-ray diffraction patterns. The variations in relative intensities of the low-angle x-ray reflections could be related to the radial density distributions seen in the electron micrographs. 3. An analysis of the preparation procedures revealed that OsO₄ fixation introduces a greater shrinkage of the layer spacings and more pronounced changes in the density distribution within the layers than KMnO₄ fixation. The effects of methacrylate and gelatin embedding are described, and their relative merits considered in relation to the preservation of myelin structure by OsO₄ fixation. 4. The experimental modifications introduced by freezing and thawing of fresh whole nerve are described, particularly the enhancement of the intermediate lines and the dissociation of the layer components in the myelin sheath. A characteristic collapsing of the radial period of the sheath is observed after subjecting fresh nerve trunks to prolonged and intense ultracentrifugation. 5. Controlled extraction of fresh nerve with acetone at 0°C., which preferentially removes cholesterol, produces characteristic, differentiated modifications of the myelin sheath structure. Electron microscopy reveals several types of modifications within a single preparation, including both expanded and collapsed layer systems, and internal rearrangements of the layer components. Alcohol extraction leads to a more extensive structural breakdown, but in certain areas collapsed layer systems can still be observed. The components of the lipide extracts could be identified by means of x-ray diffraction. These modifications emphasize the importance of cholesterol in the myelin structure, and disclose a resistance of the dense osmiophilic lines to lipide solvents. 6. The significance of these structures is discussed in relation to present concepts of the molecular organization of myelin. The available evidence is consistent with the suggestion that the primary site of osmium deposition is at the lipoprotein interfaces and that the light bands probably represent regions occupied by lipide chains. The electron microscope and x-ray diffraction data also indicate the possibility of a regular organization within the plane of the layers, probably involving units of 60 to 80 A. The myelin sheath is regarded as a favourable cell membrane model for detailed analysis by combined application of x-ray diffraction and electron microscopy.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Summary Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO₄) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO₄ darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO₄, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.