UV Micrographs and Photomicrographs from the Palynological Laboratory,Stockholm-Solna

In Equisetum both the spore and the rhizoidal and first-formed prothallial cells contain sac-like cytoplasmic particles limited by a unit membrane. After KMnO₄ fixation these bodies resemble the spherosomes described from earlier studies (e.g. Frey-Wyssling et al., 1964). They are bounded by a unit membrane, have a diameter of 0.8–1.7 μ and a fine granular content. After double fixation with buffered glutaraldehyde combined with an osmium post-fixative, or triple fixation with buffered formaldehyde added, the bodies resemble microbodies in fine structure. They have a thin unit membrane, a more coarsely granular matrix than after KMnO₄ fixation and very often a core like a 0.5 μ cluster of tubules or discs of around 200 Å in diameter. When spores are fixed in glutaraldehyde the bodies often show cytoplasmic invaginations or inclusions, which is never the case when they are fixed with KMnO₄     

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.     

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.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of Bombyx mori Linné were fixed in buffered (pH 8.2) 1% OsO₄ or 3 % KMnO₄ and thin sections of the tissue, embedded in methacrylate or epoxy Epon resin, were studied under the electron or light microscope.At the late stage of differentiation of the spermatid, the nucleus shows an elongated conical contour, being composed of fine fibrillar elements. These fibrillar elements fixed in OsO₄ measure 100 to 130 Å in diameter, while those fixed with KMnO₄ are approximately 70 Å in diameter.It has been found for the first time in the spermiogenesis of the silkworm that two bands and a tubular structure develop in close proximity to one another and attached to the plasma membrane of the spermatid. The two bands fixed in OsO₄ are electron dense, but in the material fixed with KMnO₄, one of them, situated within the cell body, is as dense as that fixed in OsO₄, while the other, outside the cell body, is much less dense. These apparently novel apparatuses develop from the caudal nuclear region along the elongating spermatid, but the dense band intertwines with the acrosome in the apical region of the nucleus along the major axis of spermatid, while the tubular structure and the clear band reach far into the nutritive cell where the dense band and acre-some are not visible.A possible relationship between the tubular structure and the nutritive cell has been discussed.This study was supported by Grant GM-8327-03 from the United States Public Health Service.     

Fine structure of Bacillus subtilis. I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO(4), OsO(4), or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO(4) in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO(4) fixation appeared to provide much better definition of the boundaries of various structures than did OsO(4). 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 KMnO(4) fixation) as two thin lines. In cells fixed first with OsO(4) solution, and then refixed with a mixture of KMnO(4) and OsO(4) 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 KMnO(4). 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.     

Fine Structure of Bacillus subtilis : II. Sporulation Progress

The sporulation process in Bacillus subtilis has been studied principally with KMnO₄ fixation, but also, for the purpose of comparison, with OsO₄ and mixtures of both fixatives. At a very early stage, the pre-spore is seen to consist of what seems to be the nuclear material and granular substance, surrounded by a layer of dense material destined to become the innermost layer of the spore coat. At a subsequent stage, a light interspace is observed that is destined to become the spore cortex. The mature spore shows a very complex structure. The spore coat is composed of three layers, the middle layer of which consisted of 5 to 8 lamellae of thin membranes and interspaces, both about 20 to 25 A thick. Between the inner layer of the spore coat and the spore cortex, a thin membrane with an affinity to the cortex can be observed. The spore coat is enclosed within two envelopes, one loosely surrounding the core, and the other adhering to it. The process of spore maturation has been studied in detail. Certain peculiar cellular structures have been observed that seemed to represent features of abnormal sporulation processes.     

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.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

Relative speed of fixation of glutaraldehyde and osmic acid in plant cells measured by grana appearance in chloroplasts

Summary Brief fixation in a mixture of glutaraldehyde and OsO₄ caused stacked chloroplast grana membranes in leaf cells of wheat, barley, tobacco, maize, cowpea, pigweed or bean plants to distend and vesiculate. Fixation with glutaraldehyde followed by OsO₄ prevented this fixation artifact. In a fixative mixture, OsO₄ apparently reacted with cell contents before glutaraldehyde.     

THE CYTOPLASMIC FINE STRUCTURE OF THE DIATOM, NITZSCHIA PALEA

The cytoplasmic fine structure of the motile, pennate diatom, Nitzschia palea was studied in thin sections viewed in the electron microscope. The cells were fixed in OsO₄, embedded in methacrylate, and immersed in 10 per cent hydrofluoric acid (HF) for 36 to 40 hours to remove the siliceous cell wall prior to sectioning. The HF treatment did not cause any obvious cytoplasmic damage. The dictyosome complex is perinuclear, and located only in the central cytoplasm. Mitochondria are sparse in the central cytoplasm, but abundant in the peripheral cytoplasm, and fill many of the transvacuolar cytoplasmic strands. Characteristic, amorphous oil bodies fill certain cytoplasmic strands and probably are not leucosin. The pyrenoid appears to be membrane limited, and oil droplets are found adjacent to the pyrenoid. The pyrenoid of another diatom, Cymbella affinis, is also membrane-limited. The membrane limiting the pyrenoid may be a composite of the terminal portions of chloroplast discs, facilitating rapid movement of photosynthate into the pyrenoid matrix, where the characteristic oil droplets may be formed. Carinal fibrils are found singly in each carinal pore, and may be involved in the locomotion of Nitzschia palea.     

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.     

THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.     

STUDIES ON THE FIXATION OF ARTIFICIAL AND BACTERIAL DNA PLASMS FOR THE ELECTRON MICROSCOPY OF THIN SECTIONS

The process of fixation of DNA-containing plasms is investigated by macroscopical and electron microscopical observations on solutions of DNA, nucleohistones, as well as on bacterial nuclei. The following treatments were found to produce a gelation of a solution of DNA or nucleohistones: (a) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺. (b) Exposure to aqueous solutions of uranyl acetate. (c) Exposure to aqueous solutions of indium chloride. Observed in the electron microscope, these gels show a fine fibrillar material. From experiments in which solutions of DNA or nucleohistones are mixed with bacteria and treated together, it is concluded that the behavior of the bacterial nucleoplasm is similar to that of the DNA solutions. The appearance of birefringence indicates that uranyl acetate and indium chloride produce an orientation of the molecules of a DNA solution during gelation. Bacterial chromosomes fixed by these agents also show a certain order, while those fixed by the OsO₄-amino acid-Ca⁺⁺ formula do not. Whether or not the order can be considered to be artificial is discussed, and a tentative conclusion is presented: (a) Uranyl acetate may induce artificial order. (b) Fixatives which do not gel DNA probably result in the grossest artifacts. (c) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺ may give the most accurate preservation of the in vivo disposition of DNA (RK⁺ fixation).     

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     

Relation Between Basophilia and Fine Structure of Cytoplasm in the Fungus Allomyces macrogynus Em

In a fungus, Allomyces macrogynus Em., staining tests have revealed changes in the location of cytoplasmic basophilia following different phases of the developmental cycle. These variations in location were used to observe which fine structures correspond to basophile and non-basophile areas of the cytoplasm. Hyphae, gametangia, zygotes, and plants were fixed at various developmental stages in OsO₄, pH 6.1, and embedded in vestopal. Sections were examined in the electron microscope. Comparison of basophile and non-basophile cytoplasms leads to the conclusion that cytoplasmic particles of 150 to 200 A in diameter are responsible for basophilia. The possibility of these particles being ribosomes is discussed and confirmed. The present paper also describes some observations on the fine structure of other cellular components of this fungus, such as nuclei, mitochondria, various granules, and flagella.     

Improved structural preservation in freeze-substituted sporidia ofUstilago avenae—a comparison with low-temperature embedding

Summary In the present study the cellular fine structure of freeze-substituted sporidia of the phytopathogenic fungusUstilago avenae is investigated by means of thin-section electron microscopy. A conventional embedding method using Spurr's low viscosity resin is compared with the recently developed methacrylate mixtures Lowicryl^® K 4 M and HM 20 resin. Generally, freeze-substitution yields improved preservation of fine structural details of the fungus compared to previously applied conventional fixation methods. Using double fixation during freeze-substitution prior to conventional embedding the fungal membrane system (plasmalemma, endoplasmic reticulum), organelles (mitochondria, nucleus etc.) and other cytoplasmic features (ribosomes, cytoskeleton) appear well resolved and smoothly contoured. Aldehyde fixed and Lowicryl embedded sporidia ofU. avenae resemble these double fixed fungal specimens fairly closely. The complete low-temperature preparation produces visualization of distinct cellular details although contrast reversal of cellular membranes (er, mitochondria etc.) is sometimes observed. In particular, fine structure resolution is enhanced in Lowicryl HM 20 embedded fungal cells. This is due also to significant improvement in staining of the cellular membranes, cytoskeleton (microfilaments and microtubules) and Golgi apparatus-like areas, using tannic acid. In case of the fungusU. avenae, freeze-substitution in combination with mild glutaraldehyde fixation and final low-temperature embedding in HM 20 resin prove suitable for improved preservation of cellular ultrastructure.Abbreviations cw cell wall - cy cytoplasm - FS freeze-substitution - FS-A GA/OsO₄ freeze-substitution and Spurr's LV-embedding - FS-B GA freeze-substitution and Lowicryl K 4 M LT-embedding - FS-C GA freeze-substitution and Lowicryl HM 20 LT-embedding - go Golgi apparatus-like body - GA glutaraldehyde - g glycogen deposit - l lipid droplet - LT low temperature - Lowicryl LT-embedding Lowicryl low-temperature embedding - Lowicryl LT-resin Lowicryl low-temperature resin - MeOH methanol - mf microfilament - mt microtubule - m mitochondrion - mvb multivesicular body - ne nuclear envelope - np nuclear pore - npl nucleoplasm - nu nucleolus - n nucleus - OsO₄ osmium tetroxide - pl plasmalemma - pr polyribosomes - Pb-citrate Reynolds' lead citrate - r ribosome - RT room temperature - rer rough endoplasmic reticulum - Spurr's LV-embedding Spurr's low viscosity embedding - Spurr's LV-resin Spurr's low viscosity resin - t tonoplast - Uac uranyl acetate - v vacuole     

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.     

ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION ON THE STRUCTURE OF NERVE MYELIN : I. Peripheral Nerve

The dehydration of frog sciatic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. Low magnification electron micrographs of OsO₄-fixed preparations showed marked tissue shrinkage which could be correlated quantitatively with the loss of water during the preliminary drying. KMnO₄-fixation appeared to cause a rehydration of the dried tissue. Higher magnification electron micrographs of the OsO₄-fixed preparations showed a sequence of modifications of the myelin layers which could be correlated with changes in the small-angle x-ray diffraction data which were recorded during drying. An intermediate stage of drying was characterised by a partial collapse of layers and a disappearance of the intraperiod dense line in some regions of the myelin sheath. Continuity between collapsed and non-collapsed layers was maintained throughout the sheath. The fully dried preparation showed two main modifications of the myelin layers. In many regions the layers (principal layers) resembled those of normal preparations, but showed an intensification and frequently a doubling of the intraperiod dense line. In addition, there was a very extensive system of fine (40 A periodicity) dense layers, some of which could be demonstrated to be continuous with the principal layers. In such cases it was observed that two of the fine layers were related to each principal layer. The correlation between diffraction data and electron microscope data is discussed, and some speculations are made concerning the molecular significance of the observations.     

INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

FINE STRUCTURES OF INTRACYTOPLASMIC ORGANELLES OF MYCOBACTERIA

The fine structure of the intracytoplasmic organelles of mycobacteria was studied by means of electron microscopy of ultrathin sections. A well-preserved nuclear apparatus was obtained by fixation with OsO₄ in acetate-veronal buffer, containing calcium and tryptone, or in collidine-HCl buffer, followed by uranyl-acetate treatment and embedding in araldite. A low density nuclear region was filled with fine fibrils, 30 A in diameter, in parallel or concentric arrangement. A membranous organelle, tentatively designated as "lamellar structure," consists of unit membranes in lamellar arrangement. The thickness of each lamella in this membranous organelle coincides with that of the three-layered cytoplasmic membrane Moreover, the continuity of this unit membrane with the cytoplasmic membrane was demonstrated.