Ultrastructure of the Acidophilic Aerobic Photosynthetic Bacterium Acidiphilium rubrum

The ultrastructure of cells of Acidiphilium rubrum, which is an acidophilic aerobic photosynthetic bacterium containing zinc-complexed bacteriochlorophyll a, was studied by electron microscopy with the rapid substitution technique. Thin-section electron microscopy indicated that any type of internal photosynthetic membranes was not present in this organism despite a relatively high content of the photopigment. The majority of cells had poly-β-hydroxybutyrate granules and electron-dense spherical bodies identified as being polyphosphate granules. When the organism was grown chemotrophically with 0.1% FeSO₄, it produced another group of electron-dense granules that were associated with the inner part of the cytoplasmic membrane. An energy-dispersive X-ray analysis showed that these membrane-bound, electron-dense granules contained iron. Received: 24 November 1999 / Accepted: 5 January 2000     

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3–11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - ER endoplasmic reticulum - HPLC high-pressure liquid chromatography - MRST Murashige and Skoog's revised tobacco medium     

Polyhydroxybutyrate particles in Synechocystis sp. PCC 6803: facts and fiction

Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Spherical inclusions inside the cell that are electron-transparent and/or slightly electron-dense and that are found in transmission electron micrographs of cyanobacteria are generally assumed to be PHB granules. The aim of this study was to test this assumption in different strains of the cyanobacterium Synechocystis sp. PCC 6803. Inclusions that resemble PHB granules were present in strains lacking a pair of genes essential for PHB synthesis and in wild-type cells under conditions that no PHB granules could be detected by fluorescence staining of PHB. Indeed, in these cells PHB could not be demonstrated chemically by GC/MS either. Based on the results gathered, it is concluded that not all the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria. Alternate assignments for these inclusions are discussed.     

The fine structural localization of acid phosphatase in the gut epithelial cells of the slug,Avion ater (L.)

Summary Acid phosphatase, generally thought of as a lysosomal enzyme and indeed widely employed as a lysosomal marker, has been found associated with the Golgi complex of all cell types from the crop, intestine and digestive gland ofArion ater. Reaction product was also detected within the multivesicular bodies and cytoplasm of columnar cells from the crop and the multivesicular bodies of mucous cells from the intestine. A vacuolar localization was obtained in the digestive cells of the intestine and digestive gland. Secretory protein granules in the calcium cells of the same gland and apical vacuoles in the so-called thin cells also showed a positive reaction.This work was undertaken as part of a slug research project under the direction and co-ordination of Dr. D. K.Roach, supported by A.R.C. Assistance was given by Mr. T. R.Mainwaring in the preparation of tissue for electron microscopy.I would like to thank Professor J.Brough and Professor D.Bellamy for providing facilities and encouragement.     

Light,fluorescence and electron microscopic studies on “neurosecretion” in tritonia diomedia bergh (Mollusca,Nudibranchia)

Summary Membrane-limited electron-dense inclusions designated as elementary neurosecretory granules have a characteristic distribution in cerebropleural ganglia of the nudibranch snail Tritonia diomedia. They occur in the neuropile and also in individual nerve fibres, connectives and commissures. These granules have been found neither in perikarya of nerve cells nor in proximal segments of their processes.Specific fluorescence obtained in Tritonia preparations with Sterba's pseudoisocyanin method for neurosecretory products has the same pattern of location.The distribution of stainable material in preparations prepared with ordinary neurosecretory procedures (chrome haematoxylin-phloxin after Gomori-Bargmann and paraldehydefuchsin after Gomori-Gabe) is similar to that described by different authors in other gastropods, but strongly differs from the locationof elementary neurosecretory granules and of pseudoisocyanin-positive material. The adequacy of different histological methods for studying neurosecretion in gastropods is discussed.     

Peroxidase and Tyrosinase Are Present in Secondary Lysosomes That Degrade Photosensory Membranes of the Crayfish Photoreceptor: Possible Role in Pigment Granule Formation

Three enzymes (acid phosphatase, peroxidase, and tyrosinase) were localized by electron microscopy within the retina of crayfish Orconectes limosus. Peroxidase activity was observed only in lamellar bodies, which are secondary lysosomes and degrade photosensory membrane. After H₂O₂ was omitted from the reaction medium, peroxidase activity in lamellar bodies was partly inhibited but was not missing completely. After addition of sodium pyruvate, which inhibits endogenous generation of H₂O₂, staining of lamellar bodies was absent. Tyrosinase activity was found in lamellar bodies and in small vesicles within the rhabdoms similar to those found positive for acid phosphatase. Granules (500–700 nm in diameter) with an electron opaque matrix and mature screening pigment granules showed tyrosinase activity. Moreover, lamellar structures within membrane-bound organelles that additionally contained screening pigment-like granules were electron dense because of tyrosinase activity. After addition of phenylthiourea (PTU) to the incubation medium, lamellar bodies did not generally contain electron dense deposits, although weak staining of single membranes still was sometimes observed. After addition of sodium pyruvate in combination with PTU, no staining was detected. The possible role of tyrosinase in ommochrome synthesis within secondary lysosomes that degrade photosensory membrane is discussed.     

Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role

The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.     

Some ultrastructural observations on the mode of association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

Summary— The ultrastructural aspects of the association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Polamon dehaani, were studied by thin-section electron microscopy combined with detergent treatment. In the tendon cell, MTs were linked laterally by anchoring filaments to the plasmalemma via a submembranous electron-dense layer called the plasmalemmal undercoat. To further clarify how such anchoring filaments are spatially related to the plasmalemma through the undercoat, we carefully examined and compared thin-section images obtained from various specimen preparations using saponin and Triton X-100. When the tissues were treated with saponin or Triton, electron-dense materials in the undercoat were extracted in varying degrees to expose internal substructures. The undercoat appeared to show a two-layer organization, the inner and outer layers. In more extracted samples, filamentous networks became prominent in the outer layer. Anchoring filaments were seen to attach to such filamentous networks, which in turn were linked to the plasmalemma proper. Thus, it may be reasonable to consider that the filamentous network constitutes the core structure of the plasmalemmal undercoat which is structurally reinforced by extractable electron-dense materials.     

Monoclonal antibodies to surface and cytoskeletal components of the spermatozoid ofPteridium aquilinum

Summary Detergent extracted spermatozoids of the fernPteridium aquilinum were used as mixed antigen preparations for raising monoclonal antibodies in order to obtain reagents for detecting as yet uncharacterized components of the plant cytoskeleton. Selected antibodies were studied by immunofluorescence microscopy of developing spermatids and mature spermatozoids. Some reacted directly with fixed cells, others required permeabilization treatments with cold methanol or Triton X-100. AntibodiesPas2D9 andPas6D7 bind to glycoprotein antigenic determinants that are exposed on the surface of the plasma membrane. Several antibodies interact with cytoskeletal components.Pas1D3,Pas5D8 andPas5F4 bind to the cytoskeleton of permeabilized cells including the flagella. Three react specifically with the flagellar band or associated components:Pas2G6 reacts with the whole flagellar band but shows a prominent binding to basal bodies,Pas5E2 binds exclusively to basal bodies, andPas5E7 detects mitochondria associated with the flagellar band. Cross-reactions to wheat root tip cells at different stages of the cell cycle are described inMarc andGunning (1988).Abbreviations MLS multilayered structure - MT microtubule - MAb monoclonal antibody - MAP microtubule associated protein     

Flagellar Morphology in Stumpy-Flagella Mutants of Chlamydomonas reinhardtii1

Sixteen new mutants of the biflagellate green alga Chlamydomonas reinhardtii with either stumpy-flagella or no flagella at all were examined by electron microscopy. Four of the mutants were found to carry short bulbous flagella containing amorphous electron-dense material which may represent unassembled flagellar protein. Basal bodies of normal ultrastructure were present in all mutants. Dikaryon dominance tests indicated that the stumpy mutations were recessive to wild-type in all cases tested. Stumpy mutations also conferred a measure of detergent resistance to Chlamydomonas, apparently by affecting the detergent-solubility of the flagellar membrane.     

Detailed analysis of pronucleus development in bovine zygotes in vivo: Ultrastructure and cell cycle chronology

The ultrastructural development of pronuclei and cytoplasm was studied in bovine zygotes developed in the oviducts. The timing of the morphological events was related to sonographically detected ovulation and to the progress of the cell cycle determined by double labelling (³H and ¹⁴C-thymidine) of newly synthesized DNA combined with autoradiographic detection. The onset of the S-phase occurred at 11–12 hr after the estimated time of ovulation (EO), and this phase of the cell cycle lasted for 7–9 hr. During the G1-phase, the pronuclei contained spheres of compact, electron-dense fibrillar material classified as nucleolus precursor bodies. Early in the S-phase (13 hr aver EO) spherical fibrillogranular bodies containing larger rounded electron-dense components were detected in the periphery of the pronuclei as well. At 15 hr, the latter bodies had become connected through electron-dense material with spherical multivacuolated fibrillar bodies of the same electron density as the nucleolus precursor bodies. At 17 hr, similar compact spherical bodies, now presenting a single large vacuole, were observed on some occasions, while in other zygotes the morphology remained unchanged throughout the rest of the S and G2-phases. © 1996 Wiley-Liss, Inc.     

Ultrastructural aspects of wheat straw degradation by Phanerochaete chrysosporium and Trametes versicolor

The ultrastructural patterns characterizing wheat straw degradation by the ligninolytic fungi Phanerochaete chrysosporium and Trametes versicolor were studied. During fungal attack, the less lignified tissues were degraded first, whereas the xylematic and sclerenchymatic fibers underwent a delayed attack. In straw samples degraded by T. versicolor, partial delignification, defibrillation and swelling of cell walls, often causing separation between primary and secondary walls, were observed. By contrast, the formation of erosions and fissures, with minor lignin removal, characterized the attack to the cell wall by P. chrysosporium. At an advanced stage of decay, KMnO₄ staining demonstrated abundant electron-dense material around hyphae and in the proximity of the cell-wall surface. In the case of P. chrysosporium, spherical black bodies were found in the erosions and fissures produced during fungal attack.     

On the existence in vivo of „Herring bodies“ and granules in the interstitial colloid of the neurohypophysis

Summary The paper deals with the question, whether the Herring bodies and the granular structures formed by the so-called Gomori-substance in the neurohypophysis are artefacts or not. It is shown that the Gomori-substance and also its globular and granular structures are present in paraffin sections of the gland prepared according to the freezing-drying technique of Altmann-Gersh. All Gomori-substance is washed away, however, if the sections are treated with toluene and alcohol. If, on the contrary, the sections are treated with Bouin before the paraffin is removed, the Gomori-substance is precipitated and stands the treatment with alcohol. After staining with Gomori's haematoxylin and phloxine such preparations give the same picture of the Gomori-substance as common, wet-fixed Bouin preparations, provided the fixation has been carefully made. This strongly suggests that the classical opinion of the Gomori-substance as a granular matter which occasionally forms larger globulets (Herring bodies) truly reflects the conditions in the living tissue. Gersh and Tarr, who used the common technique when making their freezing-drying preparations, apparently lost the Gomori-substance when they treated the sections with alcohol. Their statement that the Herring bodies and other granular matter in the neurohypophyseal interstitia are artefacts is therefore to be abandoned.     

An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.     

The proteoglycan skeleton of the Kurloff body as evidenced by Cuprolinic Blue staining

Summary This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosome organelle, metachromatic towards Toluidine Blue, of a blood cell unique to the guinea pig and called the Kurloff cell. Splenic Kurloff cell from oestrogen-treated guinea pig cells were examined after staining with Cuprolinic Blue, a cationic phthalocyanine-like dye, in the presence of MgCl₂ in a critical electrolyte concentration method. Better results were obtained when the fixation-staining by the glutaraldehyde Cuprinolinic Blue MgCl₂ mixture was preceded by a glutaraldehyde pre-fixation. On light microscopy, Kurloff bodies generally exhibited an overall pink and glassy metachromasia, sometimes with additional darker metachromatic small dots at their peripheries. At the ultrastructural level, the metachromatic central matrix of the Kurloff body usually exhibited, as a major feature, a typical network pattern of ribbon-like or stellate electron-dense precipitates suggesting the presence of a skeleton of Cuprolinic Blue-reactive filamentous structures. Taking into account their high anionicity (as shown by the stability of the dye binding in the presence of 0.3 m MgCl₂) and their susceptibility to chondroitinase ABC, these anionic structures were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell.     

Light and electron microscopic study of <Emphasis Type="Italic">Pelomyxa stagnalis</Emphasis> sp. n. (Archamoebae,pelobiontida)

The structure of a new pelomyxa species was investigated on the level fo light and electron microscopy. The length of locomotive forms of Pelomyxa stagnalis reaches 800 μm. The thin layer of amorphous glycocalyx is located on the cell surface. Numerous nonfunctioning flagellae are revealed predominantly in the uroidal zone. The axoneme has a nonstable set of microtubules. No additional structures are present in the transition zone. The length of P. stagnalis flagella kinetosomes does not exceed 150 nm. Fifteen to twenty microtubules extend from the side surface of each kinetosome at a small angle to the cell surface. One of main components of the P. stagnalis cytoplasm are structural vacuoles. Glycogen bodies in cells are surrounded by flattened ER cisterns, which are often filled with electron-dense material. Cells of P. stagnalis were found to contain two species of prokaryote endobionts that differ in the peculiarities of their fine structure. The number of nuclei in cells of the P. stagnalis adult individuals can reach 50 or more. The nuclei are surrounded by a bilayer envelope formed by the multilaminar layer and by the outer layer composed of vesicles often filled with an electron-dense material. The nucleolus is usually single and is located in the center of the nucleus. In nuclei, predominantly in connection with nucleoli, bodies are formed that are formed by interlacing electron-dense strands.     

Amorphophallus: New insights into pollen morphology and the chemical nature of the pollen wall

In pollen characters, Amorphophallus is one of the most diverse genera in the Araceae. The present work is a critical survey of contradicting reports on the impact of acetolysis treatment on Amorphophallus pollen, on the chemical nature of the outer pollen wall layer and of electron-dense (dark) granules found within it. Furthermore, we wanted to clarify the pollen polarity and to test conclusions based on different preparation techniques. Pollen morphology of 25 species is investigated by light microscopy, scanning electron microscopy and transmission electron microscopy. Our results show that Amorphophallus pollen is not resistant to acetolysis treatment. The use of different transmission electron microscopy staining methods proved the polysaccharide nature of the outer pollen wall layer and of the granules within it. Moreover, an additional thin surface layer was found in all investigated species. Microspores in early and late tetrad stages show that the less convex side of the microspore is the proximal face and the more convex side the distal face. The extrusion of pollen in strands is illustrated for the first time by light microscopy and scanning electron microscopy. Furthermore, observations of pollen in water showed that in some of the investigated species the pollen wall is shed immediately before pollen tube formation.     

Structure and Elemental Composition of the Cyst Wall of Echinosphaerium nucleofilum Barrett (Heliozoea,Actinophryida)1

The structure of the cyst wall of the heliozoon Echinosphaerium nucleofilum has been investigated using light microscopy, scanning and transmission electron microscopy, and X-ray microanalysis. The cyst wall is a composite structure of seven or eight layers. These are: an enveloping gelatinous layer; a layer of siliceous spheroidal bodies; an electron-dense supporting membrane; a broad electron-lucent zone; an electron-dense layer; a layer of helicoidally packed material; and one or two layers with a granular appearance lying next to the plasma membrane of the encysted organism. The structure of the cyst wall closely resembles that of Actinophrys sol, confirming the close relationship of these actinophryid heliozoa while emphasizing their distinctiveness from other amoeboid protista.     

Structure, morphology, and composition of silicon biocomposites in the palm tree Syagrus coronata (Mart.) Becc.

Summary.  Syagrus coronata is an economically important palm tree grown as an ornament, for the oil extracted from its seeds, and the wax from its leaves which has several applications in industry. Silicon biocomposites were analyzed in leaves of S. coronata. Silica bodies were found as extracellular silica masses between the hypodermal-layer cell walls and in granules present in the vacuoles of palisade cells. Scanning electron microscopy of the hypodermal layer of cells showed a collection of spherical bodies embedded in enveloping cavities that outlined the general structure of the bodies. Globular subunits with sharp edges formed the spherical bodies that ranged from 6 to 10 μm in diameter (average, 7.8 μm). X-ray microanalysis detected only silicon and oxygen homogeneously distributed throughout the bodies. Vacuoles of palisade cells contained a large number of granules ranging from 20 nm to 1.2 μm in size (average, 300 nm). Transmission electron microscopy associated with electron spectroscopic imaging and electron energy loss spectroscopy were used to determine the elemental composition of the granules. Vacuolar granules were amorphous and composed of silicon and oxygen, suggesting they consist of amorphous silica biominerals. No nitrogen, indicative of organic matter, was detected in the granules. Received November 26, 2001; accepted July 1, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saude, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.     

Influence of plastic embedding media on staining and morphology of lipid bodies

Summary The following light microscopy staining techniques were applied to plastic embedded tissues: Toluidine Blue, Sudan III, Sudan Black and Nile Blue sulphate. All these procedures stain the lipid bodies; however the Sudan III appears to be the most suitable.The ultramicroscopic appearance of lipid bodies of tissues embedded in Epon, Araldite and Vestopal W is presented. The effects of dehydration as well as of different electron microscopic staining techniques is also investigated.This work was supported by Grants of Consiglio Nazionale delle Ricerche of Italy: no. 115/1151/0-1247, no. 115/0815/0-1365.The excellent technical assistance of Miss Hermina Spiele is gratefully acknowledge.