Biochemical study of the adepidermal granules of Urodela and Anura—Especially on lipids

Summary The lipids of the adepidermal granules of Hynobius tokyoensis and Rana japonica were analysed qualitatively. To extract the lipids from the adepidermal granules, the epidermis of the larvae of both animals were removed from the body. Then the granules were extracted from the surface of the basal lamina by means of wiping with the cotton balls containing the solvent. It was ascertained with the electron microscope whether the adepidermal granules were extracted by the cotton balls or not.By the analysis which was made with thin layer chromatography, the same kind of phospholipids or neutral lipids were detected from the extracts of both R. Japonica and H. tokyoensis. Moreover, four spots which seemed to contain some sialic acids were detected from the both extracts.     

Fine structures on the surface of nuptial pads of male hylid and rhacophorid frogs

Nuptial pads, secondary sexual characteristics of male frogs, develop on the first digit of the hand of Hyla japonica in the family Hylidae and of Rhacophorus schlegelii in the family Rhacophoridae, and on both the first and second digits of the rhacophorids Buergeria buergeri and B. japonica. By scanning electron microscopy (SEM), it was seen that numerous mounds covered the surface of the pads. Mounds were similarly hemispherical in R. schlegelii and B. buergeri and flat in B. japonica. The mounds of H. japonica were irregular in shape and size and some of them stood in rows. Fine columnar protuberances were present all over the surfaces of the mounds in this species. Numerous peg-like protuberances projected radially upward from the top of the mounds in B. buergeri and B. japonica. Irregular-shaped, leaf-like protuberances bearing knobby or rod-shaped apical protrusions were present on the top of the mounds in R. schlegelii. In pads observed by transmission electron microscopy, the outermost epithelial monolayer of the mounds was generally thick, especially at the top, compared to that of the rest of the skin. Epidermal cells in this layer were well keratinized, devoid of organelles, and contained closely packed, fine filaments within a dense matrix. Apical accessory protuberances projecting from the outermost cells were also packed with dense filamentous materials, showing rigid comb-like structures. © 1994 Wiley-Liss, Inc.     

Comparative ultrahistochemistry of the adepidermal granules of Salmo irideus, Lebistis reticulatus and Hynobiuo tokyoensis. Enzyme digestive experiment for the epoxy-embedded sections;.

A comparative ultrahistochemical investigation of the adepidermal granules of Salmo irideus, Lebistis reticulatus and Hynobius tokyoensis was carried out using enzyme digestion methods on epoxy-embedded sections. The granules of S. irideus larvae were decomposed by periodic acid, and digested by lipase without periodic acid pretreatmenetection of the granules. The secondary postosmificated granules were digested by lipase as in S; irideus, but complete decomposition by periodic acid was not observed in this experiment; Both periodic acid and lipase changed the shape of the adepidermal granules of H. tokyoensis to suggest partial digestion, but it appeared that these granules show rather stronger resistance to periodic acid and lipase than those of S. irideus. The granules of H. tokyoensis were completely digested when the sections were treated sequentially with phospholipase C, neuraminidase and lipase.     

Cephalexin-Induced Morphological Alterations in the Surface Structures of Staphylococcus aureus and Escherichia coli Demonstrated by Scanning Electron Microscopy

The scanning electron microscope was used to investigate the alterations in surface morphology of Staphylococcus aureus 209P and Escherichia coli NIH induced by the action of cephalexin known to interfere with cell-wall synthesis. Exposure to cephalexin produced a series of changes on the surface morphology in proportion to the concentrations of cephalexin added. Untreated S. aureus cells had smooth contours. Exposure to 1 μg/ml of cephalexin during the logarithmic phase of growth in S. aureus did not produce any detectable changes. Upon exposure of S. aureus to 5 μ/ml or 10 μg/ml, some cells were larger than normal and showed abnormal cell division-like structures in part. When S. aureus was exposed to 50 μg/ml, cell division was completely inhibited, and no formation of grape-like clusters was observed. Untreated E. coli cells appeared to have smooth and regular contours. E. coli propagated almost normally upon exposure of the organisms to 1 μg/ml of cephalexin. Filamentous structures were observed with the exposure of E. coli to 12.5 μg/ml or 25 μg/ml, but spheroplast-like structures were not observed. Exposure to 100 μg/ml of cephalexin resulted in the formation of marked filamentous cells and spheroplast-like structures having multiple small saccular outpouchings. Scanning electron microscope demonstrated more completely the morphological abnormalities induced by cephalexin.     

Structural differences between the resting eggs of Brachionus plicatilis and Brachionus rotundiformis (Rotifers, Brachionidae): an electron microscopic study

Surface topography and layering of resting eggs of Brachionus plicatilis and B. rotundiformis are described, based on scanning and transmission electron microscope studies. In B. plicatilis, the resting eggs are spherical with smooth wavy ridges on the surface, whereas these ridges are small and condensed in B. rotundiformis. The distribution of pores on the egg surface clearly distinguishes the two species.Ultrastructure of the egg membranes of both species varies greatly with regard to size, shape and sculpturing of each membrane. The alveolar and dense sublayers, which constitute the outer egg membrane (S1) are very thick (10–12 µm) in B. rotundiformis compared to B. plicatilis (4–5 µm). Thus, each species has a characteristic surface and membrane architecture. The functional roles of these membranes, during the resting phase of rotifers, are discussed.     

Scanning electron microscopy of volk absorption in early chick embryos.

Fresh pullet eggs (White Leghorn Strain) were incubated from 6-19 hours. Blastoderms were fixed in situ with aldehyde fixative, post-osmicated in 2% OsO4, dehydrated in acetone, critical-point-dried, mounted ventral side up, coated with palladium-gold wire and observed in a Cambridge Stereoscan S4 scanning electron microscope. Large extracellular yolk granules had a smooth surface and appeared to break up into smaller particles. Similar particles have been observed intracellularly in transmission electron microscopy. Numerous microappendages, mostly ruffles, suggest phagocytosis as a method of absorption of yolk granules into the cells. Absorption of yolk by the cells of the blastoderm involves an initial break up of yolk granules followed by phagocytosis.     

PEARL MILLET (PENNISETUM AMERICANUM (L.) LEEKE) AND GRAIN SORGHUM (SORGHUM BICOLOR (L.) MOENCH) ULTRASTRUCTURE

Ultrastructural features of pearl millet (Pennisetum americanum (L.) Leeke) and grain sorghum (Sorghum bicolor (L.) Moench) caryospses were investigated with thin sections of the dry, mature grain in the transmission electron microscope, and fractured kernels in the scanning electron microscope. The pericarp of those grains is comprised of three distinct layers: epicarp, mesocarp of parenchyma cells, and endocarp of compressed cross and tube cells. Mesocarp cells of grain sorghum contain starch granules embedded in a cytoplasmic matrix. The major constituent of sorghum and millet aleurone cells are aleurone grains (protein bodies) and lipid bodies. Subaleurone cells contain a much higher proportion of protein bodies than starch granules, and the protein bodies are structurally distinct from those in the aleurone. The germ scutellar ultrastructures of the two grains were similar; protein bodies, lipid bodies, epidermal cells and parenchyma cells of the germ are described.     

新疆曲尾藓属10种植物蒴齿和孢子的形态观察

该研究通过光学显微镜(LM)和扫描电镜(SEM)观察了曲尾藓属(Dicranum Hedw.)10种植物的蒴齿和孢子的形态特征。结果表明:(1)曲尾藓属10种植物的孢蒴均为圆柱形。(2)孢子球形、近球形、椭圆形或三角状卵形,颜色为黄色、棕色或黄褐色;近极面向内凹,纹饰为疣状、颗粒状或芽孢状。(3)曲尾藓属10种植物的蒴齿多为披针形,但也有阔披针形,颜色为杏黄色、深红色、褐黄色或红褐色;纹饰为条状或疣状,具穿孔成网状结构,一些种无穿孔成平滑结构,表面具细疣。该研究结果为曲尾藓属属下类群划分提供了分类依据,并为苔藓植物系统分类、演化研究提供基础资料。     

Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.     

Silicon-containing granules of rat liver,kidney and spleen mitochondria

Summary Isolated rat liver mitochondria containing granule aggregates (25–75 nm in diameter) and small (5–10 nm) electron opaque granules were examined by electron probe X-ray microanalysis. The granule aggregates gave an intense Si signal, while the small granules gave both Si and P signals. Isolated mitochondria of rat liver, spleen and kidney, subjected to detergent solubilization and differential centrifugation, produced two granule fractions: (1) a 10,000g fraction consisting predominantly of granule aggregates (25–75 nm) composed of smaller granules (5–10 nm in diameter), and (2) a 10,000–30,000 g fraction of non-aggregated small granules (5–10 nm). Thin sections of isolated granule aggregates gave Si X-ray signals similar to those obtained from in situ granules. In addition S, Cl, Mg, Cr and Fe X-ray signals were observed. Cr occurred only in the large kidney granules, while Fe occurred in both fractions of the spleen and kidney granules. The presence of Si in the granules was confirmed by chemical analysis of the isolated granules and in vivo radiolabeling of the granules with ³¹Si and ⁶⁸Ge. Contamination within the electron microscope was eliminated by a liquid nitrogen anticontamination device.Supported by Grant GM-08229-13-15 from the National Institutes of Health, USPHSWe are grateful to the Perkin-Elmer Corporation and to their Western Branch Manager, Mr. Michael E. Mullen and Microscopist, Mr. Minoru Saito, for use of and assistance with the Hitachi H 500 transmission electron microscope with the scanning attachment, and to the Kevex Corporation for the use of the Kevex X-ray Spectrometer. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and X-ray spectra photographs and Darlene Lum for technical assistance in the laboratory     

Modeling effects of granules on the start-up of anaerobic digestion of dairy wastewater with Langmuir and extended Freundlich equations

The effects of granules-inocula on the start-up of anaerobic reactors treating dairy manure were studied in a batch-fed reactor. The effects of start-up period and ratio of granules to feed were analyzed. Results indicated that the effects of start-up period could be described by Langmuir model, while the Extended Freundlich model could be used to model the effects of ratio of granules to feed on cumulative biogas production. In addition, transmission electron microscopes (TEM) and scanning electron microscope analysis were conducted to elucidate the distribution of microbial population and micro-colonies in granules and manure. From the TEM micrographs analyses, the ratios the Syntrophobacter and methanogens in granule and manure were shown to be 1.57 ± 0.42 and 0.22 ± 0.20, respectively. These results demonstrated that granules-inocula could reduce the period required for onset of biogas by 25%.     

Fine structure of diffused pseudobranchial neurosecretory cells associated with carotid labyrinth in an air‐breathing catfish Clarias batrachus

Fish are known to have branchial chemoreceptors and even extrabranchial chemoreceptors to meet the challenges of aquatic environment. The pseudobranchial neurosecretory system associated with carotid labyrinth (CL) is one such example. CL – a chemosensory organ is well known in amphibians. The homologous structure also exists in fish. Clusters of neurosecretory cells, close to the CL and the first two efferent branchial arteries occur in catfish and a few other groups of teleosts. These cells belong to the pseudobranchial neurosecretory system (PNS). To reveal the ultrastructure of CL and the pseudobranchial neurosecretory cells (PNSCs), environmental scanning electron microscope (ESEM) and transmission electron microscope (TEM) investigations were made in an Asian air‐breathing catfish Clarias batrachus. Under ESEM, the PNS appeared as a mass of cells innervated by nerves and supplied by blood capillaries. The CL appeared to have a network of blood capillaries. The transmission electron microscopic investigations showed pear shaped PNSCs having different sizes of dense cored vesicles (DCVs), numerous mitochondria, nerve varicosities, indicating a secretory function of the cells. The CL shows a close association with PNSCs and smooth muscles. Although the exact function of the CL and associated PNSCs in the biology of fish is far from clear, their morphology suggests they are involved in a stress response such as to hypoxia.     

Classification of Streptomyces Spore Surfaces into Five Groups

Streptomyces spores surfaces have been classified into five groups, smooth, warty, spiny, hairy, and rugose, by examination of carbon replicas of spores with the transmission electron microscope and by direct examination of spores with the scanning electron microscope.     

Fine structure of the egg envelopes of one proturan and two Collembolan genera (apterygota)

The eggs of one proturan and two representatives of Collembola (Heteromurus nitidus and Hypogastrura succinea) were studied by scanning and transmission electron microscopy. The proturan (Eosentomon) eggs are covered by 2 envelopes. The surface of the outer one is equipped with numerous hemispherical projections. The eggs of H. nitidus possess an envelope, which is studded with numerous granules. In H. succinea, eggs are covered with 2 envelopes. The outer is studded with characteristic complex structures. Neither micropyles nor aeropyles were observed in either proturan or collembolan eggs.     

Scanning electron microscopic studies of cilia

Summary A simple method for the preparation of ciliated epithelia for study with the scanning electron microscope is described. Ciliary groups are well preserved and it is possible to discern individual cilia and work out their numbers and orientation. Following scanning electron microscopical study some of the material was prepared for transmission electron microscopy and the ultrastructure of the tissue was found to be surprisingly well preserved. The tracheal epithelium of the rabbit, the olfactory epithelia of the goldfish and the rabbit, and the sensory epithelia in the statocyst of a cephalopod mollusc were examined with the scanning electron microscope to demonstrate the possibilities of the method. Acknowledgements. We would like to thank Professor J. Z. Young for his continued interest and support. The scanning electron microscope was purchased with a grant provided by the Science Research Council to Dr. Boyde, Mr. R. Willis helped in the initial stages of the study, Mr. G. Savage provided help with the goldfish material, Mr. S. Waterman provided much photographic assistance, and Mrs. N. Finney the secretarial assistance.     

The ultrastructure of hemocytes inDactylopius confusus (Cockerell), and the role of granulocytes in the synthesis of cochineal dye

Summary The ultrastructural study of free circulating hemocytes in the adult cochineal scale,Dactylopius confusus (Cockerell), demonstrated five cell types: prohemocytes, typical granulocytes (T-granulocytes), oenocytoids, plasmatocytes, and granulocytes with modified sub-cellular structure to perform a special synthetic and secretory function, which we refer to as modified granulocytes (M-granulocytes). Prohemocytes showed undifferentiated sub-cellular structure of the basic stem cell type (i.e., high cytoplasmic density with numerous ribosomes, centrally located large nucleus with a distinct nucleolus, and poorly developed endoplasmic reticulum). The commonly observed typical granulocytes (T-granulocytes) had several smooth endoplasmic reticulum (SER) with dilated cisternae and many SER-derived membrane bounded granules of different sizes and electron density. Oenocytoids were identified by the presence of many crystals, RER-originated fine secretory granules, and an eccentric nucleus. Plasmatocytes were easily characterized by their variable shapes and irregular outline with pseudopodia-like cytoplasmic extensions, possession of an elongated lobed nucleus, multivesicular bodies, RER-derived membrane bounded, electron-dense, lysosomelike vacuoles, well-developed SER cisternae, and numerous pinocytic and SER-originated vesicles of different sizes along the peripheral region. M-granulocytes comprised the largest proportion of hemocytes in all samples observed. M-granulocytes were distinguished not only by the presence of membrane bounded granules of different sizes and electron density, but by the possession of large nuclei with distinct nucleoli, many mitochondria, and a highly developed network of rough endoplasmic reticulum (RER). M-granulocytes had abundant, rosette-shaped, RER-derived chains of fine secretory granules, which accumulated in the cytoplasm and vacuoles, and were ultimately deposited into the hemolymph by exocytosis. These fine granules gave a positive result with periodic acid-Schiff (PAS) test. Based on RER-synthesized fine secretory granules (M-granulocytes), their ultimate deposition into hemolymph, the red pigmentation of hemolymph, positive PAS histochemical test of these granules, and the high population of these hemocytes, no such cell type has been described in previous studies in insects. The sub-cellular structure of the granulocyte in this insect has been modified to perform a special synthetic and secretory function (i.e., possibly the synthesis of the red pigment found in hemolymph, which has been the source of commercially important cochineal dye).Abbreviations EM electron microscope - ER endoplasmic reticulum - LM light microscopy - MVB multivesicular body - PAS periodic acid-Schiff - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum - SG secretory granules - TEM transmission electron microscopy - UA uranyl acetate     

Structure and colour-production of the blue barbs of Agapornis roseicollis and Cotinga maynana

Summary The spongy structure in medullary cells responsible for the colour of blue barbs in rump feathers of Agapornis roseicollis and back feathers of Cotinga maynana is studied with the scanning and the transmission electron microscope. The Agapornis structure is an irregular three-dimensional network of connected keratin rods which in many places form rings with outer diameters 0.25–0.3 . The air-filled space likewise consists of an irregular network of connected channels. The Cotinga structure consists of spherical cavities fairly evenly distributed in a keratin matrix.Earlier spectrophotometric measurements on the Agapornis structure have shown that the colour production has to be ascribed to the interference of light, not to Rayleigh (Tyndall) scattering as previously held. The optical path lengths corresponding to the outer diameter of the Agapornis rings and to the Cotinga cavities including their horny walls both are c. 0.4 . It is suggested that the colours are produced by the interference of light reflected from the front and rear surfaces of the rings, respectively the spherical cavities.I wish to thank Miss Annelise Nørgaard Jensen, Institute of Historical Geology and Palaeontology, University of Copenhagen, for operating the scanning electron microscope for me.     

Localization of α-galactomannan and of wheat germ agglutinin receptors inSchizosaccharomyces pombe

The location of galactomannan on the surface ofSchizosaccharomyces pombe cells was reexamined by scanning electron microscopy by an indirect but specific method using gold markers. The polysaccharide was found on the cell surface and at the end beginning to grow but not on the wall established by division. Galactomannan was also localized onS. pombe thin sections by transmission electron microscopy using the same method. The polysaccharide was found deposited in two layers in the cell wall, i.e. at the periphery of the wall and near the plasmalemma. The septum was also marked but mainly near the plasmalemma. These results indicated that the polysaccharide is elaborated onto the outside of the wall during extension but not during septum formation. When thin sections ofS. pombe were marked with gold granules labeled with wheat germ agglutinin, marking was found in vacuoles but not in the cell wall. This confirmed thatS. pombe cell wall is devoid of chitin.Non-Standard Abbreviations Au gold colloid - RCA_I Ricinus communis lectin - SEM scanning electron microscopy - TEM transmission electron microscopy - WGA wheat germ agglutinin     

Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii

Cells ofSaccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KCl), fixed in 3% glutaraldehyde fortified with 2 M KCl, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCl. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation inSaccharomyces rouxii is contrasted with otherSaccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic β-fructofuranosidase inS. rouxii is briefly discussed.     

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.