Characterization of a Potato Starch-digesting Bacterium and Its Production of Amylase

A bacterium which can utilize potato starch granules as sole carbon source was isolated and identified as Bacillus circulans from its physiological and biochemical properties. Scanning electron microscopic observation of potato starch granules recovered from the culture broth revealed that granules were degraded gradually from their surface resulting in elongated granules with layered structures on their surface. This bacterium produced extracellular amylase which can digest potato starch granules in vitro. The amylase has a unique property in that it produces only maltohexaose from gelatinized starch in the early stage of the reaction. For the production of this amylase potato starch was found to be most effective while soluble sugars including gelatinized starch and maltose had little effect.     

Bacterial and Chemical Iron Oxide Deposition in a Shallow Bay on Palaea Kameni,Santorini, Greece: Microscopy,Electron Probe Microanalysis,and Photometry of in situ Experiments

The present study was aimed at determining whether microorganisms mediate iron oxide deposition in a shallow bay (Iron Bay) on the still volcanically active island of Palaea Kameni, Santorini, Greece, north of Crete in the Aegean Sea. The results of in situ experiments showed that iron oxide deposition in this marine environment occurs at an exceptionally high rate. It was also found that deposition in the central part of the bay proceeded within specific vertical zones in the water column, showing that the bay may be regarded as a stratified system. Using light and scanning electron microscopic methods, including electron probe microanalysis, it was shown that deposition (binding and oxidation) of Fe(II) was mediated by bacteriological processes, which differed in intensity depending on their specific location in the bay. The most intensive iron oxidations and depositions were found in the hot volcanic springs at the toe of the bay, where rates more than 60 times those found in freshwater iron-oxidizing environments were measured. Bacteria that excreted extracellular slime (EPS), which apparently catalysed the process and itself became encrusted with tiny globules of hydrated iron(III) oxide, were responsible for this extremly high rate of iron deposition. The same process of oxidation catalysed by bacterial EPS was also responsible for iron oxide deposition in the main oxidation zone in the central part of the bay. Thus, precipitation of Fe(II) in Iron Bay is not, as was previously assumed, a purely chemical, Eh- and pH-dependent process, but results from the interaction of water chemistry with a high level of bacterial activity, especially surface reactions on bacterial excretion products.     

Fine Structure Observations of Phagotrophic Activity By Plasmodia of Physarum Polycephalum

ABSTRACT. Scanning electron microscopic observations of feeding plasmodia show three characteristic features: 1) extension of multilobed pseudopodia protruding from the leading edge of the plasmodium as it advances onto the surface of a food particle, 2) confluence of the lobes to form a sheath-like pseudopodium attached to the surface of the food particle, and 3) protrusion of small nodules with thin lamellar projections from the leading edge of the plasmodium. Sections through freeze-dried preparations of the feeding plasmodium exhibit a highly convoluted under surface in contact with loosened starch grains that appear to be released by extracellular digestion. the cytoplasm, viewed by transmission electron microscopy, contains branched, internally penetrating canals (ca. 2 μm wide) enclosing engulfed starch grains. Starch grains in the deeper part of the canals are more electron dense and appear to be digested. Micropseudopodia (70-80 nm dia.), projecting from the surface of the canals, protrude toward and into the ingested starch grains. Digestive marker enzyme (acid phosphatase) activity was detected cytochemically in food particles penetrated by micropseu-dopodia indicating a digestive role for these structures not reported previously.     

X-ray microanalysis and electron microscopy of platinum complex in the epithelium of proximal renal tubules of the cisplatin administered rabbits

The initial phase of endocytosis of cisplatin, an anti-tumor platinum agent, into epithelial cells of the proximal renal tubules of rabbits was studied using an X-ray microanalyser at the electron microscopic level. After one to 11 intravenous injections of cisplatin (1 mg/kg/daily), each rabbit was sacrificed with overdose of pentobarbital and small pieces of renal cortex were fixed with 4% glutaraldehyde and 2% osmium tetroxide. After binding on the surface of brush border, dense substance was taken up in endocytic vacuoles that proceeded into the center of epithelial cells leaving empty or scanty vacuoles in apical area. Both platinum and iron were detected in such intracellular dense substance. This shows the transcellular pathways of platinum complex. On the other hand, intercellular pathways were not found in this experiment.     

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.     

An electron microscopic study of surface polysaccharides in Bacteroides

The electron microscopic appearance of the cell surface of Bacteroides strains and Klebsiella pneumoniae stained with ruthenium red or colloidal iron is described. The effect of polymyxin B (PMB) was also registered. It was found that all Bacteroides strains have a polysaccharide lined 'micro-capsule' external to the outer membrane which could aggregate and form blebs. The blebs so formed were distinct from other types of bleb formed in Klebsiella involving the outer membrane and induced by PMB. Such types of PMB alterations were not induced in Bacteroides.     

Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

Histochemical study on the bile duct system of normal Mongolian gerbils

The bile duct system of normal Mongolian gerbils was examined histochemically. The luminal surface membrane and apical cytoplasm of the biliary and gallbladder epithelial cells were stained with periodic acid-Schiff (PAS), alcian blue, pH 2.5 (AB) and high iron diamine (HID)-AB, and many epithelial cells of the common bile duct and gallbladder had weakly PAS-positive granular material in their supranuclear cytoplasm. Lectin-histochemically, these cells had binding sites to Concanavalia ensiformis (ConA), Dolichos biflorus (DBA), Glycine max (SBA), Ulex europeas-I (UEA-I), and Triticum vulgaris (WGA). On the other hand, the periductal glandular epithelial cells were not stained by any histochemical stainings. In addition to these light microscopic findings, the electron microscopic findings based on the periodic acid-silver methenamine method and avidin-biotin colloidal gold method for DBA and WGA suggested that the biliary and gallbladder epithelial cells of Mongolian gerbils secreted mucin with terminal sialic and sulfonic acid residues and that the lectin binding activity of mucin secreted from these cells was similar to that of mucin secreted from the periductal glandular epithelial cells of mice and rats.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

Immunohistochemical detection of dysbindin at the astroglial endfeet around the capillaries of mouse brain

Dysbindin was first identified by the yeast two hybrid assay as a binding partner of dystrobrevin which is a cytoplasmic member of dystrophin glycoprotein complex. Immunolocalization of dystrobrevin in the astrocyte endfeet and endothelial cells in the rat cerebellum was reported. Therefore, we were interested in the expression and localization of dystrobrevin binding protein dysbindin in the mouse brain capillary wall and its surrounding astroglial endfeet. We examined whether the dysbindin expression is present in astroglial endfeet and/or capillary endothelial cells at light and electron microscopic levels. Using brain samples from five normal mice (C57BL/6ScSn), we prepared the anti-dysbindin antibody stained brain samples with immunoperoxidase method at light microscopic level and with immunogold method at ultrastructural level. Immunohistochemistry showed that dysbindin was located in the brain capillary at light microscopic level. Immunogold electron microscopy revealed that dysbindin signal was observed at the inside surface of plasma membrane of glial endfeet which surrounded the brain capillary endothelial cells and pericytes.     

Fermentation RS3 derived from sago and rice starch with Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629

Resistant starch type 3 (RS3) is retrograded starch which is not digested by human starch degrading enzyme, and will thus undergo bacterial degradation in the colon. The main fermentation products are the Short Chain Fatty Acid (SCFA): acetate, propionate and butyrate. SCFA has significant benefit impact on the metabolism of the host. The objectives of this research were to study the SCFA profile produced by colonic butyrate producing bacteria grown in medium containing RS3. RS3 was made from sago or rice starch treated with amylase, pullulanase and the combination of amylase and pullulanase. Fermentation study was performed by using Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629, which has been identified as capable of degradation of starch residue and also regarded as beneficial bacteria. Experimental result revealed that enzyme hydrolysis of retrograded sago or rice starch was beneficial to RS formation. RS3 derived from sago contained higher RS (31-38%) than those derived from rice starch (21-26%). This study indicated that C. butyricum BCC B2571 produced acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1, when the medium was supplemented with RSSA at concentration 1%. In the medium containing similar substrate, E. rectale DSM 17629 produced acetate, propionate and butyrate at molar ratio of 1.7 : 1 : 1.2. High levels of acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1.1 was also produced by E. rectale DSM 17629 in medium supplemented with RSSP at concentration 1%. The results showed that both bacteria responded differently to the RS3 supplementation. Such result provided insight into the possibility of designing RS3 as prebiotic with featured regarding SCFA released in the human colon with potential health implication.     

Microscopic analysis and seasonality of gemma production in the freshwater red alga Hildenbrandia angolensis (Hildenbrandiales, Rhodophyta)

The development and release of the unique vegetative propagules of the freshwater encrusting alga Hildenbrandia angolensis Welwitsch ex West et West, gemmae, were studied using several different microscopic and histochemical techniques. In addition, the seasonality of gemma production was monitored bimonthly over a 12‐month period in two spring‐fed streams in Texas, USA. Gemmae differentiate within the thallus and are subsequently released from the surface of the crust. Release of the gemmae most likely occurs by digestion of surrounding cells, as suggested by the presence of starch granules and lipid globules in the region between the released gemma and the thallus. The initial separation of the gemmae from the thallus occurs from the sides of the gemma or the bottom, or possibly simultaneously. Contrary to previous studies, we have observed that gemma production occurs endogenously within the thallus of freshwater Hildenbrandia, rather than on the surface of the crust in raised structures. Histochemical tests and electron microscopic examination indicate that the cells of the gemmae contain a large amount of floridean starch. The starch granules frequently form rings surrounding the nuclei of both gemma and thallus cells; a feature infrequently reported for florideophyte red algae. Our seasonality investigations indicate that large fluctuations in gemma production occur over 1 year, but at least some gemma production continues year‐round in the streams examined.     

Biofilm-specific cross-species induction of antimicrobial compounds in bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10(T) and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10(T) and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Effects of storage temperature and sucrose on bulblet growth, starch and protein contents in in vitro cultures of Hyacinthus orientalis

The scale segments of the bulblets of Hyacinthus orientalis L. cv. Anna Marie were examined to improve their growth and development with cold-pretreatment and sucrose. The cold-pretreated (4 °C for 4 months) segments showed higher growth and better development of the bulblets on medium without sucrose than ones stored at 20 °C. A rapid decrease in starch content of bulb pieces was found during the first 2 weeks in all cultures and thereafter the content decreased gradually. A scanning electron microscopic observation during the bulblet growth and development showed a gradual decreasing trend of the starch granules from 2 to 16 weeks of the cultures. SDS-PAGE electrophoresis revealed the presence of a characteristic polypeptide of approximately 45 kD, which is assumed to be a major storage protein in the bulblets.     

Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.     

rRNA sequence-based scanning electron microscopic detection of bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

High nitrate removal by starch‐stabilized Fe0 nanoparticles in aqueous solution in a controlled system

This study was conducted to investigate biodenitrification efficiency with starch‐stabilized nano zero valent iron (S‐nZVI) as the additional electron donor in the presence of S₂O₃ in aqueous solutions, under anaerobic conditions. The main challenge for nZVI application is their tendency to agglomeration, thereby resulting in loss of reactivity that necessitates the use of stabilizers to improve their stability. In this study, S‐nZVI was synthesized by chemical reduction method with starch as a stabilizer. The synthesized nanoparticles were characterized by TEM, XRD, and FTIR. Transmission electron microscopy (TEM) image shows S‐nZVI has a size in the range of 5–27.5 nanometer. Temperature and S‐nZVI concentration were the important factors affecting nitrate removal. Biodenitrification increased at 35°C and 500 mg/L of S‐nZVI, in these conditions, biodenitrification efficiency increased from 40.45 to 78.84%. Experimental results suggested that biodenitrification increased by decreasing initial nitrate concentration. In the bioreactor biodenitrification rate was 94.07% in the presence of S‐nZVI. This study indicated that, Fe²⁺ could be used as the only electron donor or as the additional electron donor in the presence of S₂O₃ to increase denitrification efficiency.     

Role of extracellular transaldolase from Bifidobacterium bifidum in mucin adhesion and aggregation

The ability of bifidobacteria to establish in the intestine of mammals is among the main factors considered to be important for achieving probiotic effects. The role of surface molecules from Bifidobacterium bifidum taxon in mucin adhesion capability and the aggregation phenotype of this bacterial species was analyzed. Adhesion to the human intestinal cell line HT29 was determined for a collection of 12 B. bifidum strains. In four of them-B. bifidum LMG13195, DSM20456, DSM20239, and A8-the involvement of surface-exposed macromolecules in the aggregation phenomenon was determined. The aggregation of B. bifidum A8 and DSM20456 was abolished after treatment with proteinase K, this effect being more pronounced for the strain A8. Furthermore, a mucin binding assay of B. bifidum A8 surface proteins showed a high adhesive capability for its transaldolase (Tal). The localization of this enzyme on the surface of B. bifidum A8 was unequivocally demonstrated by immunogold electron microscopy experiments. The gene encoding Tal from B. bifidum A8 was expressed in Lactococcus lactis, and the protein was purified to homogeneity. The pure protein was able to restore the autoaggregation phenotype of proteinase K-treated B. bifidum A8 cells. A recombinant L. lactis strain, engineered to secrete Tal, displayed a mucin- binding level more than three times higher than the strain not producing the transaldolase. These findings suggest that Tal, when exposed on the cell surface of B. bifidum, could act as an important colonization factor favoring its establishment in the gut.     

Surface iron polymers and hydroxy acids. A model of iron supply in sideramine-free fungi

Biochemical and electron microscopic evidence is presented that sideramine-free fungi form iron hydroxide polymer layers on the cell surface when grown in an iron containing medium.Iron hydroxide polymer formation on the cell surface is completely prevented in sideramine producing strains of Neurospora crassa. After feeding a sideramine-free mutant of Neurospora crassa with ornithine in order to restore the sideramine synthesis the iron hydroxide coat is gradually dissolved.The addition of excess citrate and malate to the incubation medium also prevents iron polymer adsorption, suggesting that hydroxy acids may be involved in iron supply, when sideramine-free organisms are grown in iron containing media.In order to study the interaction between iron hydroxide polymer deposition upon the cell surface and iron chelating acids in Neurospora crassa, the amount and the proportion of excreted acids was studied under various experimental conditions. Gas chromatographic analysis of the acids produced under iron deficient conditions revealed that succinate, malate and citrate were present within the cells in the early growth phase. The acids were sequentially excreted into the medium in the order succinate, malate and citrate. The amount of succinate decreased after 2 days of cultivation, whereas the amount of malate and citrate continually increased. Although citrate was present within the cells from the 1st day, excretion occurred very late, generally after the 3rd day.It is suggested that sideramine-free fungi first adsorb iron as a hydroxide polymer on the cell surface, and that it is gradually solubilized by excreted hydroxy acids such as citrate or malate. Thus high local concentrations of iron chelated by hydroxy acids provide sideramine-free fungi with a continuous iron supply.Abbreviations BSTFA N,O-Bis(trimethylsilyl)-trifluoracetamide - GC Gaschromatography - EGTA Ethylenglykol-bis(2-aminoethylether) N,N-tetraacetic acid - TMS Trimethylsilyl