Cytochemical localization of mercury in Saccharomyces cerevisiae treated with mercuric chloride

We describe a cytochemical method for localizing mercury at the electron microscopic level in the yeast Saccharomyces cerevisiae. After addition of a lethal concentration of mercuric chloride to growing yeast cells, mercury was associated with the cell wall and cytoplasmic membrane. Little or no mercury was present in the cytoplasm. Electron probe X-ray microanalysis (EPMA) confirmed that the cytochemical reaction, visualized as mercury-silver complexes, was localized in dense bodies consisting of a core of mercury sulfide polymers surrounded by a shell of silver atoms.     

Cytochemical Localization of Urease in a Rumen Staphylococcus sp. by Electron Microscopy

We describe a technique whereby intracellular urease activity can be localized by transmission electron microscopy. The ammonia produced from the enzymatic hydrolysis of urea is first precipitated with sodium tetraphenylboron and then replaced with silver to produce electron-dense silver tetraphenylboron. This direct reaction product deposition procedure was used to demonstrate the presence of membrane-bound urease of Staphylococcus sp. H3-22, a gram-positive ruminal bacterium.     

ELECTRON MICROSCOPY OF THE AMMONIACAL SILVER REACTION FOR HISTONES IN THE ERYTHROPOIETIC CELLS OF THE CHICK

The product of the postformalin ammoniacal silver reaction, which has been claimed to distinguish lysine-rich from arginine-rich histones with the light microscope on the basis of a color difference, was examined in developing erythroid cells of chick bone marrow with the electron microscope. Stem cells and early erythroblasts exhibit no, or little, ammoniacal silver reaction product, while small basophilic erythroblasts, polychromatophilic erythrocytes, and reticulocytes exhibit an increasing amount of reaction product as maturation proceeds. The reaction product is in the form of discrete electron-opaque particles associated with heterochromatin. The ammoniacal silver reaction in the erythroid cell series is interpreted as reflecting either the accumulation of newly synthesized arginine-rich histones or changes in the availability of reactive sites in preformed histones.     

Green synthesis of silver nanoparticles using Rhodobacter Sphaeroides

The use of microorganisms in the synthesis of nanoparticles emerges as an eco-friendly and exciting approach. In this study, silver nanoparticles were successfully synthesized from AgNO₃ by reduction of aqueous Ag⁺ ions with the cell filtrate of Rhodobacter sphaeroides. Nanoparticles were characterized by means of UV–vis absorption spectroscopy, X-Ray Diffraction (XRD), transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). Crystalline nature of the nanoparticles in the fcc structure are confirmed by the peaks in the XRD pattern corresponding to (111), (200), (220) and (311) planes, bright circular spots in the selected are a electron diffraction (SAED) and clear lattice fringes in the high-resolution TEM image. Also, the size of silver nanoparticles was controlled by the specific activity of nitrate reductase in the cell filtrate.     

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3′-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.     

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.     

Metal compound intensification of the electron-density of diaminobenzidine

Diaminobenzidine (DAB), commonly used in immunocytochemistry as the substrate for peroxidase, has a low electron density. DAB has a known affinity for the salts of some metals and therefore an examination of the ability of six metal compounds (including osmium tetroxide) to increase the electron density associated with DAB deposits has been undertaken. Ultra-thin sections of unosmicated rat pituitary gland, embedded in L. R. White resin, were immunostained by a hapten sandwich immunoperoxidase method, using antibodies to ACTH and TSH. The unintensified electron density of the DAB polymer reaction product on the specific endocrine granules was compared with the electron density resulting from the use of each of the six metal compounds. Lead and silver nitrate gave unsatisfactory results, while phosphotungstic acid and uranyl acetate produced a limited increase in specific electron density under the conditions used. Gold chloride was found to give the highest electron density to the specific endocrine granules, followed closely by osmium tetroxide. Background staining was greater when osmium was used. We conclude that several metal compounds may be used to intensify the electron density of DAB, but of the ones tested, gold chloride, which is safer, more stable, and cheaper than osmium tetroxide, was clearly the best. This approach not only increases the electron density of the DAB reaction product, but allows of the possibility of quantitation using energy dispersive X-ray analysis.     

Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii.

A lysosomal system was demonstrated in hyphal tip cells of Sclerotium rolfsii by light and electron microscopy observations of the sites of acid phosphatase activity visualized by a modified Gomori lead nitrate method. The cytochemical reaction product was found to be present in numerous vacuoles, each aout 0.5 mum in diameter, which were seen as chains of spheres when viewed with the light microscope. They usually did not occur in the first 30 to 40 mum of the hyphal tip cell, but were concentrated in a zone extending from 30 to 200 mum from the hyphal apex. As shown by the electron microscope, the vacuoles were sometimes interconnected by narrow channels. Acid phosphatase reaction product was also occasionally localized in vacuoles of the older hyphal cells, but never in apical vesicles, lipid bodies, or microbodies. It is proposed that this vacuolar system may orginate from the endoplasmic reticulum.     

Argyrophilic proteins in Dinoflagellates: An ultrastructural,cytochemical and immunocytochemical study

Summary— Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR-like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates (Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non-synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo-granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reaction's temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti-B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co-locating with end-products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23-like protein is probably a DNA partner.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Cytochemical localization of high-affinity Ca2(+)-ATPase activity in synaptic terminals

High-affinity Ca2(+)-ATPase activity in the optic tectum of the brain of cichlid fish was cytochemically localized using cerium ions to precipitate phosphate. Activation of the enzyme with micromolar instead of millimolar calcium concentrations (i.e., physiological cytoplasmic instead of extracellular concentrations) resulted in intracellular localization of reaction product attached to the cytoplasmic side of plasma membranes and to synaptic vesicles. The plasmalemmal enzyme activity was concentrated in synaptic regions. Synaptic vesicles in some terminals exhibited high amounts of ATPase activity, whereas others were free of reaction product. By use of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) techniques, even small amounts of cerium-containing precipitates could be analyzed and precisely localized. The cytochemical observations are in good agreement with biochemical findings and therefore indicate that the calcium pump of neuronal plasma membranes can be successfully localized.     

The ultrastructural effect and subcellular localization of mercuric chloride and methylmercuric chloride in insect cells (Aedes albopictusC6/36)

Abstract.The ultrastructural effects of mercuric chloride (Hg) and methylmercuric chloride (MeHg) were studied in theAedes albopictusC6/36 cell line. Both metal salts caused nuclear indentations, chromatin clumping and proliferation of the nucleolus. The mitochondria became pleomorphous. An increase of both free and membrane-bound ribosomes, swelling of the rough endoplasmic reticulum caused by accumulated protein and the appearance of well developed Golgi stacks all indicated the activation of protein synthesis. The activation was probably a cellular response to general stress, and the synthesized proteins may be members of the heat shock protein family. Apart from these common ultrastructural features, Hg-treated cells showed typical clusters of small electron-lucent vacuoles near the Golgi stacks. In cells exposed to MeHg, cytoplasmic tube-like structures were often observed and the disorganization of the organelles together with the appearance of blebs suggested disruption of the microtubules. Mercury accumulation was localized by an autometallographical silver staining technique both at the light and electron microscopic level; silver deposits were quantified by image analysis. For both Hg- and MeHg-treated cells, the degree of silver staining increased rapidly with increasing exposure time, but a considerable heterogeneity within the cell population was found. Lysosomes proved to be the major mercury storage sites in theAedescells and silver deposits could already be found after 30 min of Hg treatment. At sublethal concentrations, Hg inhibited the lysosomal marker enzyme acid phosphatase to some extent. For MeHg, no effect on this enzyme was found.     

Electron-microscopic-histochemical demonstration of succinic-dehydrogenase activity in root cells of yellow lupine

Summary Succinic dehydrogenase (SDH) activity was demonstrated in unfixed root segments from Lupinus luteus at the ultrastructural level by the use of ferricyanide as electron acceptor. The specificity of the reaction was proven by malonate inhibition. The reaction product was found to be localized in the mitochondria and to a lesser extent on the membranes of plastids. Different mitochondria of the same cell often showed different intensity of the staining reaction. Different cells of the same tissue exhibited varying degrees of enzyme activity. An increase was found in the number of cells exhibiting the SDH reaction, as well as in the intensity of the reaction itself, from the meristematic zone of the root to the more differentiated regions.     

Silk I structure in Bombyx mori silk foams.

Single crystals of Bombyx mori silk fibroin in the metastable silk I polymorph have been produced using a new foaming technique. Foams of silk protein are generated by bubbling pure nitrogen gas through an aqueous solution of regenerated silk fibroin. The foamed material is collected, dried, and then sonicated to yield individual crystals which were examined using transmission electron microscopy and electron diffraction. It is found that slightly acidic conditions in the solution from which the foam was generated favor the formation of silk II while neutral to slightly basic solutions favor silk I formation. More dilute solutions favor the formation of silk II while more concentrated solutions (about 7 wt.% or greater) favor the formation of silk I. X-ray powder diffraction patterns from the dried silk I foams displayed features highly indicative of silk I. We also report the first single crystal electron diffraction patterns of silk I. These patterns indicate a large unit cell, possibly 22.66 x 5.70 x 20.82 A. with six chains of six residues, Gly-Ala-Gly-Ala-Gly-Ser. Although we have not fully characterized this complex structure it appears that the chain is nearly fully extended and thus our data is consistent with models possessing general features similar to those proposed by Fossey SA, Nemethy G, Gibson KD, Scheraga HA. (Biopolymers 1991;31:1529-1541).     

Fruit extract capped colloidal silver nanoparticles and their application in reduction of methylene blue dye

Green synthesis of silver nanoparticles (AgNPs) has become a promising environmentally benign synthetic route in nanoscience and nanotechnology during recent years. In the present work, we have developed an environment-friendly and low-cost method for synthesis of silver nanoparticles from silver nitrate using aqueous fruit extract of Dillenia indica. The as-synthesized nanoparticles were characterized by UV-Vis spectrophotometer, transmission electron microscopy (TEM) and X-ray diffraction (XRD). FTIR study was performed to know the interaction of bio-molecules present in the fruit extract with AgNPs. The catalytic application of the as-synthesized AgNPs was demonstrated against degradation of methylene blue (MB) in aqueous system. The absorption spectra of colloidal suspension of AgNPs showed characteristic surface plasmon resonance (SPR) band centred at a wavelength of 416?nm. TEM image showed that the AgNPs were almost spherical in shape having an average diameter of 10.78?±?.48?nm. XRD pattern and selected area electron diffraction (SAED) pattern with bright spots signify the crystalline nature of nanoparticles. The fruit extract-capped AgNPs was highly stable and have showed the effective catalytic activity in reduction of MB dye.     

Subcellular localization of cadmium in the root cells of<Emphasis Type="Italic">Allium cepa</Emphasis> by electron energy loss spectroscopy and cytochemistry

The ultrastructural investigation of the root cells ofAllium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.     

Ultrastructural localization of Lucifer Yellow and endocytosis in plant cells

Summary The fluorescent dye Lucifer Yellow CH (LYCH) was localized at the ultrastructural level with a precipitation method using barium chloride. Applying this technique, endocytosis of LYCH was examined in the nutrient absorptive trichomes of a carnivorous bromeliad. After a two hour incubation, the electron dense reaction product was localized in the membrane compartments of the endocytotic system. These structures included coated regions of the plasma membrane, coated and smooth vesicles, dictyosomes, partially coated reticulum, and smooth endoplasmic reticulum. This procedure demonstrates for the first time at the ultrastructural level endocytosis in whole plant cells, using a non-toxic compound.Abbreviations ER endoplasmic reticulum - BaCl₂ barium chloride - LYCH Lucifer Yellow CH - PCR partially coated reticulum     

Chromosomal localization of genes by scanning electron microscopy using in situ hybridization with biotinylated probes: Y chromosome repetitive sequences

Summary The feasibility of using scanning electron microscopy (SEM) to identify the position of specific DNA sequences was examined using a Y chromosome specific probe (pHY2.1). Tests were carried out on chromosome spreads hybridizedin situ with biotinylated pHY2.1. Chromosomal sites of hybridization of the probe were localized by an indirect immunohistochemical procedure which resulted in a gold product which could be amplified by silver precipitation. In the SEM, the specific location of the probe was easily identified due to the enhanced signal produced by the gold—silver complex. The probe was localized both on the long arm of the Y chromosome and within interphase nuclei. It was found that SEM was more sensitive than light microscopy since the probe could be identified without silver amplification. With refinements to the technique, SEM could provide a useful method for high resolution localizing of unique DNA sequences (i.e. single copy genes).