Subcellular localization of chromium and nickel in root cells of Allium cepa by EELS and ESI

The ultrastructural investigation of the root cells of Allium cepa L. exposed to two different concentrations of chromium + nickel (Cr+Ni) (10 micromol/L and 100 micromol/L) revealed that toxic symptoms were induced by increasing heavy metal concentration and treatment time. Several significant ultrastructural changes were caused by 100 micromol/L Cr+Ni - deposition of electron dense material in cell walls; larger vacuolar precipitates surrounded by membranes inside vacuoles; increment of disintegrated organelles and high vacuolization in cytoplasm. The localization of the precipitates in which the metal ions were detected by electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) was investigated. Chromium and nickel were localized in the electron dense precipitates of the root cells exposed to only 100 micromol/L Cr+Ni. None were found in the root cells exposed to 10 micromol/L Cr+Ni. Higher amounts of Cr+Ni were mainly accumulated in the cell walls and vacuoles of the fourth or fifth cortical layer.     

Cometabolism of Cr(VI) by Shewanella oneidensis MR-1 produces cell-associated reduced chromium and inhibits growth

Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR-1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR-1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked. Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI). The 48-h transformation capacity (Tc) was 0.78 mg (15 micromoles) Cr(VI) reduced. [mg protein](-1) for high levels of Cr(VI) added as a single spike. For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 micromoles) Cr(VI) reduced. [mg protein](-1), indicating that it is limited by toxicity at higher concentrations. During denitrification and aerobic growth, MR-1 reduced Cr(VI), with much faster rates under denitrifying conditions. Cr(VI) had no effect on nitrate reduction at 6 microM, was strongly inhibitory at 45 microM, and stopped nitrate reduction above 200 microM. Cr(VI) had no effect on aerobic growth at 60 microM, but severely inhibited growth above 150 microM. A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium. Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron-dense round globules inside cells.     

Isolation and characterization of a chromium-reducing bacterium from a chromated copper arsenate-contaminated site

A Gram-negative bacterium (CRB5) was isolated from a chromium-contaminated site that was capable of reducing hexavalent chromium to an insoluble precipitate, thereby removing this toxic chromium species from solution. Analysis of the 16S rRNA from the isolate revealed that it was a pseudomonad with high similarity to Pseudomaonas synxantha . CRB5 was tolerant to high concentrations of chromate (500 mg l⁻¹) and can reduce Cr(VI) under aerobic and anaerobic conditions. It also exhibited a broad range of reduction efficiencies under minimal nutrient conditions at temperatures between 4°C and 37°C and at pH levels from 4 to 9. As reduction increased, so did total cellular protein, indicating that cell growth was a requirement for reduction. Under low nutrient conditions with CRB5 or when using non-sterile contaminated groundwater from the site, reduction of Cr(VI) was followed by a increase in solution turbidity as a result of the formation of fine-grained Cr(III) precipitates, most probably chromium hydroxide mineral phases such as Cr(OH)₃. Chromium adsorption and precipitation, as observed by transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (TEM/EDS), revealed that the surfaces of the cells were uniformly stained with bound Cr(III) and amorphous precipitates (as determined by selected area electron diffraction; SAED). A mass balance of chromium in a batch bioreactor revealed that up to 30% of the total Cr was as settable precipitates or bound to cells.     

3‐D analysis of bacterial cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches

The formation of cell‐(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2‐D microscopy techniques, the 3‐D and internal structure remain obscure. In this study, we examined the 3‐D structure of cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3‐D microscopy techniques. We obtained 3‐D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4–200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike‐ or platelet‐shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell‐(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell‐(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3‐D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X‐ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe‐precipitates and their association with bacterial cells and EPS.     

Chromium(VI) resistance and removal by <Emphasis Type="Italic">Acinetobacter haemolyticus</Emphasis>

The present work highlighted the studies on Cr(VI) reduction by cells of Acinetobacter haemolyticus (A. haemolyticus). The strain tolerated 90 mg Cr(VI) l⁻¹ in LB broth compared to only 30 mg Cr(VI) l⁻¹ in LB agar. From the FTIR analysis, the Cr(III) species formed was also most likely to form complexes with carboxyl, hydroxyl, and amide groups from the bacteria. A TEM study showed the absence of precipitates on the cell wall region of the bacteria. Instead, microprecipitates were observed in the cytoplasmic region of the cells, suggesting the transportation of Cr(VI) into the cells. Intracellular reduction of Cr(VI) was supported by a reductase test using soluble crude cell-free extracts. The specific reductase activity obtained was 0.52 μg Cr(VI) reduced per mg of protein an hour at pH 7.2 and 37°C. Our results indicated that A. haemolyticus can be used as a promising microorganism for Cr(VI) reduction from industrial wastewaters.     

Extracellular reduction of hexavalent chromium by cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

To characterize the roles of cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 in Cr(VI) reduction, the effects of deleting the mtrC and/or omcA gene on Cr(VI) reduction and the cellular locations of reduced Cr(III) precipitates were investigated. Compared to the rate of reduction of Cr(VI) by the wild type (wt), the deletion of mtrC decreased the initial rate of Cr(VI) reduction by 43.5%, while the deletion of omcA or both mtrC and omcA lowered the rate by 53.4% and 68.9%, respectively. In wt cells, Cr(III) precipitates were detected by transmission electron microscopy in the extracellular matrix between the cells, in association with the outer membrane, and inside the cytoplasm. No extracellular matrix-associated Cr(III) precipitates, however, were found in the cytochrome mutant cell suspension. In mutant cells without either MtrC or OmcA, most Cr(III) precipitates were found in association with the outer membrane, while in mutant cells lacking both MtrC and OmcA, most Cr(III) precipitates were found inside the cytoplasm. Cr(III) precipitates were also detected by scanning election microscopy on the surfaces of the wt and mutants without MtrC or OmcA but not on the mutant cells lacking both MtrC and OmcA, demonstrating that the deletion of mtrC and omcA diminishes the extracellular formation of Cr(III) precipitates. Furthermore, purified MtrC and OmcA reduced Cr(VI) with apparent k(cat) values of 1.2 ± 0.2 (mean ± standard deviation) and 10.2 ± 1 s(-1) and K(m) values of 34.1 ± 4.5 and 41.3 ± 7.9 μM, respectively. Together, these results consistently demonstrate that MtrC and OmcA are the terminal reductases used by S. oneidensis MR-1 for extracellular Cr(VI) reduction where OmcA is a predominant Cr(VI) reductase.     

The effects of hexavalent chromium on thioredoxin reductase and peroxiredoxins in human bronchial epithelial cells

Inhalational exposure to hexavalent chromium (Cr(VI)) compounds (e.g., chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells, where they are reduced to reactive Cr species, which may also contribute to the generation of reactive oxygen species. The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trx’s keep many intracellular proteins reduced, including the peroxiredoxins (Prx’s). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trx’s. Prx oxidation is therefore probably the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR’s) keep the Trx’s largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 was reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trx’s and Prx’s. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trx’s, Prx’s, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance of oxidant insults.     

Hexavalent chromium reduction by bacteria from tannery effluent

Chromium is generated from several industrial processes. It occurs in different oxidation states, but Cr(III) and Cr(VI) are the most common ones. Cr(VI) is a toxic, soluble environmental contaminant. Some bacteria are able to reduce hexavalent chromium to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of considerable interest. An indigenous chromium-reducing bacterial strain, Rb-2, isolated from a tannery water sample, was identified as Ochrobactrum intermedium, on the basis of 16S rRNA gene sequencing. The influence of factors like temperature of incubation, initial concentration of Cr, mobility of bacteria, and different carbon sources were studied to test the ability of the bacterium to reduce Cr(VI) under variable environmental conditions. The ability of the bacterial strain to reduce hexavalent chromium in artificial and industrial sewage water was evaluated. It was observed that the mechanism of resistance to metal was not due to the change in the permeability barrier of the cell membrane, and the enzyme activity was found to be inductive. Intracellular reduction of Cr(VI) was proven by reductase assay using cell-free extract. Scanning electron microscopy revealed chromium precipitates on bacterial cell surfaces, and transmission electron microscopy showed the outer as well as inner distribution of Cr(VI). This bacterial strain can be useful for Cr(VI) detoxification under a wide range of environmental conditions.     

Ultrastructural localization of calcium and Ca(2+)-ATPase activity in gonadotropes and stellate cells of the catfish pituitary

In the pituitary of the African catfish, Clarias gariepinus, calcium precipitates were ultrastructurally visualized with the oxalate-pyroantimonate procedure (OPP). The presence of calcium in these precipitates was validated with several methods, including "Electron Energy Loss Spectrometry" (EELS). In the OPP-treated tissue calcium precipitates were seen in a) non-secretory stellate cells and b) gonadotropic (GTH-) cells. In the latter the amount of precipitate is generally low, but stimulation of the gonadotropin release, either in vivo or in vitro, resulted in a considerable increase. This increase is discussed in relation to the role of calcium as second messenger in the GTH-cells. Ca(2+)-ATPase was exclusively represented in stellate cells and GTH-cells, its strongest activity associated with the plasma membrane and with the membranes of the endoplasmic reticulum. The localization of this enzyme is discussed in relation to its role in the regulation of the intracellular calcium concentration in the GTH-cells. The stellate cells are considered to be involved in the regulation of extracellular calcium concentrations in the pituitary.     

Effects of chromium(VI) and chromium(III) on energy charge and oxygen consumption in rat thymocytes

The cytotoxic effects of chromium compounds in two oxidation states have been studied in rat thymocytes. endogenous nucleotide levels and oxygen consumption were examined as relevant parameters of the physiological state of the cell. Incubation of rat thymocytes with Cr(VI) produced a marked unbalance of endogenous purine nucleotide pool and a parallel decrease in oxygen consumption. A close correlation between the reduction of oxygen consumption and ATP level in rat thymocytes treated with increasing concentrations of Cr(VI) has been found. In rat thymocytes permeabilized with digitonin and in isolated rat liver mitochondria both Cr(VI) and Cr(III) showed, at different range of concentrations, a marked inhibition of maximal oxygen consumption rate (uncoupled respiration). The effects observed were depending on chromium oxidation state and on different mitochondrial sites of substrate oxidation.     

Interference ofSalmonella enteritidis andLactobacillus spp. with IL-8 levels and transepithelial electrical resistance of enterocyte-like caco-2 cells

Caco-2 cells (exhibiting characteristics of mature villus enterocytes) were used to determine bacteria (Salmonella enteritidis causing human gastroenteritis)-intestinal cell interactions. The interference of bacteria with the transepithelial electrical resistance (TEER) of filter-grown Caco-2 cells and the production of IL-8 after exposure of the cells to S. enteritidis 857 and/or Lactobacillus strains (L. gasseri LF221 and L. rhamnosus BGT10) was evaluated. The strain 857 decreased TEER of filter-grown Caco-2 cells; in contrast, lactobacilli had a little or no effect. The effect of S. enteritidis on the TEER decreased if Caco-2 cells were pre-incubated with lactobacilli. This strain induced high levels of IL-8 (which can lead to cell damage). Compared to the IL-8 synthesis after exposure of Caco-2 cells to S. enteritidis 857, simultaneous exposure of Caco-2 cells to S. enteritidis and lactobacilli inhibited the IL-8 synthesis after short recovery periods.     

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.     

Subcellular localization of copper in the root cells of Allium sativum by electron energy loss spectroscopy (EELS)

Ultrastructural investigation of the root cells of Allium sativum L. exposed to three different concentrations of Cu (1, 10 and 100 microM) for 9 days was carried out using transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS). The results presented here indicate that excess Cu induces ultrastructural changes such as strong vacuolization, condensed nuclear chromatin, decreased endoplasmic reticulum (ER) and ribosome and serious plasmolysis. EELS analysis indicated that electron dense granules containing Cu appeared in the cells after Cu treatment. The vacuoles of the root tip cells were the main Cu-accumulation site. Small amounts of copper were also localized to cytoplasmic vesicles or cell walls of cortical cells. The results of the present investigation have significant importance in further understanding the mechanisms of absorption, transportation and accumulation of heavy metals in plants grown in polluted soil.     

Treatment of Alkaline Cr(VI)-Contaminated Leachate with an Alkaliphilic Metal-Reducing Bacterium

Chromium in its toxic Cr(VI) valence state is a common contaminant particularly associated with alkaline environments. A well-publicized case of this occurred in Glasgow, United Kingdom, where poorly controlled disposal of a cementitious industrial by-product, chromite ore processing residue (COPR), has resulted in extensive contamination by Cr(VI)-contaminated alkaline leachates. In the search for viable bioremediation treatments for Cr(VI), a variety of bacteria that are capable of reduction of the toxic and highly soluble Cr(VI) to the relatively nontoxic and less mobile Cr(III) oxidation state, predominantly under circumneutral pH conditions, have been isolated. Recently, however, alkaliphilic bacteria that have the potential to reduce Cr(VI) under alkaline conditions have been identified. This study focuses on the application of a metal-reducing bacterium to the remediation of alkaline Cr(VI)-contaminated leachates from COPR. This bacterium, belonging to the Halomonas genus, was found to exhibit growth concomitant to Cr(VI) reduction under alkaline conditions (pH 10). Bacterial cells were able to rapidly remove high concentrations of aqueous Cr(VI) (2.5 mM) under anaerobic conditions, up to a starting pH of 11. Cr(VI) reduction rates were controlled by pH, with slower removal observed at pH 11, compared to pH 10, while no removal was observed at pH 12. The reduction of aqueous Cr(VI) resulted in the precipitation of Cr(III) biominerals, which were characterized using transmission electron microscopy and energy-dispersive X-ray analysis (TEM-EDX) and X-ray photoelectron spectroscopy (XPS). The effectiveness of this haloalkaliphilic bacterium for Cr(VI) reduction at high pH suggests potential for its use as an in situ treatment of COPR and other alkaline Cr(VI)-contaminated environments.     

Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe–metal interactions studies

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H₂S and by a direct enzymatic process involving hydrogenases and c₃ cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria–metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.     

A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.     

Changes of calcium distribution in ovules ofTorenia fournieri during pollination and fertilization

Summary Calcium distribution in ovules ofTorenia fournieri was studied by electron energy loss spectroscopy and transmission electron microscopic visualization of calcium antimonate precipitates. High calcium levels were found in the ovules ofT. fournieri. Calcium is situated mainly in extracellular regions before fertilization, including the surface of embryo sac, in the mucilage, and among the cells of the egg apparatus. Intracellular calcium was found only in the nucellar cells around the embryo sac and in the epidermis of the central axis and funiculus. After pollination, a labyrinthine structure (coralloid-like cell wall formation) develops on the micropylar surfaces of the egg apparatus that contain high levels of calcium. Calcium levels increase in the degenerating synergid after the penetration of the pollen tube. Calcium-antimonate precipitates are abundant in vacuoles of the disrupted synergid and pollen tube cytoplasm.Abbreviations EELS electron energy loss spectroscopy - EDX energy-dispersive X-ray microanalysis - LS labyrinthine structure     

The differential stress response of adapted chromite mine isolates Bacillus subtilis and Escherichia coli and its impact on bioremediation potential

In the current study, indigenous bacterial isolates Bacillus subtilis VITSUKMW1 and Escherichia coli VITSUKMW3 from a chromite mine were adapted to 100 mg L^?1 of Cr(VI). The phase contrast and scanning electron microscopic images showed increase in the length of adapted E. coli cells and chain formation in case of adapted B. subtilis. The presence of chromium on the surface of the bacteria was confirmed by energy dispersive X-ray spectroscopy (EDX), which was also supported by the conspicuous Cr–O peaks in FTIR spectra. The transmission electron microscopic (TEM) images of adapted E. coli and B. subtilis showed the presence of intact cells with Cr accumulated inside the bacteria. The TEM–EDX confirmed the internalization of Cr(VI) in the adapted cells. The specific growth rate and Cr(VI) reduction capacity was significantly higher in adapted B. subtilis compared to that of adapted E. coli. To study the possible role of Cr(VI) toxicity affecting the Cr(VI) reduction capacity, the definite assays for the released reactive oxygen species (ROS) and ROS scavenging enzymes (SOD and GSH) were carried out. The decreased ROS production as well as SOD and GSH release observed in adapted B. subtilis compared to the adapted E. coli corroborated well with its higher specific growth rate and increased Cr(VI) reduction capacity.     

Temperature sensitivity on proliferation and morphologic alteration of human esophageal carcinoma cells in culture

Summary As basic studies of hyperthermia and hypothermia on malignant tumor, the kinetics of proliferative activity, the morphologic changes in the two cell lines, SGF-3 and SGF-5, established in our department after the change of culture temperature were examined. The results obtained were: a) A significant difference was found in the sensitivity to temperatures between the two cell lines originated from human esophageal squamous cell carcinoma. The temperature range allowing cultured cell to proliferate were from 31° to 39° C in SGF-3 and from 29° to 41° C in SGF-5. b) Minor difference occurred in the results between the two cell lines examined during the recovery of proliferative activity, but no proliferative activity was discovered after the cells were exposed to 42° C for 72 h. Two cell lines resumed their proliferation after having been exposed to 27° or 28° C for 72 h. c) Morphologic changes of the cell lines cultured at high temperature were cytoplasmic vacuolation and cell aggregation by phase contrast microscope and the increase of heterochromatin, the decrease of granular formation in nucleoli, and nucleolar vacuolation by transmission electron microscopy (TEM). At low temperatures the changes observed included cytoplasmic ballooning and circumnuclear halo formation by phase contrast microscope, and the increase of heterochromatin, nucleolar segregation, swelling of mitochondria, and dilatation of rough endoplasmic reticulum (rER) by TEM.     

Electron Microscopy and X-ray Analysis of Cr-Containing Precipitates Synthesized by Newly Isolated Actinobacterium,Flexivirga alba ST13T

Chromium(Cr) precipitate synthesized by Cr(VI)-reducing bacterium Flexivirga alba ST13^T was examined using transmission electron microscopy (TEM) and the energy dispersive X-ray (EDX). The strain showed altered-morphology after exposing to Cr(VI) in minimal medium. The resultant precipitate included bacterial pellet and needle-like structure which was similar to the structure made from Cr(OH)₃ precipitate. Cr was observed in bacterial cells using TEM–EDX. Bacteria with high electron density showed the precipitation of Ca in addition to Cr. The isolated strain would be useful to precipitate Cr from Cr(VI)-containing environment.