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Epifluorescence Microscopy (epifluorescence + microscopy)
Selected AbstractsPolysaccharide hydrolysis in aggregates and free enzyme activity in aggregate-free seawater from the north-eastern Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Kai Ziervogel Summary Marine snow aggregates represent hotspots of carbon remineralization in the ocean. Various aspects of bacterial dynamics have been investigated on marine snow. To date, extracellular enzymatic activities in aggregates have been measured using small substrate proxies that do not adequately reflect the complexity of biomacromolecules such as polysaccharides, proteins and lipids. To address this issue, we used six structurally distinct, fluorescently labelled polysaccharides to measure enzymatic hydrolysis on aggregates formed with a roller table and in aggregate-free (ambient) seawater from two near-coast sites, north-eastern Gulf of Mexico. A single polysaccharide was incubated in aggregates and ambient seawater. Changes in polysaccharide molecular weight were monitored over time to measure the course of enzymatic hydrolysis. All six polysaccharides were hydrolysed in aggregates, indicating a broad range of enzyme activities in aggregate-associated bacteria. Four substrates were also hydrolysed in ambient waters. Epifluorescence microscopy revealed that nearly all of the bacteria present in original waters were incorporated into aggregates. Therefore hydrolytic activities in ambient waters were presumably due to enzymes spatially disconnected from cells and aggregates. Our results show substantial enzymatic activity in cell/aggregate-free seawater, suggesting a significant role of free enzymes in hydrolytic activity in waters from the north-eastern Gulf of Mexico. [source] Epifluorescence microscopy and image analysis of high-level polycyclic aromatic hydrocarbon contamination in soilsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2006J. Chadwick Roper Abstract Interactions between polycyclic aromatic hydrocarbons (PAHs) and soil are an important determinant of their chemical availability and transport. Laboratory examination of microscale PAH,soil interaction is limited by the availability of methods for particle-scale observation. Inverted epifluorescence microscopy, combined with digital photography and computer image analysis, was evaluated for specificity and linearity using dissolved PAHs. A pyrene filter (excitation wavelength, 360,400 nm; emission wavelength, 450,520 nm) gave nonspecific PAH fluorescence, and bias for fluoranthene, benzo[b]fluoranthene, benzo[g, h, i]perylene, and benz[a]anthracene was quantified in comparison to that for pyrene. Concentrations ranging from 1 to 10 mM for anthracene, fluoranthene, and pyrene and from 1 to 50 mM for naphthalene produced a linear response with low interpixel variability. Liquid-phase analyses validated use of the technique for the descriptive analysis of PAH distribution in solid samples, but liquid-phase calibration was not quantitative for spiked or field-contaminated soils. The mean luminance for three field soils was proportional to the values predicted from their chemically measured concentrations and to values from spiked, aged, uncontaminated materials. Image analysis of laboratory- and field-contaminated samples determined the area distribution of fluorescent intensity and the size of fluorescent areas exceeding a threshold luminance. These qualitative descriptions of the microscale spatial distribution of PAH contamination are presented as potential endpoints for future research on biogeochemical interactions in heavily contaminated solids. [source] Single-cell image analysis to assess ABC-transporter,mediated efflux in highly purified hematopoietic progenitorsCYTOMETRY, Issue 4 2002H.G.P. Raaijmakers Abstract Background Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. Methods We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter,mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). Results The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein,mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 ± 0.35 and 1.14 ± 0.11, respectively; P = 0.01). P-glycoprotein,mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 ± 0.19 and 1.28 ± 0.18, respectively). Conclusion The described method is a valuable tool for assessing ABC-transporter,mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells. Cytometry 49:135,142, 2002. © 2002 Wiley-Liss, Inc. [source] Effect of environmental variables on eukaryotic microbial community structure of land-fast Arctic sea iceENVIRONMENTAL MICROBIOLOGY, Issue 3 2010Brian Eddie Summary Sea ice microbial community structure affects carbon and nutrient cycling in polar seas, but its susceptibility to changing environmental conditions is not well understood. We studied the eukaryotic microbial community in sea ice cores recovered near Point Barrow, AK in May 2006 by documenting the composition of the community in relation to vertical depth within the cores, as well as light availability (mainly as variable snow cover) and nutrient concentrations. We applied a combination of epifluorescence microscopy, denaturing gradient gel electrophoresis and clone libraries of a section of the 18S rRNA gene in order to compare the community structure of the major eukaryotic microbial phylotypes in the ice. We find that the community composition of the sea ice is more affected by the depth horizon in the ice than by light availability, although there are significant differences in the abundance of some groups between light regimes. Epifluorescence microscopy shows a shift from predominantly heterotrophic life styles in the upper ice to autotrophy prevailing in the bottom ice. This is supported by the statistical analysis of the similarity between the samples based on the denaturing gradient gel electrophoresis banding patterns, which shows a clear difference between upper and lower ice sections with respect to phylotypes and their proportional abundance. Clone libraries constructed using diatom-specific primers confirm the high diversity of diatoms in the sea ice, and support the microscopic counts. Evidence of protistan grazing upon diatoms was also found in lower sections of the core, with implications for carbon and nutrient recycling in the ice. [source] Bacteria associated with the rapid tissue necrosis of stony coralsENVIRONMENTAL MICROBIOLOGY, Issue 7 2007G. M. Luna Summary The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio -specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for >,15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology. [source] Epifluorescence microscopy and image analysis of high-level polycyclic aromatic hydrocarbon contamination in soilsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2006J. Chadwick Roper Abstract Interactions between polycyclic aromatic hydrocarbons (PAHs) and soil are an important determinant of their chemical availability and transport. Laboratory examination of microscale PAH,soil interaction is limited by the availability of methods for particle-scale observation. Inverted epifluorescence microscopy, combined with digital photography and computer image analysis, was evaluated for specificity and linearity using dissolved PAHs. A pyrene filter (excitation wavelength, 360,400 nm; emission wavelength, 450,520 nm) gave nonspecific PAH fluorescence, and bias for fluoranthene, benzo[b]fluoranthene, benzo[g, h, i]perylene, and benz[a]anthracene was quantified in comparison to that for pyrene. Concentrations ranging from 1 to 10 mM for anthracene, fluoranthene, and pyrene and from 1 to 50 mM for naphthalene produced a linear response with low interpixel variability. Liquid-phase analyses validated use of the technique for the descriptive analysis of PAH distribution in solid samples, but liquid-phase calibration was not quantitative for spiked or field-contaminated soils. The mean luminance for three field soils was proportional to the values predicted from their chemically measured concentrations and to values from spiked, aged, uncontaminated materials. Image analysis of laboratory- and field-contaminated samples determined the area distribution of fluorescent intensity and the size of fluorescent areas exceeding a threshold luminance. These qualitative descriptions of the microscale spatial distribution of PAH contamination are presented as potential endpoints for future research on biogeochemical interactions in heavily contaminated solids. [source] Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transferEQUINE VETERINARY JOURNAL, Issue 1 2008B. A. BALL Summary Reasons for performing study: Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objectives: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647 -pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively. Results: Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647 -pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed. Conclusions: The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7,10 embryos, there was no evidence of expression of EGFP in these embryos. Potential relevance: Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids. [source] Abundance, diversity, and activity of microbial assemblages associated with coral reef fish guts and fecesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2010Steven Smriga Abstract Feces and distal gut contents were collected from three coral reef fish species. Bacteria cell abundances, as determined via epifluorescence microscopy, ranged two orders of magnitude among the fishes. Mass-specific and apparent cell-specific hydrolytic enzyme activities in feces from Chlorurus sordidus were very high, suggesting that endogenous fish enzymes were egested into feces. Denaturing gradient gel electrophoresis profiles of 16S rRNA genes were more similar among multiple individuals of the surgeonfish Acanthurus nigricans than among individuals of the parrotfish C. sordidus or the snapper Lutjanus bohar. Analyses of feces-derived 16S rRNA gene clones revealed that at least five bacterial phyla were present in A. nigricans and that Vibrionaceae comprised 10% of the clones. Meanwhile, C. sordidus contained at least five phyla and L. bohar three, but Vibrionaceae comprised 71% and 76% of the clones, respectively. Many sequences clustered phylogenetically to cultured Vibrio spp. and Photobacterium spp. including Vibrio ponticus and Photobacterium damselae. Other Vibrionaceae -like sequences comprised a distinct phylogenetic group that may represent the presence of ,feces-specific' bacteria. The observed differences among fishes may reflect native gut microbiota and/or bacterial assemblages associated with ingested prey. [source] Abundance and production of bacteria, and relationship to phytoplankton production, in a large tropical lake (Lake Tanganyika)FRESHWATER BIOLOGY, Issue 6 2009STEPHANE STENUITE Summary 1. Abundance and bacterial production (BP) of heterotrophic bacteria (HBact) were measured in the north and south basins of Lake Tanganyika, East Africa, during seasonal sampling series between 2002 and 2007. The major objective of the study was to assess whether BP can supplement phytoplankton particulate primary production (particulate PP) in the pelagic waters, and whether BP and particulate PP are related in this large lake. HBact were enumerated in the 0,100 m surface layer by epifluorescence microscopy and flow cytometry; BP was quantified using 3H-thymidine incorporation, usually in three mixolimnion layers (0,40, 40,60 and 60,100 m). 2. Flow cytometry allowed three subpopulations to be distinguished: low nucleic acid content bacteria (LNA), high nucleic acid content bacteria (HNA) and Synechococcus -like picocyanobacteria (PCya). The proportion of HNA was on average 67% of total bacterial abundance, and tended to increase with depth. HBact abundance was between 1.2 × 105 and 4.8 × 106 cells mL,1, and was maximal in the 0,40 m layer (i.e. roughly, the euphotic layer). Using a single conversion factor of 15 fg C cell,1, estimated from biovolume measurements, average HBact biomass (integrated over a 100-m water column depth) was 1.89 ± 1.05 g C m,2. 3. Significant differences in BP appeared between seasons, especially in the south basin. The range of BP integrated over the 0,100 m layer was 93,735 mg C m,2 day,1, and overlapped with the range of particulate PP (150,1687 mg C m,2 day,1) measured in the same period of time at the same sites. 4. Depth-integrated BP was significantly correlated to particulate PP and chlorophyll- a, and BP in the euphotic layer was on average 25% of PP. 5. These results suggest that HBact contribute substantially to the particulate organic carbon available to consumers in Lake Tanganyika, and that BP may be sustained by phytoplankton-derived organic carbon in the pelagic waters. [source] Living under an atomic force microscopeGEOBIOLOGY, Issue 3 2005An optimized approach for in vivo investigations on surface alterations towards biomineral nucleation on cyanobacterial cells ABSTRACT An approach for long-term in vivo investigations on cyanobacterial cell surface changes at high spatial resolution by Atomic Force Microscopy (AFM) was developed in this study. Until recently, changes of bacterial cell surfaces due to changes of the chemical environment could neither be investigated in situ nor in vivo. However, in vivo investigations give insights into kinetics of cell response to environmental changes and mineral nucleation at the cell's surface. Continuously cultured cyanobacteria of the representative freshwater strain Synechococcus leopoliensis (PCC 7942) were washed and artificially immobilized on poly-l-lysine-coated glass slides. Both immobilization and environmental conditions were optimized in order to facilitate long-term experiments (> 100 h) with living cells. AFM samples were investigated in situ in two different solutions: Culture medium was used for cultivation experiments and nutrient-free NaHCO3/CaCl2 solutions (supersaturated with respect to calcite) for long-term characterizations of the changes in cell surface topography. Cell viability under these conditions was investigated by AFM, TEM and epifluorescence microscopy, independently. No indications for extended starvation were found within the relevant timescales. Analysing the influence of Ca2+ on the surface of S. leopoliensis, we found significant changes compared to a Ca-free solution. Few hours after CaCl2 was added to the circumfluent solution, small protuberances were observed on the cell surface. These are promising results to environmental scientists for a wide range of applications, as cell response to environmental changes can now be monitored online and in vivo at timescales, which are relevant for natural processes. Most especially studies of biomineralization and mineral nucleation on bacterial cell surfaces will profit from this new approach. [source] Detecting microdamage in boneJOURNAL OF ANATOMY, Issue 2 2003T. C. Lee Abstract Fatigue-induced microdamage in bone contributes to stress and fragility fractures and acts as a stimulus for bone remodelling. Detecting such microdamage is difficult as pre-existing microdamage sustained in vivo must be differentiated from artefactual damage incurred during specimen preparation. This was addressed by bulk staining specimens in alcohol-soluble basic fuchsin dye, but cutting and grinding them in an aqueous medium. Nonetheless, some artefactual cracks are partially stained and careful observation under transmitted light, or epifluorescence microscopy, is required. Fuchsin lodges in cracks, but is not site-specific. Cracks are discontinuities in the calcium-rich bone matrix and chelating agents, which bind calcium, can selectively label them. Oxytetracycline, alizarin complexone, calcein, calcein blue and xylenol orange all selectively bind microcracks and, as they fluoresce at different wavelengths and colours, can be used in sequence to label microcrack growth. New agents that only fluoresce when involved in a chelate are currently being developed , fluorescent photoinduced electron transfer (PET) sensors. Such agents enable microdamage to be quantified and crack growth to be measured and are useful histological tools in providing data for modelling the material behaviour of bone. However, a non-invasive method is needed to measure microdamage in patients. Micro-CT is being studied and initial work with iodine dyes linked to a chelating group has shown some promise. In the long term, it is hoped that repeated measurements can be made at critical sites and microdamage accumulation monitored. Quantification of microdamage, together with bone mass measurements, will help in predicting and preventing bone fracture failure in patients with osteoporosis. [source] Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperaturesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004E. van Rensburg Abstract Aims:, To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. Methods and Results:,Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, ,18 and ,75°C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain,heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. Conclusions:,Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. Significance and Impact of the Study:, Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen. [source] A 210-min solid phase cytometry test for the enumeration of Escherichia coli in drinking waterJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2000S.O. Van Poucke A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies. The test involves membrane filtration through a 25-mm black polyester filter, induction of ,-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-,- d -glucuronide as an enzyme substrate and laser scanning of the membrane filter. Scan results can be confirmed on-line by epifluorescence microscopy. Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated ,,90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar. In 5·4% of all samples examined, SPC detected between 1 and 11 E. coli per 100 ml, while the two plate methods yielded negative results. Cases of a negative SPC result but a positive E. coli count on both reference media were not observed. This test would primarily be useful for ,emergency' monitoring of drinking water when rapid results are crucial. [source] INTRACELLULAR CYANOBACTERIAL SYMBIONTS IN THE MARINE DIATOM CLIMACODIUM FRAUENFELDIANUM (BACILLARIOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 3 2000Edward J. Carpenter The diatom Climacodium frauenfeldianum Grunow was collected in the tropical Atlantic and Pacific Oceans. Observations with epifluorescence microscopy revealed that this diatom contained coccoid symbionts (2.5,3.5 ,m) with a typical cyanobacterial fluorescence in addition to that of their own chloroplasts. Mean concentration of C. frauenfeldianum for 28 stations in the SW tropical Pacific Ocean was 530 x 103 (SE = 1372) cells·m,2, with highest concentration (mean 17.5 cells·L,1) at 40-m depth. The symbiosis was only observed at water temperatures between 26.3 and 28.9° C, with highest concentrations at 27.7° C. Three almost complete 16S rDNA sequences from one sample were determined, and they were identical. The phylogenetic analysis of this 16S rDNA sequence and those from other cyanobacteria and plastids revealed that it was closely related to the 16S rDNA sequence from Cyanothece sp. ATCC 51142. Cyanothece sp. ATCC 51142 is a unicellular nitrogen-fixing cyanobacterium isolated from a coastal marine environment and has ultrastructural features similar to the symbionts of C. frauenfeldianum. The close relationship between Cyanothece sp. and the cyanobacterial symbiont in C. frauenfeldianum suggests the potential for nitrogen fixation in the symbiosis. [source] Ecological implications of biomass and morphotype variations of bacterioplankton: an example in a coastal zone of the Northern Adriatic Sea (Mediterranean)MARINE ECOLOGY, Issue 2 2005Rosabruna La Ferla Abstract This study had the objective of quantifying the variability in abundance, cell volume, morphology and C content of a natural bacterioplankton community in a coastal zone of the North Adriatic Sea during two periods (February and June) of two consequent years (1996 and 1997). We used epifluorescence microscopy with Acridine Orange staining procedures and a microphotographic technique. Low variability in bacterial abundance (range 0.3,3.1 × 105 cells ml,1) occurred between summer and winter periods. Conversely, the cell volume and the calculated carbon content changed greatly with warm and cold periods (ranges: 0.015,0.303 ,m3 and 5.83,42.17 fg C cell,1, respectively). Elongated bacteria were dominant while coccoid cells prevailed only in February 1997. Biomass showed high variability (range 0.12,10.21 ,g C l,1) whilst the abundance did not show noticeable differences among the sampling periods. As a consequence, quantification of bacterial biomass based solely on cell abundance must be considered with caution because the true biomass could depend on variability in cell volumes and morphotypes. [source] Mitochondrial morphology transition is an early indicator of subsequent cell death in ArabidopsisNEW PHYTOLOGIST, Issue 1 2008Iain Scott Summary ,,Mitochondrial morphology and dynamics were investigated during the onset of cell death in Arabidopsis thaliana. Cell death was induced by either chemical (reactive oxygen species (ROS)) or physical (heat) shock. ,,Changes in mitochondrial morphology in leaf tissue, or isolated protoplasts, each expressing mitochondrial-targeted green fluorescent protein (GFP), were observed by epifluorescence microscopy, and quantified. ,,Chemical induction of ROS production, or a mild heat shock, caused a rapid and consistent change in mitochondrial morphology (termed the mitochondrial morphology transition) that preceded cell death. Treatment of protoplasts with a cell-permeable superoxide dismutase analogue, TEMPOL, blocked this morphology change. Incubation of protoplasts in micromolar concentrations of the calcium channel-blocker lanthanum chloride, or the permeability transition pore inhibitor cyclosporin A, prevented both the mitochondrial morphology transition and subsequent cell death. ,,It is concluded that the observed mitochondrial morphology transition is an early and specific indicator of cell death and is a necessary component of the cell death process. [source] The role of lateral roots in bypass flow in rice (Oryza sativa L.)PLANT CELL & ENVIRONMENT, Issue 5 2010BUALUANG FAIYUE ABSTRACT Although an apoplastic pathway (the so-called bypass flow) is implicated in the uptake of Na+ by rice growing in saline conditions, the point of entry of this flow into roots remains to be elucidated. We investigated the role of lateral roots in bypass flow using the tracer trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS) and the rice cv. IR36. PTS was identified in the vascular tissue of lateral roots using both epifluorescence microscopy and confocal laser scanning microscopy. Cryo-scanning electron microscopy and epifluorescence microscopy of sections stained with berberine-aniline blue revealed that the exodermis is absent in the lateral roots. We conclude that PTS can move freely through the cortical layers of lateral roots, enter the stele and be transported to the shoot via the transpiration stream. [source] High Numbers of Naked Amoebae in the Planktonic Waters of a Mangrove Stand in Southern Florida, USATHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2000ANDREW ROGERSON ABSTRACT. This is the first study to examine the abundance of naked amoebae in the water column of a mangrove stand. A total of 37 different morphotypes was noted and at least 13 of these are probably new species. Over a one-year sampling interval, amoebae averaged 35,400 cells liter,1 (range 2,000,104,000) by an indirect enrichment cultivation method. Densities in the upper end of this range arc the highest ever reported for any planktonic habitat. Variation between samples was related to the quantity of suspended aggregates (floes) in the water column emphasizing that amoebae are usually floc-associated. The study also showed that it is essential to disrupt floc material prior to withdrawing sample aliquots for the indirect counting method since several amoebae can occupy the interstices of aggregates. There is concern that indirect enumeration methods that require organisms to be cultured in the laboratory seriously underestimate the true count. A direct counting method using acridine orange staining and epifluorescence microscopy was attempted to assess the possible magnitude of the error associated with indirect counting. While this direct method had limitations, notably the difficulty of unambiguously differentiating between small amoebae and nanoflagellates, the results suggested that the indirect method gave estimates that were close to the true count (within a factor of two). Mangrove waters are rich in heterotrophic protozoa (,3 × 106 liter1) and while the heterotrophic flagellates are by far the dominant group, naked amoebae outnumber ciliates some 20-foid. The ecological consequences of high numbers of amoebae, particularly the common small forms less than 10 ,m in length, need to be examined for these important coastal sites. [source] SURGICAL ADHESIONS: EVIDENCE FOR ADSORPTION OF SURFACTANT TO PERITONEAL MESOTHELIUMANZ JOURNAL OF SURGERY, Issue 6 2000Y. Chen Background: It has been speculated that the formation of surgical adhesions must be preceded by physical adhesion of the two surfaces, a process normally prevented by a lining of adsorbed surface-active phospholipid (surfactant) acting as both a superb boundary (solid-to-solid) lubricant and a release (antistick) agent. Animal trials administering exogenous surfactant as a dry powder (ALECÔ) have previously demonstrated a reduction of 80% in abdominal adhesions. Methods: Incubation of rat peritoneum (both live and excised) with radiolabelled dipalmitoyl phosphatidylcholine (DPPC) has been used to demonstrate adsorption; while the normal lining of surfactant in the human abdominal cavity has been confirmed by epifluorescence microscopy using Phosphin E as the hydrophobic probe. Aims: The overall aim is to confirm that peritoneal mesothelium has a lining of surfactant known for its lubricating and release properties, and that this lining can be enhanced by the adsorption of exogenous material. Results: Adsorption of DPPC to peritoneal mesothelium was 470 ng/cm 2 (n = 8) ex vivo and 598 ng/cm 2 (n = 18) in vivo, these rates being enhanced by EggPG by 62%ex vivo and 47%in vivo to reach the equivalent of almost three close-packed monolayers. Conclusions: These results can explain the reduction in surgical adhesions previously reported in animals by administering ALECÔ (7:3 DPPC:EggPG) as a highly surface-active dry powder, although it is now used in saline suspension to treat respiratory distress syndrome in newborns, in whom it has no side-effects. These findings would appear to justify clinical trials for dry ALECÔ in suppressing surgical adhesions with minimal risk of an adverse reaction. The results of these trials are also discussed and found to be compatible with the known ability of surfactant to resist physical adhesion by fibronectin, the tacky ,glue' by which fibroblasts attach to surfaces as the first step in formation of fibrinous adhesions. [source] In vitro tracheal hyperresponsiveness to muscarinic receptor stimulation by carbachol in a rat model of bleomycin-induced pulmonary fibrosisAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2006J. Barrio Summary 1 Bleomycin-induced lung injury is widely used as an experimental model to investigate the pathophysiology of pulmonary fibrosis but the alterations in the pharmacological responsiveness of airways isolated from bleomycin-exposed animals has been scarcely investigated. The aim of this study was to examine the in vitro tracheal responses to muscarinic receptor stimulation with carbachol in a rat bleomycin model. 2 Concentration,response curves to carbachol (10 nm to 0.1 mm) were obtained in tracheal rings isolated from Sprague,Dawley rats 14 days after endotracheal bleomycin or saline. The intracellular calcium signal in response to carbachol (10 ,m) was measured by epifluorescence microscopy using fura-2 in primary cultures of tracheal smooth muscle cells from bleomycin- and saline-exposed rats. Circulating plasma tumour necrosis factor (TNF)- ,/interleukin (IL)-1, levels were measured by enzyme-linked immunosorbent assay. 3 Maximal contraction in response to carbachol was significantly greater in tracheal rings from bleomycin-exposed rats compared with controls (15.8 ± 1.3 mN vs. 11.8 ± 1.4 mN; n = 19, P < 0.05). 4 Carbachol (10 ,m) elicited a transient increase of intracellular calcium with greater increment in tracheal smooth muscle cells from bleomycin-exposed rats compared with controls (372 ± 42 nmvs. 176 ± 20 nm; n = 7, P < 0.01). 5 Circulating plasma levels of TNF- ,/IL-1, were augmented in bleomycin-exposed rats compared with controls. Tissue incubation with TNF- , (100 ng ml,1)/IL-1, (10 ng ml,1) increased in vitro tracheal responsiveness to carbachol. 6 In conclusion, tracheal contraction in response to muscarinic receptor stimulation with carbachol was increased in bleomycin-exposed rats. This in vitro cholinergic hyperresponsiveness may be related to the augmented levels of inflammatory cytokines in bleomycin-exposed rats. [source] Assessment of physiological conditions in E. coli fermentations by epifluorescent microscopy and image analysisBIOTECHNOLOGY PROGRESS, Issue 3 2009Sónia Carneiro Abstract The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | |||||||||||||||||||||||||