A contamination reducing method by ion beam bombardment of the specimen in high resolution electron microscopy

The theory of contamination and a contamination reducing method are discussed on the basis of time dependent micrograph series and their tilted images for determining contamination.For a high current density of an electron probe in the field emission scanning electron microscope, it is observed that contaminated cones are formed in proportion to the exposure time of an electron beam. From the measurement of the contamination layer thickness and its area, the contamination rate and time dependent shape are formulated, mainly depending on the cross-section and current density together with the average lifetime of adsorption molecules.It is found that the contamination rate and stray contamination of outgassing molecules forming part of the specimen are effectively reduced by a pre-bombardment of argon ions on the surfaces of specimens. The contamination rate is reduced to a small extent (5%) using the present method.     

A tale of two fluids: does storing specimens together in liquid preservative cause DNA cross‐contamination in molecular gut‐content studies?

The study of food webs and trophic interactions increasingly relies on PCR‐based molecular gut‐content analysis. However, this technique may be prone to error from contamination of minute quantities of DNA; i.e., simply storing specimens together in a liquid medium may lead to cross‐contamination. In this study, we used PCR to determine the contamination rate when (1) specimens were stored together in 95% ethanol for various time periods, and (2) predators fall into ethylene glycol‐filled pitfall traps where the dying predator may inadvertently consume prey DNA‐contaminated liquid. We designed experiments and PCR primers to quantify the risk of contamination for both situations and found no contamination by storing specimens together in 95% ethanol. Furthermore, zero predators contained prey DNA in their gut contents from imbibing prey DNA‐contaminated ethylene glycol. These results support the use of mass sampling techniques, like wet pitfall traps, for molecular gut‐content analysis.     

Canopy seed bank structure in relation to: fire,tree size and density

To assess the canopy seed bank structure of Pinus halepensis, we measured the level of serotiny and the seed bank size and density of trees in unburned stands and post-fire regenerated stands in Israel. We analysed the effects of tree size, tree density and fire history on the level of serotiny. The level of serotiny decreased with an increase in tree height. The high level of serotiny in short trees could be explained by selection to increase regeneration chances after burning at pre-mature age. Also, limitation of long-distance seed dispersal opportunities in short trees may favour high serotiny levels. The level of serotiny was higher in post-fire stands than in unburned stands, suggesting a fast selection for serotiny by fire. Unburned stands had a higher total stand seed density than post-fire regenerated stands, but the proportion of seeds in serotinous cones of the total stand seed density was higher in post-fire regenerated stands. The fact that P. halepensis bears simultaneously serotinous and non-serotinous cones reflects its dual strategy as both a post-fire obligate seeder, mainly from serotinous cones and an early coloniser during fire-free periods, mainly from non-serotinous cones. The relative investment in these strategies is dependent on fire history and varies with tree height. Furthermore, mature brown cones can contribute to post-fire regeneration in case of spring fires, and serotinous cones are known to open partially also in dry spell events. Thus, post-fire regeneration and invasion are strategies, which seem to complement each other.     

The biofiltration permeable reactive barrier: Practical experience from Synthesia

The paper refers to utilization of biological elements within permeable reactive barriers. The concept of a biofiltration permeable barrier has been tested in the laboratory and in pilot-scale. Oxyhumolite (oxidized young lignite) was examined as an absorption material and a biofilm carrier. Laboratory tests performed before the pilot verification confirmed that oxyhumolite adsorbs organic pollutants at a minimum value, but that it can be used for biofilm attachment. An experimental barrier was built on premises of a chemical factory contaminated mainly by various organic pollutants [benzene, toluene, ethylbenzene, and xylenes (BTEX), chlorobenzenes, naphthalene, nitro-derivatives, phenols, trichloroethylene (TCE), and total petroleum hydrocarbon (TPH)]. Before the barrier was installed, a preliminary survey of the unsaturated zone, hydrogeological investigation, and a microbiological survey had been performed. The barrier was designed as a trench-and-gate system with an in situ bioreactor. During the year 2004, measurements of groundwater flux and retention time under current hydrological conditions, together with chemical and microbiological monitoring, were carried out on the site. The results showed high effectiveness of organic contamination removal. Average elimination varied from 57.3% (naphthalene) to 99.9% (nitro-derivatives, BTEX); microbial density in the bioreactor was approx. 10⁵ CFU mL⁻¹.     

Evaluation of decontamination process of heart valve and artery tissues in European Homograft Bank (EHB): a retrospective study of 1,055 cases

To evaluate the efficiency of decontamination practice in European Homograft Bank (EHB), the data of the cardiovascular tissues received during recent 2?years were retrospectively analysed in this study. After initial assessment, the tissues were incubated in a 3-antibiotics?? cocktail at 4°C for 20?C48?h. The states of contamination were evaluated before and after incubation with the focus on the differences in donor type, tissue type, germ type and incubation time. Amongst 1,055 eligible tissues, 77.2% were hearts and 22.8% were arteries. 82.2% of the tissues were retrieved from the multi-organ donors (MOD), 15.4% from the recipients of heart transplantation (RHT) and 2.4% from the non-heart beating donors (NHBD). The initial contamination rate was 27.4% with a significantly higher incidence in arteries. The RHT tissues had the lowest contamination rate comparing to that of MOD and NHBD. Staphylococcus species was the major source of contamination. After antibiotic incubation, 76.8% of the contaminated tissues were disinfected, which was significantly higher for the hearts than the arteries. The RHT tissues had the highest decontamination rate than that of MOD and NHBD tissues. Propionibacterium acnes was detected in 48.1% of the remaining contaminated cases. The average incubation time of the Propionibacterium-positive tissues was significantly shorter than that of decontaminated tissues. In conclusion, the current decontamination protocol of EHB is sufficient for most of the initially contaminated bacteria, whereas it is inadequate for Propionibacterium acnes. This may be related to the slow-growing nature of this bacterium and thereby the relative shorter antibiotic incubation time.     

An in-use study of the relationship between bacterial contamination of food preparation surfaces and cleaning cloths

The relationship between bacterial contamination of food preparation surfaces and the cleaning cloths associated with these surfaces was investigated in a busy college kitchen. Cleaning cloths used in conjunction with detergent became heavily contaminated within hours of first use. Following 'cleaning' with these cloths with detergent, both surfaces and cloths were found to be more heavily contaminated and there was evidence of contamination transfer to and from cloths and surfaces. Following 'cleaning' with cloths impregnated with quaternary ammonium disinfectant, there was a significant reduction in contamination on both surfaces and cloths, together with a reduction in the numbers of enterobacteria and pseudomonads. The results suggest that significant improvements in kitchen hygiene could be achieved by ensuring that contaminated cloths are not used for food preparation activities.     

A lagged, density-dependent relationship between jack pine budworm Choristoneura pinus pinus and its host tree Pinus banksiana

Abstract. 1. Survival of newly emerged jack pine budworm Choristoneura pinus pinus is related to the density of available pollen cones (microsporangiate strobili) produced by its host tree, jack pine Pinus banksiana. 2. A 7‐year time series of observations from a plot network in Ontario, Canada, compared the propensity of jack pine to produce pollen cones, τ, on trees that were either defoliated or undisturbed by the jack pine budworm. 3. Non‐defoliated jack pine trees have a high propensity to produce pollen cones. More than one‐third of these trees produced pollen cones in every year of the series. Propensity varied significantly among plots and trees. Temporal patterns in propensity were also highly variable but within a plot propensity was often autocorrelated in time. 4. Defoliation by the jack pine budworm was associated with forest plots composed of the oldest and the largest trees and with the fewest trees per hectare. Within a plot, outbreaks lasted 3 or 4 years although individual trees were only defoliated in 1 or 2 years. 5. The propensity to produce pollen cones in jack pine was reduced in the years after defoliation. The most pronounced reductions in propensity occurred where defoliation was most severe. 6. The reduction in propensity to produce pollen cones resulting from previous defoliation, coupled with the dependence of jack pine budworm survival on the availability of pollen cones, induces a lagged, negative feedback between the density of the consumer and that of its resource. 7. The lagged, density‐dependent relationship between jack pine budworm and its jack pine host contributes to oscillatory dynamics of the jack pine budworm. Comparison of the outbreak behaviour of jack pine budworm with that of the closely related eastern spruce budworm C. fumiferana suggests that differences in the strength of the host‐plant interaction may account for differences in the relative frequency of outbreaks in the respective systems.     

Bleached pigment activates transduction in salamander cones

We have used suction electrode recording together with rapid steps into 0.5 mM IBMX solution to investigate changes in guanylyl cyclase velocity produced by pigment bleaching in isolated cones of the salamander Ambystoma tigrinum. Both backgrounds and bleaches accelerate the time course of current increase during steps into IBMX. We interpret this as evidence that the velocity of the guanylyl cyclase is increased in background light or after bleaching. Our results indicate that cyclase velocity increases nearly linearly with increasing percent pigment bleached but nonlinearly (and may saturate) with increasing back-ground intensity. In cones (as previously demonstrated for rods), light-activated pigment and bleached pigment appear to have somewhat different effects on the transduction cascade. The effect of bleaching on cyclase rate is maintained for at least 15-20 min after the light is removed, much longer than is required after a bleach for circulating current and sensitivity to stabilize in an isolated cone. The effect on the cyclase rate can be completely reversed by treatment with liposomes containing 11-cis retinal. The effects of bleaching can also be partially reversed by beta-ionone, an analogue of the chromophore 11- cis-retinal which does not form a covalent attachment to opsin. Perfusion of a bleached cone with beta-ionone produces a rapid increase in circulating current and sensitivity, which rapidly reverses when the beta-ionone is removed. Perfusion with beta-ionone also causes a partial reversal of the bleach-induced acceleration of cyclase velocity. We conclude that bleaching produces an "equivalent background" excitation of the transduction cascade in cones, perhaps by a mechanism similar to that in rods.     

Influence of precipitation and soil on transport of fecal enterococci in fractured limestone aquifers

Limestone aquifers provide the main drinking water resources of southern Italy. The groundwater is often contaminated by fecal bacteria because of the interaction between rocks having high permeability and microbial pollutants introduced into the environment by grazing and/or manure spreading. The microbial contamination of springwater in picnic areas located in high mountains can cause gastrointestinal illness. This study was carried out in order to analyze the interaction between Enterococcus faecalis and the soil of a limestone aquifer and to verify the influence of this interaction on the time dependence of groundwater contamination. E. faecalis was chosen because, in the study area involved, it represents a better indicator than Escherichia coli. The research was carried out through field (springwater monitoring) and laboratory experiments (column tests with intact soil blocks). The transport of bacterial cells through soil samples was analyzed by simulating an infiltration event that was monitored in the study area. Comparison of laboratory results with data acquired in the field showed that discontinuous precipitation caused an intermittent migration of microorganisms through the soil and produced, together with dispersion in the fractured medium (unsaturated and saturated zones), an articulated breakthrough at the spring. The short distances of bacterial transport in the study area produced a significant daily variability of bacterial contamination at the field scale.     

Highly Purified Micro- and Macronuclei from Tetrahymena thermophila Isolated by Percoll Gradients1

ABSTRACT. A new procedure is described that utilizes Percoll gradients for purifying micronuclei (MIC) and macronuclei (MAC) from Tetrahymena thermophila. Separation of MIC from MAC during certrifugation in Percoll gradients occurs as a result of their difference in size rather than density. Three kinds of tests were used to evaluate the purity of the nuclei: visualization of the nuclei by light microscopy; examination of the nuclei by electron microscopy; and Southern blots of MIC and MAC DNA probed with the 5s rRNA genes or a fragment from the MAC extrachromosomal rDNA molecule. When examined under the light microscope, the isolated MIC and MAC have much lower nuclear cross contamination levels than previous methods have reported. MIC's contaminated with less than 1 MAC in 1000 MIC and MAC's contaminated with less than 1 MIC in 500 MAC can be routinely prepared. Quantitative analyses of electron micrographs gave higher estimates of cross contamination in our purified nuclei, which may, in part, be explained by the difficulty in identifying small MIC or MAC fragments. Southern blots of MIC and MAC DNA probed with 5s rDNA confirmed the level of MAC contamination in the MIC estimated by light microscopy during purification of the nuclei. The level of nucleolar contamination in the MIC was estimated at 10% by Southern blots of MIC and MAC DNA, derived from a heterokaryon with distinctive MIC and MAC Bam HI sites, using an rDNA probe.     

Turning cones off: the role of the 9-methyl group of retinal in red cones

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than approximately 0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.     

Influence of Precipitation and Soil on Transport of Fecal Enterococci in Fractured Limestone Aquifers

Limestone aquifers provide the main drinking water resources of southern Italy. The groundwater is often contaminated by fecal bacteria because of the interaction between rocks having high permeability and microbial pollutants introduced into the environment by grazing and/or manure spreading. The microbial contamination of springwater in picnic areas located in high mountains can cause gastrointestinal illness. This study was carried out in order to analyze the interaction between Enterococcus faecalis and the soil of a limestone aquifer and to verify the influence of this interaction on the time dependence of groundwater contamination. E. faecalis was chosen because, in the study area involved, it represents a better indicator than Escherichia coli. The research was carried out through field (springwater monitoring) and laboratory experiments (column tests with intact soil blocks). The transport of bacterial cells through soil samples was analyzed by simulating an infiltration event that was monitored in the study area. Comparison of laboratory results with data acquired in the field showed that discontinuous precipitation caused an intermittent migration of microorganisms through the soil and produced, together with dispersion in the fractured medium (unsaturated and saturated zones), an articulated breakthrough at the spring. The short distances of bacterial transport in the study area produced a significant daily variability of bacterial contamination at the field scale.     

Morphometric Indices of Scots Pine Needle under Chronic Radiation Exposure

We have studied the morphometric indices of needles in Scots pine populations that grow in Bryansk oblast in sites with radioactive contamination long after the Chernobyl accident. The variability in needle weight and length, as well as the fluctuating asymmetry indices and occurrence of necroses and morphoses, were studied in four contaminated and two reference populations of Scots pine in 2011, 2013, and 2014. The exposure of needles in radioactively contaminated sites varied from 7 to 130 mGy/yr. We found brachyblasts with three needles in contaminated Scots pine populations; this morphosis was absent in the reference populations. A dependence of the occurrence of needles damaged by necrosis on the levels of radiation exposure in 2011 was found. However, it was statistically insignificant in other years. The length and weight of needles in contaminated populations differed from the control values; however, the dependence of these indices on the level of radiation exposure was not revealed in the studied range of doses. In 2011 and 2013, the index of fluctuating asymmetry in needle length exceeded the control levels in sites where the absorbed doses were 90 and 130 mGy; this index also tended to grow (statistically significantly in 2011) with an increase in the characteristics of radioactive contamination of the studied plots: the exposure dose rate and the specific activity of ⁹⁰Sr and ¹³⁷Cs in cones and of ¹³⁷Cs in the soil. Therefore, one can observe the consequences of chronic radiation exposure at the organismic level in populations of Scots pine (one of the most radiosensitive plant species) even 25 years after the accident. The data that we obtained in natural habitats confirm the international estimates, according to which the annual chronic radiation exposure of 100 mGy can be considered the limit of safe radiation exposure of natural populations according to morphological and ontogenetic parameters.     

Contamination and bacterial retention capacity of beef carcasses at the abattoir

The contamination of beef carcasses was studied together with the capacity of meat surfaces to retain bacteria along the processing line in the slaughter hall. The results showed that the contamination varied along the processing line, but that this pattern was essentially dependent on the contamination at the dressing station. It decreased or remained unchanged during the first 12 min and then increased, even without additional contamination. The contamination varied according to carcasses and micro-organisms studied and was not greatly affected by spray cleaning. The number of bacteria retained changed at a rate similar to that of the contaminants. The attachment was instantaneous. The results are discussed and compared with the various hypotheses about contamination and bacterial attachment processes.     

Optimizing cell arrays for accurate functional genomics

ABSTRACT: BACKGROUND: Cellular responses emerge from a complex network of dynamic biochemical reactions. In order to investigate them is necessary to develop methods that allow perturbing a high number of gene products in a flexible and fast way. Cell arrays (CA) enable such experiments on microscope slides via reverse transfection of cellular colonies growing on spotted genetic material. In contrast to multi-well plates, CA are susceptible to contamination among neighboring spots hindering accurate quantification in cell-based screening projects. Here we have developed a quality control protocol for quantifying and minimizing contamination in CA. RESULTS: We imaged checkered CA that express two distinct fluorescent proteins and segmented images into single cells to quantify the transfection efficiency and interspot contamination. Compared with standard procedures, we measured a 3-fold reduction of contaminants when arrays containing HeLa cells were washed shortly after cell seeding. We proved that nucleic acid uptake during cell seeding rather than migration among neighboring spots was the major source of contamination. Arrays of MCF7 cells developed without the washing step showed 7-fold lower percentage of contaminant cells, demonstrating that contamination is dependent on specific cell properties. CONCLUSIONS: Previously published methodological works have focused on achieving high transfection rate in densely packed CA. Here, we focused in an equally important parameter: The interspot contamination. The presented quality control is essential for estimating the rate of contamination, a major source of false positives and negatives in current microscopy based functional genomics screenings. We have demonstrate that a washing step after seeding enhances CA quality for HeLA but is not necessary for MCF7. The described method provides a way to find optimal seeding protocols for cell lines intended to be used for the first time in CA.     

Respiration rate of male and female cones of Pinus contorta

Summary The effect of light on the efflux of CO₂ from reproductive structures and vegetative buds of Pinus contorta (Dougl.) was measured 12 times during the life cycle of female cones and three times towards the end of the life cycle of male cones. The net efflux of CO₂ from female cones, vegetative and male cone-bearing buds decreased exponentially with increasing quantum flux density. A simple model was derived relating temperature and quantum flux density to the net CO₂ efflux from female cones. This model was used to estimate the net respiratory losses from female cones from a few weeks after pollination until the cones senesced with the light regime normally experienced at Edinburgh (56° N). It was estimated that during that period the net respiration rate of female cones was 25% less than their dark respiration rate. This model was also used to estimate the net respiratory losses from an estimated crop of female cones in two forest stands and the simulation model MAESTRO was used to estimate the CO₂ assimilated by the needles on the trees over the same days. These simulations suggest that female cones normally respire carbon equivalent to only a small proportion (<3%) of a tree's daily assimilated CO₂.     

Functional stability of microbial communities in contaminated soils

Functional stability, measured in terms of resistance and resilience of respiration and growth rate of bacteria and fungi, was studied in soils that have been exposed to copper and low pH for more than twenty years. We used treatments, consisting of soil with no or high copper load (0 or 750 kg ha^?1) and low or neutral pH (4.0 or 6.1). Stability was examined by applying an additional stress in the form of lead or salt. After addition of lead, respiration (decomposition of freshly added lucerne meal) showed lower resistance at low than at neutral pH and at high copper than at low copper. The most acid and contaminated soil was the least resistant. Respiration showed no resilience after addition of lead. Bacterial growth rate (thymidine incorporation) also showed resistance at low pH but only in soils that were not contaminated with copper. After addition of salt, respiration showed no differences in resistance but the soils without copper contamination showed higher resilience. Bacterial growth rate showed lower resistance at low pH than at neutral pH, the latter in which the growth rate increased by on average 123%. This increase at high pH was faster in soil without copper than in soil with copper contamination in which the growth rate initially decreased and then increased. The effects of secondary stress depended on the nature of the stress (lead or salt) and on the parameter measured (respiration or bacterial growth rate). In general the highest resistance and/or resilience were found in the least contaminated soils with neutral pH and/or no copper contamination. Thus, the microbial communities in the cleaner soils showed the highest functional stability. The results seem to confirm the notion that environmental stress alters ecosystems such that supplementary stress will have stronger impacts than in an unstressed system. The results may also confirm the insurance‐hypothesis that reduced biodiversity due to the first stress negatively affected community stability. As an alternative, we discuss the observed effects in terms of altered energy budget.     

RACK1 is necessary for the formation of point contacts and regulates axon growth

Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal scaffolding protein that can interact with multiple signaling molecules concurrently through its seven WD40 repeats. We recently found that RACK1 is localized to mammalian growth cones, prompting an investigation into its role during neural development. Here, we show for the first time that RACK1 localizes to point contacts within mouse cortical growth cones. Point contacts are adhesion sites that link the actin network within growth cones to the extracellular matrix, and are necessary for appropriate axon guidance. Our experiments show that RACK1 is necessary for point contact formation. Brain‐derived neurotrophic factor (BDNF) stimulates an increase in point contact density, which was eliminated by RACK1 shRNA or overexpression of a nonphosphorylatable mutant form of RACK1. We also found that axonal growth requires both RACK1 expression and phosphorylation. We have previously shown that the local translation of β‐actin mRNA within growth cones is necessary for appropriate axon guidance and is dependent on RACK1. Thus, we examined the location of members of the local translation complex relative to point contacts. Indeed, both β‐actin mRNA and RACK1 colocalize with point contacts, and this colocalization increases following BDNF stimulation. This implies the novel finding that local translation is regulated at point contacts. Taken together, these data suggest that point contacts are a targeted site of local translation within growth cones, and RACK1 is a critical member of the point contact complex and necessary for appropriate neural development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1038–1056, 2017     

Faulty washers and soiled micropipettors may generate false positive serological results

Background: Serological markers such as HBsAg and anti-HIV may be present in serum at very high concentrations and this may give rise to erroneous diagnoses due to cross-contamination.Objectives: To investigate poor equipment maintenance and use, including contamination with human serum, as a potential source of erroneous assay results.Study design: The potential of microtitre plate washers and micropipettors to transfer material between microplate wells and between specimens was examined. For the study of micropipettors we recruited 19 UK diagnostic laboratories.Results: Four out of seven plate washers in use, until adjusted, had the potential to cause false positive HBsAg reactions. The centering of the probes that delivered the wash fluid, delivery pressure, wash volume and the use of a pre-programmed card to direct the washing procedure were important variables. We investigated soiling of tip cones of micropipettors. In every laboratory human IgG could be detected in at least a third of eluates from micropipettor tip cones; only 31 (14%) of 222 showed no evidence of contamination with human serum. Only one laboratory submitted eluates devoid of specific antibodies. Anti-HAV was the marker most commonly found (n = 68), followed by HBsAg (n = 27) and anti-HIV (n = 20). Seven micropipettor eluates from two laboratories were radioactively contaminated.Conclusions: Recommended precautions are regular checking, cleaning and servicing of equipment, care in interpreting weak reactions, reference back to serum left on the clot of the original specimen and testing of a follow up specimen. Poorly maintained immunoassay equipment can readily generate false positive results due to low-level cross-contamination, particularly with the current highly sensitive HBsAg and anti-HIV assays.