Multistage Electrophoretic Separators

A multistage electrophoresis apparatus has been invented for use in the separation of cells, protein molecules, and other particles and solutes in concentrated aqueous solutions and suspensions. The design exploits free electrophoresis but overcomes the deficiencies of prior free-electrophoretic separators by incorporating a combination of published advances in mathematical modeling of convection, sedimentation, electro-osmotic flow, and the sedimentation and aggregation of droplets. In comparison with other electrophoretic separators, these apparatuses are easier to use and are better suited to separation in relatively large quantities characterized in the art as preparative (in contradistinction to smaller quantities characterized in the art as analytical). In a multistage electrophoretic separator according to the invention, an applied vertical steady electric field draws the electrically charged particles of interest from within a cuvette to within a collection cavity that has been moved into position of the cuvette. There are multiple collection cavities arranged in a circle; each is aligned with the cuvette for a prescribed short time. The multistage, short-migration-path character of the invention solves, possibly for the first time, the fluid-instability problems associated with free electrophoresis. The figure shows a prototype multistage electrophoretic separator that includes four sample stations and five collection stages per sample. At each sample station, an aqueous solution or suspension containing charged species to be separated is loaded into a cuvette, which is machined into a top plate. The apparatus includes a lower plate, into which 20 collection cavities have been milled. Each cavity is filled with an electrophoresis buffer solution. For the collection of an electrophoretic fraction, the lower plate is rotated to move a designated collection cavity into alignment with the opening of the cuvette. An electric field is then applied between a non-gassing electrode in the collection cavity and an electrolyte compartment, which is separated from the cuvette by a semipermeable membrane. The electrolyte is refreshed by circulation by use of a peristaltic pump. In subsequent steps, the lower plate is rotated to collect other electrophoretic fractions. Later, the collected fractions are removed from the collection cavities through ports that have threaded plugs. The base of the apparatus contains power supplies and a computer interface. The design includes provisions for monitoring and feedback control of cavity position, electric field, and temperature. The operation of the apparatus can easily be automated, as demonstrated by use of software that has already been written for this purpose

Multistage Magnetic Separator of Cells and Proteins

The multistage electromagnetic separator for purifying cells and magnetic particles (MAGSEP) is a laboratory apparatus for separating and/or purifying particles (especially biological cells) on the basis of their magnetic susceptibility and magnetophoretic mobility. Whereas a typical prior apparatus based on similar principles offers only a single stage of separation, the MAGSEP, as its full name indicates, offers multiple stages of separation; this makes it possible to refine a sample population of particles to a higher level of purity or to categorize multiple portions of the sample on the basis of magnetic susceptibility and/or magnetophoretic mobility. The MAGSEP includes a processing unit and an electronic unit coupled to a personal computer. The processing unit includes upper and lower plates, a plate-rotation system, an electromagnet, an electromagnet-translation system, and a capture-magnet assembly. The plates are bolted together through a roller bearing that allows the plates to rotate with respect to each other. An interface between the plates acts as a seal for separating fluids. A lower cuvette can be aligned with as many as 15 upper cuvette stations for fraction collection during processing. A two-phase stepping motor drives the rotation system, causing the upper plate to rotate for the collection of each fraction of the sample material. The electromagnet generates a magnetic field across the lower cuvette, while the translation system translates the electromagnet upward along the lower cuvette. The current supplied to the electromagnet, and thus the magnetic flux density at the pole face of the electromagnet, can be set at a programmed value between 0 and 1,400 gauss (0.14 T). The rate of translation can be programmed between 5 and 2,000 m/s so as to align all sample particles in the same position in the cuvette. The capture magnet can be a permanent magnet. It is mounted on an arm connected to a stepping motor. The stepping motor rotates the arm to position the capture magnet above the upper cuvette into which a fraction of the sample is collected. The electronic unit includes a power switch, power-supply circuitry that accepts 110-Vac input power, an RS-232 interface, and status lights. The personal computer runs the MAGSEP software and controls the operation of the MAGSEP through the RS-232 interface. The status of the power, the translating electromagnet, the capture magnet, and the rotation of the upper plate are indicated in a graphical user interface on the computer screen

Engineering Support of Microgravity Life Science Research: Development of an Avian Development Facility

The Avian Development Facility (ADF) is designed to provide a 'window' for the study of embryogenesis in space. It allows researchers to determine and then to mitigate or nullify the forces of altered gravity upon embryos when leaving and re-entering the Earth's gravity. The ADF design will allow investigations to begin their incubation after their experiments have achieved orbit, and shut down the experiment and fix specimens before leaving orbit. In effect, the ADF makes every attempt to minimize launch and re-entry effects in order to isolate and preserve the effects of the experimental variable(s) of the space environment

Apparatus and method for centrifugation and robotic manipulation of samples

A device for centrifugation and robotic manipulation of specimen samples, including incubating eggs, and uses thereof are provided. The device may advantageously be used for the incubation of avian, reptilian or any type of vertebrate eggs. The apparatus comprises a mechanism for holding samples individually, rotating them individually, rotating them on a centrifuge collectively, injecting them individually with a fixative or other chemical reagent, and maintaining them at controlled temperature, relative humidity and atmospheric composition. The device is applicable to experiments involving entities other than eggs, such as invertebrate specimens, plants, microorganisms and molecular systems

Phenotypic determinants of inter-individual variability of litter consumption rate in a detritivore population

The metabolic theory of ecology predicts resource consumption rates of animals from their body mass, but other phenotypic traits might affect individual resource consumption rate. In this paper, we used a hierarchical framework to examine relation- ships between phenotypic traits thought to constrain variation in per capita resource consumption rate. Physiological and behavioural traits were assumed to be impor- tant in mediating the control of morphology and sex on consumption. We conducted a longitudinal study aiming to relate the consumption rate of submerged leaf litter to sex, morphological, physiological and behavioural traits in an aquatic detritivore population. Then, we analysed the pattern of trait covariation using structural equa- tion modelling (SEM). We observed broad and repeatable inter-individual variation in leaf consumption rate and other phenotypic traits. We found that expressing litter consumption rate relative to the time individuals spent feeding revealed and increased the effect of body mass and sex differences, respectively. Accordingly, SEM analyses showed that time allocated to resource acquisition mediated body mass and sex effects on apparent litter consumption rate whose variation was also accounted for by an indicator of activity-specific metabolic rate. Substantial variation in resource consump- tion rate was due to sex difference whereas body mass was of secondary importance. Individual phenotypic trait variations strongly altered consumer–resource relation- ships. Therefore, we encourage studies on consumers’ intraspecific variability to advance knowledge about phenotypic determinants of individual resource consump- tion, an important link between individuals and ecosystems

New Applications of Gamma Spectroscopy: Characterization Tools for D&D Process Development, Inventory Reduction Planning & Shipping, Safety Analysis & Facility Management During the Heavy Element Facility Risk Reduction Program

Novel applications of gamma ray spectroscopy for D&D process development, inventory reduction, safety analysis and facility management are discussed in this paper. These applications of gamma spectroscopy were developed and implemented during the Risk Reduction Program (RPP) to successfully downgrade the Heavy Element Facility (B251) at Lawrence Livermore National Laboratory (LLNL) from a Category II Nuclear Facility to a Radiological Facility. Non-destructive assay in general, gamma spectroscopy in particular, were found to be important tools in project management, work planning, and work control (''Expect the unexpected and confirm the expected''), minimizing worker dose, and resulted in significant safety improvements and operational efficiencies. Inventory reduction activities utilized gamma spectroscopy to identify and confirm isotopics of legacy inventory, ingrowth of daughter products and the presence of process impurities; quantify inventory; prioritize work activities for project management; and to supply information to satisfy shipper/receiver documentation requirements. D&D activities utilize in-situ gamma spectroscopy to identify and confirm isotopics of legacy contamination; quantify contamination levels and monitor the progress of decontamination efforts; and determine the point of diminishing returns in decontaminating enclosures and glove boxes containing high specific activity isotopes such as {sup 244}Cm and {sup 238}Pu. In-situ gamma spectroscopy provided quantitative comparisons of several decontamination techniques (e.g. TLC-free Stripcoat{trademark}, Radiac{trademark} wash, acid wash, scrubbing) and was used as a part of an iterative process to determine the appropriate level of decontamination and optimal cost to benefit ratio. Facility management followed a formal, rigorous process utilizing an independent, state certified, peer-reviewed gamma spectroscopy program, in conjunction with other characterization techniques, process knowledge, and historical records, to provide information for work planning, work prioritization, work control, and safety analyses (e.g. development of hold points, stop work points); and resulted in B251 successfully achieving Radiological status on schedule. Gamma spectroscopy helped to define operational approaches to achieve radiation exposure ALARA, e.g. hold points, appropriate engineering controls, PPE, workstations, and time/distance/shielding in the development of ALARA plans. These applications of gamma spectroscopy can be used to improve similar activities at other facilities

Behavioural and Physiological Responses of Gammarus pulex Exposed to Cadmium and Arsenate at Three Temperatures: Individual and Combined Effects

This study aimed at investigating both the individual and combined effects of cadmium (Cd) and arsenate (AsV) on the physiology and behaviour of the Crustacean Gammarus pulex at three temperatures (5, 10 and15°C). G. pulex was exposed during 96 h to (i) two [Cd] alone, (ii) two [AsV] alone, and (iii) four combinations of [Cd] and [AsV] to obtain a complete factorial plane. After exposure, survival, [AsV] or [Cd] in body tissues, behavioural (ventilatory and locomotor activities) and physiological responses (iono-regulation of [Na+] and [Cl−] in haemolymph) were examined. The interactive effects (antagonistic, additive or synergistic) of binary mixtures were evaluated for each tested temperature using a predictive model for the theoretically expected interactive effect of chemicals. In single metal exposure, both the internal metal concentration in body tissues and the mortality rate increased along metallic gradient concentration. Cd alone significantly impaired both [Na+] and [Cl−] while AsV alone had a weak impact only on [Cl−]. The behavioural responses of G. pulex declined with increasing metal concentration suggesting a reallocation of energy from behavioural responses to maintenance functions. The interaction between AsV and Cd was considered as ‘additive’ for all the tested binary mixtures and temperatures (except for the lowest combination at 10°C considered as “antagonistic”). In binary mixtures, the decrease in both ventilatory and locomotor activities and the decline in haemolymphatic [Cl−] were amplified when respectively compared to those observed with the same concentrations of AsV or Cd alone. However, the presence of AsV decreased the haemolymphatic [Na+] loss when G. pulex was exposed to the lowest Cd concentration. Finally, the observed physiological and behavioural effects (except ventilation) in G. pulex exposed to AsV and/or Cd were exacerbated under the highest temperature. The discussion encompasses both the toxicity mechanisms of these metals and their interaction with rising temperature

Environmental Report 1998

This summary provides an overview of LLNL's environmental activities in 1998, including radiological and nonradiological surveillance, effluent, and compliance monitoring, remediation, assessment of radiological releases and doses, and determination of the impact of LLNL operations on the environment and public health

The impact of natural and anthropogenic Dissolved Organic Carbon (DOC), and pH on the toxicity of triclosan to the crustacean Gammarus pulex (L.).

Regulatory ecotoxicology testing rarely accounts for the influence of natural water chemistry on the bioavailability and toxicity of a chemical. Therefore, this study identifies whether key omissions in relation to Dissolved Organic Carbon (DOC) and pH have an impact on measured effect concentrations (EC). Laboratory ecotoxicology tests were undertaken for the widely used antimicrobial compound triclosan, using adult Gammarus pulex (L.), a wild-type amphipod using synthetic fresh water, humic acid solutions and wastewater treatment works effluent. The toxicity of triclosan was tested at two different pHs of 7.3 and 8.4, with and without the addition of DOC and 24 and 48hour EC values with calculated 95% confidence intervals calculated. Toxicity tests undertaken at a pH above triclosan's pKa and in the presents of humic acid and effluent, containing 11 and 16mgL(-1) mean DOC concentrations respectively, resulted in significantly decreased triclosan toxicity. This was most likely a result of varying triclosan speciation and complexation due to triclosan's pKa and high hydrophobicity controlling its bioavailability. The mean 48hour EC50 values varied between 0.75±0.45 and 1.93±0.12mgL(-1) depending on conditions. These results suggest that standard ecotoxicology tests can cause inaccurate estimations of triclosan's bioavailability and subsequent toxicity in natural aquatic environments. These results highlight the need for further consideration regarding the role that water chemistry has on the toxicity of organic contaminants and how ambient environmental conditions are incorporated into the standard setting and consenting processes in the future