Microbial source tracking in a rural watershed dominated by cattle.

Antibiotic resistance analysis (ARA), frequency of sampling, and seasonality were evaluated in a rural Virginia watershed dominated by cattle. The selected watershed (Mill Creek) was 3767 ha in size, included two small communities (one sewered and one unsewered), and several farms that when combined contained over 3800 beef and dairy cattle. Monthly monitoring of fecal coliforms at two sampling sites in Mill Creek from January to December, 2001, revealed that the recreational standard (1000 colony forming units, CFUs/100 ml) was exceeded a total of eight times for a 33% violation rate at each site. In addition, stream samples were collected weekly for 4 consecutive weeks during seasonal high flows (March) and seasonal low flows (September-October), plus daily for 7 consecutive days within the weekly schedules for a combined total of 60 stream samples (30 at each of two sites). The recreational standard was exceeded once during seasonal high flow and nine times during seasonal low flow. Microbial source tracking (MST) was performed by ARA to assess the impact of cattle on water quality within the different sampling routines. The resistance patterns of 2880 water isolates and 1158 known source (host-origin) isolates were determined with seven antibiotics at 28 different concentrations. The 1158 isolate database was reduced to 562 unique isolates when clonal ARA patterns were removed. This database of 562 unique isolates had an average rate of correct classification (ARCC) of 95.4%, and several statistical procedures confirmed the library as accurate and representative. Sixty-five percent of 50 challenge-set isolates from sources, but not samples, used in the library were correctly identified. The 562 unique pattern database was used to classify Escherichia coli isolates from water samples into six host source categories. The ARA results showed that cattle were the major source of pollution in the stream and cattle were the dominant source in over 60% of the water samples. Sampling frequency and seasonality had no effect on the MST results, as cattle dominated both seasons and samplings. Deer were a minor contributor in the summer (high water demand), and geese were a minor contributor in the winter when migratory flocks were observed moving through the watershed. An unexpected human allocation was found, especially under seasonal high flow conditions. The exact origin of this human allocation is not known. This project demonstrated that a host-origin library, based on a phenotypic method, could be developed for a well-defined watershed and was both representative of the sources in the watershed and performed reasonably well against a challenge set.

Malaria early warnings based on seasonal climate forecasts from multi-model ensembles.

The control of epidemic malaria is a priority for the international health community and specific targets for the early detection and effective control of epidemics have been agreed. Interannual climate variability is an important determinant of epidemics in parts of Africa where climate drives both mosquito vector dynamics and parasite development rates. Hence, skilful seasonal climate forecasts may provide early warning of changes of risk in epidemic-prone regions. Here we discuss the development of a system to forecast probabilities of anomalously high and low malaria incidence with dynamically based, seasonal-timescale, multi-model ensemble predictions of climate, using leading global coupled ocean-atmosphere climate models developed in Europe. This forecast system is successfully applied to the prediction of malaria risk in Botswana, where links between malaria and climate variability are well established, adding up to four months lead time over malaria warnings issued with observed precipitation and having a comparably high level of probabilistic prediction skill. In years in which the forecast probability distribution is different from that of climatology, malaria decision-makers can use this information for improved resource allocation.

Probabilistic prediction of climate using multi-model ensembles: from basics to applications.

The development of multi-model ensembles for reliable predictions of inter-annual climate fluctuations and climate change, and their application to health, agronomy and water management, are discussed.

Trace formation during locomotion of L929 mouse fibroblasts continuously recorded by interference reflection microscopy (IRM).

The recently reported formation of highly ordered traces by migrating cells has been studied on L929 fibroblasts in time lapse experiments by means of interference reflection microscopy (IRM) as well as by conventional microscopy. Formation of pronounced traces on glass substrates correlates to migration after cell division, and the trace arrangement on the substrate depends on migration velocity: slow migration results in a highly branched, broad, and relatively short trace, while fast migration yields a slim and long trace with few branches. IRM-irradiation caused cessation of locomotion and trace formation and accelerated degradation of existing traces. Traces consist of cord-like cytoplasmic strands, which contain F-actin filaments and they seem to be enveloped by a membrane. It is supposed that cell traces are homologous to filopodia. Traces arise mainly from non-retracted filopodia at the rear margin of the migrating cell. The branches within the traces are the result of the repeated stretching out of a backwardly directed lamellipodium. They arise from the formation of new filopodia that emerge at the actin ribs of the lamellipodium.

Topography of cell traces studied by atomic force microscopy.

Migrating adherent cells release material onto artificial substrates like glass and silicon while moving. Traces of mouse fibroblasts (L929) have been visualised by atomic force microscopy (AFM). "Non-contact" mode AFM in a liquid environment can extract topographic information from these traces. This dynamic mode allows the study of these soft structures without damage or compression. The AFM images show crossing and branching networks (with specific angles of branching), structured patches, nodular elements, linear elements with irregular height and other features. Fourier analysis of segment spacing in the strands is presented. These spatial features of fibroblast traces are strong indications that actin linked to structural proteins is involved in the formation of cell traces. We also give methods for trace preparation and undistorted imaging and discuss further perspectives.

Single micro electrode dielectrophoretic tweezers for manipulation of suspended cells and particles.

Cells or particles in aqueous suspension close to a single capacitively coupled micro electrode (CCME) driven with high frequency electric fields experience dielectrophoretic forces. The effects near the CCME can be used for trapping and manipulation of single cells using externally metallised glass pipettes and might be used to develop a microscope based on force or capacitance measurements in conductive media.

Cell traces--footprints of individual cells during locomotion and adhesion.

Animal cells release traces of material onto glass or silicon surfaces during adhesion and migration. This little studied phenomenon is a widespread and normal concomitant of cell migration. The paper introduces the study of such material. The traces can be visualised by different microscopic techniques (e.g. TIRF, IRM, CLSM, AFM, SEM). Cell traces typical for different cell lines (NIH 3T3 and L929 mouse fibroblasts, mouse macrophages, mouse sarcoma cells and human osteosarcoma cells) are shown and discussed. There are well organised structures such as different linear and nodular elements as well as patches. Traces can extend up to some hundred micrometers from the cell, but the dimensions of the linear elements are in the submicron range. Cell traces are not identical with focal contacts but can include them. A first classification of basic elements is proposed. It allows an estimation of the total volume and surface in comparison to the donor cell. Higher order structures are discussed and a first insight into the protein composition of traces produced by mouse fibroblasts is given. Our observations, together with the cell adhesion literature suggest that the amount of released material, its extent and chemical and structural properties depend on cell type and physiology as well as other external influences. Cell traces in combination with the adhesion pattern of the donor cell should give information about the activity and physiological status of individual cells, the mechanisms of cell locomotion and the molecular composition of the donor cell membrane. The traces might possibly be used as submicron elements for passive electric characterisation and biotechnological applications.

pH-regulated electroretention chromatography: towards a new method for the separation of proteins according to their isoelectric points.

The pH-dependent electroretention behavior of model proteins cytochrome c and ribonuclease A was studied in a hollow fiber arrangement, similar to that used in electrical field-flow fractionation. Field-induced immobilization of the proteins at the inner wall of the fiber was a function of the pH adjusted in the solution surrounding it, indicating that the pH inside the fiber lumen, relevant for protein migration, quickly equilibrates to the regulated value outside. A complete separation of the model proteins was achieved. Advantages of the principle as well as prospects for the development of a technique separating more than two protein species according to their isoelectric points are discussed.

Amperometric pH regulation--a flexible tool for rapid and precise temporal control over the pH of an electrolyte solution.

Temporal control over both pH and ionic strength of an electrolyte solution with high accuracy was achieved with a dynamic, computer feedback-controlled amperometric pH-stat device consisting of four pH-regulating electrodes placed in electrolyte reservoirs that are separated by dialysis membranes from a central compartment. Theoretical predictions of the behavior of this arrangement, obtained by computer simulation, were validated by running temporal pH programs such as step functions, oscillations, and linear pH gradients. Deviations from nominal values given by the computer program are within the limits of accuracy of the pH-measuring electrodes. No volume changes accompany a change of pH or conductivity since ions are forced to leave or enter the central compartment through the membranes by the electrical force applied between the pH-regulating electrodes. The device is flexible, easy to use and easily miniaturized. We discuss a wide range of possible applications in biochemistry and cell science. These include automated pH adjustment, isoelectric protein separation, amperometric measurement of enzyme kinetics and the response of cell cultures to well-defined pH changes.

Characterisation of cell movement by impedance measurement on fibroblasts grown on perforated Si-membranes.

Mouse fibroblasts grown on perforated Si-membranes (pore diameter approximately 10 microns have been studied to clarify cell locomotive ability. The cell motility was microscopically monitored by a time-lapse video system and, simultaneously, the impedance of the growing cells was measured every 5 s. The correlations between observed cell activities and measured impedance events are discussed and classified. The method is sensitive and allows discrimination between signals arising from translocation of single cells and those arising from filopodia activities. Both cell and filopodia motion could be detected. Designs of microdevices fabricated in semiconductor technology are presented.

Radio-frequency microtools for particle and liver cell manipulation.

Single particles can be manipulated by applying high frequencies to ultramicro electrode arrays fabricated on planar structures. Heat production can be reduced to the extent that intense electric fields can be applied even to unmodified cell culture media. Animal cells grow normally in the high field (up to 100 kV/m) between such continuously energized multielectrodes. As with laser tweezers [1-3], this technique can capture particles and cells in field traps, generate linear movement, and permit cell cultivation. It can also produce micropatterns of pH gradients, field-cast objects, and control cell adhesion. These microtools may be combined to develop cell separators, microsensors, and controlled-biocompatibility surfaces.

A traveling-wave micropump for aqueous solutions: comparison of 1 g and microgram results.

The operation of a micro-chip-based fluid circulator based on the interaction of a high-frequency traveling wave with a thermally generated inhomogeneity in an aqueous medium is investigated. The profiles of the electric field, the thermal gradient and the driving force within the device are derived numerically, and the dependence upon applied voltage under different experimental conditions is derived. The importance of convection in the operation of the device is assessed by operating it in various orientations, as well as under microgravity, and also by investigating the reversal and turn-on characteristics. The pump showed the unexpected ability to trap microparticles present in the driven fluid.

Three-dimensional electric field traps for manipulation of cells--calculation and experimental verification.

The forces acting on dielectric particles and living cells exposed to alternating and rotating fields generated by three-dimensional multi-electrode arrangements are investigated. Numerical procedures are described for the calculation of the electric field distribution and forces. The physical treatment considers electrodes of any shape and dielectric particles of complex structure. Particle and cell trapping are based on negative dielectrophoretic forces produced by high-frequency a.c. or rotating electric fields up to 400 MHz. Various multi-electrode systems were realised in commercially fabricated microelectrode systems, and tested for their ability to move and assemble microparticles or living cells without contact with the electrodes. The field distribution and accuracy of phase-controlled power application was tested using individual artificial particles trapped in the electric field cage. Position and trajectories of particle motion were measured. The paper gives an overview of electrode and field cage design in the microscale range.

Levitation, holding, and rotation of cells within traps made by high-frequency fields.

Biological cells and other particles can be electrically manipulated by means of negative dielectrophoresis within microchambers whose electrode geometry is of the order of the cell size. Very-high-frequency fields (50 MHz and above) and media of increased relative permittivity are especially suitable for the purpose, as shown by experimental data on levitation and rotation. It appears to be possible to move and rotate cells or particles at will using this technology.

Traveling-wave dielectrophoresis of microparticles.

The traveling-wave-induced linear transfer of dielectric particles like living cells and artificial objects of microscopic dimensions is analyzed. It is shown that the electrode geometries must correspond to particle sizes to allow an effective manipulation of particles immersed in weakly electrolytic solutions by high frequency traveling waves. The theoretical model elaborated in this paper is in good agreement with experimental results obtained in microfabricated chambers of linearly arranged electrodes. It explains the behavior of homogeneous cellulose spheres as well as that of membrane-covered pine polls. The traveling-wave-driven electrodes are described by a superposition of time-dependent point charges. Subsequently, each of these point charges has to be considered as polarizing the dielectric particle and interacting with the polarized particle. This results in forces which effectively translocate the particle.

Steady state electrolysis and isoelectric focusing.

The properties of pH gradients formed by stationary electrolysis of weak mobile or fixed electrolytes are analyzed. The model uses the appropriate balance equations and those of chemical equilibria. It is shown how the equation of the current density has to be modified for considering that fraction of current that is associated with the diffusion of neutral buffer molecules within a pH gradient. Furthermore it is shown that the pH gradients themselves give rise to water production within the gradient and that essential properties of the steady state are related to chemical reactions between the electrolyte constituents. The differential equations describing the gradients of the concentration of a given component, the pH, conductivity and potential are explicitly formulated in relation to those reactions. The equations are solved numerically and the significance of the results for isoelectric focusing is discussed. The experimental conditions to reach shallow and smooth pH gradients exhibiting sufficient ionic strength are formulated.

Reversible and irreversible rotating field-induced membrane modifications.

We analyse the charge distribution as submitted by additionally induced transmembrane potentials in rotating electric fields. In contrast to d.c. and a.c. fields, in rotating fields the induced peak potential differences across the membrane systems are phase shifted with respect both to each other and to the external field vector. These phase differences are strongly frequency-dependent but were also influenced by the electrical properties of both the cell and the surrounding medium. We extend our investigation up to the non-linear field strength range of electrorotation and found reversible and irreversible changes in the rotational behavior of several cells. The most convenient variables for describing non-linear electrorotation are the characteristic frequency (fc1) and the corresponding angular velocity (omega c) of the cells. With increasing field strength the observed rotational behavior becomes more and more irreversible and finally rupture of the membrane occurs.

Differences in the rotation spectra of mouse oocytes and zygotes.

Rotation spectra of mouse oocytes, zygotes and embryos in the two-cell stage under the influence of high-frequency rotating fields were studied. The characteristic frequency (fc1) of cells isolated from superovulated + mated mice is different from that of oocytes. This was attributed to an increase in the membrane resistance and, less probably, to a change in the zona pellucida conductivity. The rotation spectra can be used to differentiate between non-fertilized and fertilized eggs. A theoretical interpretation of the measured spectra and simulation of the changes caused by fertilization is given.

Rotation of dielectrics in a rotating electric high-frequency field. Model experiments and theoretical explanation of the rotation effect of living cells.

Model experiments are carried out to clarify the mechanism of rotation of living cells in a rotating electric field. According to classical investigations of the rotation of macroscopic bodies in external fields, the rotation of spherical glass vessels or metal cylinder filled with electrolyte solutions was investigated. The relation of the calculations of Lertes (1921a,b) to the recent paper of Arnold and Zimmerman (1982) and our new derivations lead to equations explaining the rotation of objects. The results are compared with measurements using mesophyll protoplasts and data from the literature.

Evaluation of colony-forming ability experiments using normal and DNA repair-deficient human fibroblast strains and an automatic colony counter.

An automated procedure for evaluation of colony-forming ability experiments has been developed using normal and xeroderma pigmentosum fibroblast strains. Technically this consists of an image analyzer equipped with a contrast-intensifying video camera, monitor, and a desk-top computer. Complex structures composed of overlapping colonies could be reduced to the constituent single colonies by special correction programs. Eighteen experiments were evaluated, and the D0 values for colony-forming ability were determined. A systematic but correctable deviation between visual and automated counting was found. The source of this deviation is described and a solution for correction is provided. The automatic procedure reduced the evaluating time by a factor of five to ten.