Differential release of manure-borne bioactive phosphorus forms to runoff and leachate under simulated rain.

Limited information exists on the unhindered release of bioactive phosphorus (P) from a manure layer to model the partitioning and transport of component P forms before they reach an underlying soil. Rain simulations were conducted to quantify effects of intensity (30, 60, and 90 mm h-1) on P release from an application of 60 Mg ha-1 of dairy manure. Runoff contained water-extractable- (WEP), exchangeable and enzyme-labile bioactive P (TBIOP), in contrast to the operationally defined "dissolved-reactive P" form. The released P concentrations and flow-weighed mass loads were described by the log-normal probability density function. At a reference condition of 30 mm h-1 and maintaining the surface at a 5% incline, runoff was minimal, and WEP accounted for 20.9% of leached total P (TP) concentrations, with an additional 25-30% as exchangeable and enzyme-labile bioactive P over the 1-h simulation. On a 20% incline, increased intensity accelerated occurrence of concentrationmax and shifted the skewed P concentration distribution more to the left. Differences in trends of WEP, TBIOP, or net enzyme-labile P (PHPo) cumulative mass released per unit mass of manure between intensities were attributable to the higher frequency of raindrops striking the manure layer, thus increasing detachment and load of colloidal PHPo of the water phases. Thus, detailed knowledge of manure physical characteristics, bioactive P distribution in relation to rain intensity, and attainment of steady-state of water fluxes were critical factors in improved prediction of partitioning and movement of manure-borne P under rainfall.

Depth-Dependent Survival of Escherichia coli and Enterococci in Soil after Manure Application and Simulated Rainfall.

Once released, manure-borne bacteria can enter runoff via interaction with the thin mixing layer near the soil surface. The objectives of this work were to document temporal changes in profile distributions of manure-borne Escherichia coli and enterococci in the near-surface soil layers after simulated rainfalls and to examine differences in survival of the two fecal indicator bacteria. Rainfall simulations were performed in triplicate on soil-filled boxes with grass cover and solid manure application for 1 h with rainfall depths of 30, 60, and 90 mm. Soil samples were collected weekly from depth ranges of 0 to 1, 1 to 2, 2 to 5, and 5 to 10 cm for 1 month. Rainfall intensity was found to have a significant impact on the initial concentrations of fecal indicator bacteria in the soil. While total numbers of enterococci rapidly declined over time, E. coli populations experienced initial growth with concentration increases of 4, 10, and 25 times the initial levels at rainfall treatment depths of 30, 60, and 90 mm, respectively. E. coli populations grew to the approximately the same level in all treatments. The 0- to 1-cm layer contained more indicator bacteria than the layers beneath it, and survival of indicator bacteria was better in this layer, with decimation times between 12 and 18 days after the first week of growth. The proportion of bacteria in the 0- to 1-cm layer grew with time as the total number of bacteria in the 0- to 10-cm layer declined. The results of this work indicate the need to revisit the bacterial survival patterns that are assumed in water quality models.

Response of coliform populations in streambed sediment and water column to changes in nutrient concentrations in water.

As sediments increasingly become recognized as reservoirs of indicator and pathogen microorganisms, an understanding of the persistence of indicator organisms becomes important for assessment and predictions of microbial water quality. The objective of this work was to observe the response of water column and sediment coliform populations to the change in nutrient concentrations in the water column. Survival experiments were conducted in flow-through chambers containing sandy sediments. Bovine feces were collected fresh and introduced into sediment. Sixteen days later, the same fecal material was autoclaved and diluted to provide three levels - 1×, 0.5×, and 0.1× of nutrient concentrations - spike in water column. Total coliforms, Escherichia coli, and total aerobic heterotrophic bacterial concentrations were monitored in water and sediment. Bacteria responded to the nutrient spike with initial growth both in the water column and in sediment. The response of bacterial concentrations in water column was nonlinear, with no significant changes at 0.1 and .5× spikes, but a substantial change at 1× spike. Bacteria in sediment responded to the spikes at all added nutrient levels. Coliform inactivation rates both in sediment and in water after the initial growth occurred, were not significantly different from the inactivation rates before spike. These results indicate that introduction of nutrients into the water column results in nonlinear response of E. coli concentrations both in water and in sediments, followed by the inactivation with the same rate as before introduction of nutrients.

Comparing temperature effects on Escherichia coli, Salmonella, and Enterococcus survival in surface waters.

UNLABELLED: The objective of this study was to compare dependency of survival rates on temperature for indicator organisms Escherichia coli and Enterococcus and the pathogen Salmonella in surface waters. A database of 86 survival datasets from peer-reviewed papers on inactivation of E. coli, Salmonella and Enterococcus in marine waters and of E. coli and Salmonella in lake waters was assembled. The Q10 model was used to express temperature effect on survival rates obtained from linear sections of semi-logarithmic survival graphs. Available data were insufficient to establish differences in survival rates and temperature dependencies for marine waters where values of Q10  = 3 and a survival rate of 0·7 day(-1) could be applied. The Q10 values in lake waters were substantially lower in marine waters, and Salmonella inactivation in lake water was, on average, twice as fast as E. coli; data on E. coli substantially outnumber data on Enterococcus and Salmonella. The relative increase in inactivation with increase in temperature is higher in marine waters than lake water, and differences in inactivation between Salmonella and E. coli at a given temperature were significant in lake water but not in marine waters. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiological quality of surface waters is of paramount importance for public health. The novelty of this work is using a large compendium of published data to develop the first comparison of temperature effects on survival of the pathogen Salmonella and water quality indicator micro-organisms Escherichia coli and Enterococcus in natural waters. The existing relatively large body of knowledge on E. coli survival appears to be useful to assess the effect of temperature on survival of Salmonella. Moreover, results of this work constitute an essential input in models to support environmental management decisions on the use of surface water sources in agriculture, aquaculture and recreation.

Performance of Weibull and Linear Semi-logarithmic Models in Simulating Inactivation in Waters.

Modeling inactivation of indicator microorganisms is a necessary component of microbial water quality forecast and management recommendations. The linear semi-logarithmic (LSL) model is commonly used to simulate the dependencies of bacterial concentrations in waters on time. There were indications that assumption of the semi-logarithmic linearity may not be accurate enough in waters. The objective of this work was to compare performance of the LSL and the two-parametric Weibull inactivation models with data on survival of indicator organism in various types of water from a representative database of 167 laboratory experiments. The Weibull model was preferred in >99% of all cases when the root mean squared errors and Nash-Sutcliffe statistics were compared. Comparison of corrected Akaike statistic values gave the preference to the Weibull model in only 35% of cases. This was caused by (i) a small number of experimental points on some inactivation curves, (ii) closeness of the shape parameter of the Weibull equation to one, and (iii) piecewise log-linear inactivation dynamic that could be well described by neither of the two models compared. Based on the Akaike test, the Weibull model was favored in agricultural, lake, and pristine waters, whereas the LSL model was preferred for groundwater, wastewater, rivers, and marine waters. The decimal reduction time parameter of both the LSL and Weibull models exhibited an Arrhenius-type dependence on temperature. Overall, the existing inactivation data indicate that the application of the Weibull model can improve the predictive capabilities of microbial water quality modeling.

Modeling transport of Escherichia coli in a creek during and after artificial high-flow events: three-year study and analysis.

Escherichia coli is the leading indicator of microbial contamination of natural waters, and so its in-stream fate and transport needs to be understood to eventually minimize surface water contamination by microorganisms. To better understand mechanisms of E. coli release and transport from soil sediment in a creek the artificial high-water flow events were created by releasing 60-80 m(3) of city water on a tarp-covered stream bank in four equal allotments in July 2008, 2009 and 2010. A conservative tracer difluorobenzoic acid (DFBA) was added to the released water in 2009 and 2010. Water flow rate, E. coli and DFBA concentrations as well as water turbidity were monitored with automated samplers at three in-stream weirs. A one-dimensional model was applied to simulate water flow, and E. coli and DFBA transport during these experiments. The Saint-Venant equations were used to calculate water depth and discharge while a stream solute transport model accounted for release of bacteria by shear stress from bottom sediments, advection-dispersion, and exchange with transient storage (TS). Reach-specific model parameters were estimated by evaluating observed time series of flow rates and concentrations of DFBA and E. coli at all three weir stations. Observed DFBA and E. coli breakthrough curves (BTC) exhibited long tails after the water pulse and tracer peaks had passed indicating that transient storage (TS) might be an important element of the in-stream transport process. Comparison of simulated and measured E. coli concentrations indicated that significant release of E. coli continued when water flow returned to the base level after the water pulse passed and bottom shear stress was small. The mechanism of bacteria continuing release from sediment could be the erosive boundary layer exchange enhanced by changes in biofilm properties by erosion and sloughing detachment.

Escherichia coli survival in waters: temperature dependence.

Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 model. This suggestion was made 34 years ago based on 20 survival curves taken from published literature, but has not been revisited since then. The objective of this study was to re-evaluate the accuracy of the Q10 equation, utilizing data accumulated since 1978. We assembled a database of 450 E. coli survival datasets from 70 peer-reviewed papers. We then focused on the 170 curves taken from experiments that were performed in the laboratory under dark conditions to exclude the effects of sunlight and other field factors that could cause additional variability in results. All datasets were tabulated dependencies "log concentration vs. time." There were three major patterns of inactivation: about half of the datasets had a section of fast log-linear inactivation followed by a section of slow log-linear inactivation; about a quarter of the datasets had a lag period followed by log-linear inactivation; and the remaining quarter were approximately linear throughout. First-order inactivation rate constants were calculated from the linear sections of all survival curves and the data grouped by water sources, including waters of agricultural origin, pristine water sources, groundwater and wells, lakes and reservoirs, rivers and streams, estuaries and seawater, and wastewater. Dependency of E. coli inactivation rates on temperature varied among the water sources. There was a significant difference in inactivation rate values at the reference temperature between rivers and agricultural waters, wastewaters and agricultural waters, rivers and lakes, and wastewater and lakes. At specific sites, the Q10 equation was more accurate in rivers and coastal waters than in lakes making the value of the Q10 coefficient appear to be site-specific. Results of this work indicate possible sources of uncertainty to be accounted for in watershed-scale microbial water quality modeling.

Persistence of Escherichia coli introduced into streambed sediments with goose, deer and bovine animal waste.

AIMS: The focus of this work was to compare the survival of Escherichia coli introduced into streambed sediments from goose, deer and bovine faeces vs indigenous E. coli. METHODS AND RESULTS: The survival experiments were conducted in flow-through chambers for 32days using two sediments (mineral and organic) obtained from a first-order creek in Maryland. Bovine, goose and deer faeces were collected fresh and diluted or enriched so that added E. coli and indigenous populations were equivalent. Escherichia coli and total coliforms were enumerated using the Colilert-18 Quanti-Tray system. Patterns of E. coli survival and inactivation rates were virtually identical for indigenous strains in both mineral and organic sediments. The addition of E. coli strains from bovine, goose or deer faeces had relatively little impact on final E. coli concentrations, with the exception of deer-borne E. coli populations in the organic sediment. CONCLUSION: These results indicate that indigenous sediment-borne E. coli strains are generally, or more, persistent than those deposited into sediments, including wildlife. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the survival of E. coli originating from wildlife faeces, in sediments, as opposed to bovine faeces or laboratory-cultured strains. As wildlife are likely to be the primary source of E. coli in most non agricultural watersheds, an understanding of the persistence of these strains is important to understanding microbial water quality.

Use of zero-valent iron biosand filters to reduce Escherichia coli O157:H12 in irrigation water applied to spinach plants in a field setting.

AIMS: Zero-valent iron (ZVI) filters may provide an efficient method to mitigate the contamination of produce crops through irrigation water. METHODS: A field-scale system was utilized to evaluate the effectiveness of a biosand filter (S), a biosand filter with ZVI incorporated (ZVI) and a control (C, no treatment) in decontaminating irrigation water. An inoculum of c.8·5log CFU100ml(-1) of Escherichia coli O157:H12 was introduced to all three column treatments in 20-l doses. Filtered waters were subsequently overhead irrigated to 'Tyee' spinach plants. Water, spinach plant and soil samples were obtained on days 0, 1, 4, 6, 8, 10, 13 and 15 and analysed for E. coli O157:H12 populations. RESULTS: ZVI filters inactivated c.6logCFU100ml(-1) E. coli O157:H12 during filtration on day 0, significantly (P<0·05) more than S filter (0·49CFU100ml(-1)) when compared to control on day 0 (8·3log CFU100ml(-1)). On day 0, spinach plants irrigated with ZVI-filtered water had significantly lower E. coli O157 counts (0·13logCFUg(-1)) than spinach irrigated with either S-filtered (4·37logCFUg(-1)) or control (5·23logCFUg(-1)) water. Soils irrigated with ZVI-filtered water contained E. coli O157:H12 populations below the detection limit (2logCFUg(-1)), while those irrigated with S-filtered water (3·56logCFUg(-1)) were significantly lower than those irrigated with control (4·64logCFUg(-1)). CONCLUSIONS: ZVI biosand filters were more effective in reducing E. coli O157:H12 populations in irrigation water than sand filters. SIGNIFICANCE AND IMPACT OF THE STUDY: Zero-valent ion treatment may be a cost-effective mitigation step to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.

Survival of manure-borne E. coli in streambed sediment: effects of temperature and sediment properties.

Escherichia coli bacteria are commonly used as indicator organisms to designate of impaired surface waters and to guide the design of management practices to prevent fecal contamination of water. Stream sediments are known to serve as a reservoir and potential source of fecal bacteria (E. coli) for stream water. In agricultural watersheds, substantial numbers of E. coli may reach surface waters, and subsequently be deposited into sediments, along with fecal material in runoff from land-applied manures, grazing lands, or wildlife excreta. The objectives of this work were (a) to test the hypothesis that E. coli survival in streambed sediment in the presence of manure material will be affected by sediment texture and organic carbon content and (b) to evaluate applicability of the exponential die-off equation to the E. coli survival data in the presence of manure material. Experiments were conducted at three temperatures (4 degrees C, 14 degrees C, and 24 degrees C) in flow-through chambers using sediment from three locations at the Beaverdam Creek Tributary in Beltsville, Maryland mixed with dairy manure slurry in the proportion of 1000:1. Indigenous E. coli populations in sediments ranged from ca. 10(1) to 10(3)MPNg(-1) while approx 10(3) manure-borne E. coli MPNg(-1) were added. E. coli survived in sediments much longer than in the overlaying water. The exponential inactivation model gave an excellent approximation of data after 6-16 days from the beginning of the experiment. Slower inactivation was observed with the increase in organic carbon content in sediments with identical granulometric composition. The increase in the content of fine particles and organic carbon in sediments led not only to the slower inactivation but also to lower sensitivity of the inactivation to temperature. Streambed sediment properties have to be documented to better evaluate the role of sediments as reservoirs of E. coli that can affect microbiological stream water quality during high flow events.

Uncertainty evaluation of coliform bacteria removal from vegetated filter strip under overland flow condition.

Vegetated filter strips (VFS) have become an important component of water quality improvement by reducing sediment and nutrients transport to surface water. This management practice is also beneficial for controlling manure-borne pathogen transport to surface water. The objective of this work was to assess the VFS efficiency and evaluate the uncertainty in predicting the microbial pollutant removal from overland flow in VFS. We used the kinematic wave overland flow model as implemented in KINEROS2 coupled with the convective-dispersive overland transport model which accounts for the reversible attachment-detachment and surface straining of infiltrating bacteria. The model was successfully calibrated with experimental data obtained from a series of simulated rainfall experiments at vegetated and bare sandy loam and clay loam plots, where fecal coliforms were released from manure slurry applied on the top of the plots. The calibrated model was then used to assess the sensitivity of the VFS efficiency to the model parameters, rainfall duration, and intensity for a case study with a 6-m VFS placed at the edge of 200-m long field. The Monte Carlo simulations were also performed to evaluate the uncertainty associated with the VFS efficiency given the uncertainty in the model parameters and key inputs. The VFS efficiency was found to be <95% in 25%, <75% in 23%, and <25% in 20% of cases. Relatively long high-intensity rainfalls, low hydraulic conductivities, low net capillary drives of soil, and high soil moisture contents before rainfalls caused the partial failure of VFS to retain coliforms from the infiltration excess runoff.

Storm water pollutant removal performance of compost filter socks.

In 2005, the U.S. Environmental Protection Agency (USEPA) National Menu of Best Management Practices (BMPs) listed compost filter socks (FS) as an approved BMP for controlling sediment in storm runoff on construction sites. The objectives of this study were to determine if FS with or without the addition of a flocculation agent to the FS system can significantly remove (i) suspended clay and silt particulates, (ii) ammonium nitrogen (NH(4)-N) and nitrate-nitrite nitrogen (NO(3)-N), (iii) fecal bacteria, (iv) heavy metals, and (v) petroleum hydrocarbons from storm water runoff. Five separate (I-V) 30-min simulated rainfall-runoff events were applied to soil chambers packed with Hartboro silt loam (fine-loamy, mixed, active, nonacid, mesic fluvaquentic Endoaquepts) or a 6-mm concrete veneer on a 10% slope, and all runoff was collected and analyzed for hydraulic flow rate, volume, pollutant concentrations, pollutant loads, and removal efficiencies. In corresponding experiments, runoff was analyzed for (i) size of sediment particles, (ii) NH(4)-N and NO(3)-N, (iii) total coliforms (TC) and Escherichia coli, (iv) Cd, Cr, Cu, Ni, Pb and Zn, and (v) gasoline, diesel, and motor oil, respectively. Results showed that: (i) FS removed 65% and 66% of clay (<0.002 mm) and silt (0.002-0.05 mm), respectively; (ii) FS removed 17%, and 11% of NH(4)-N and NO(3)-N, respectively and when NitroLoxx was added to the FS, removal of NH(4)-N load increased to 27%; (iii) total coliform and E. coli removal efficiencies were 74 and 75%, respectively, however, when BactoLoxx was added, removal efficiency increased to 87 and 99% for TC and 89 and 99% for E. coli, respectively; (iv) FS removal efficiency for Cd, Cr, Cu, Ni, Pb, and Zn ranged from 37 to 72%, and, when MetalLoxx was added, removal efficiency ranged from 47 to 74%; and (v) FS removal efficiency for the three petroleum hydrocarbons ranged from 43 to 99% and the addition of PetroLoxx increased motor oil and gasoline removal efficiency in the FS system.

Survival of Escherichia coli in cowpats in pasture and in laboratory conditions.

AIMS: To compare survival of Escherichia coli and faecal coliforms (FC) in bovine faeces deposited in a pasture or incubated in a controlled laboratory environment at temperatures within the same range. METHODS AND RESULTS: Faecal samples from three cow herds were deposited as shaded and nonshaded cowpats in a field and incubated in a laboratory for one month at 21.1, 26.7 and 32.2 degrees C. Both FC and E. coli concentrations increased as much as 1.5 orders of magnitude both in the field and in the laboratory during the 1st week and subsequently decreased. In shaded cowpats, the die-off of E. coli and FC was significantly slower, and the proportion of E. coli in FC was significantly larger as compared with nonshaded cowpats. The die-off was faster in the field than in the laboratory at similar temperatures. CONCLUSIONS: FC and E. coli die-off rates were substantially lower in laboratory conditions than in the field within the same range of temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: This study underscores the importance of field data on survival of manure-borne FC and E. coli, and indicates that laboratory die-off rates have to be corrected to be used for field condition simulations.

Modeling manure-borne bromide and fecal coliform transport with runoff and infiltration at a hillslope.

Hillslope vegetated buffers are recommended to prevent water pollution from agricultural runoff. However, models to predict the efficacy of different grass buffer designs are lacking. The objective of this work was to develop and test a mechanistic model of coupled surface and subsurface flow and transport of bacteria and a conservative tracer on hillslopes. The testing should indicate what level of complexity and observation density might be needed to capture essential processes in the model. We combined the three-dimensional FEMWATER model of saturated-unsaturated subsurface flow with the Saint-Venant model for runoff. The model was tested with data on rainfall-induced fecal coliforms (FC) and bromide (Br) transport from manure applied at vegetated and bare 6-m long plots. The calibration of water retention parameters was unnecessary, and the same manure release parameters could be used both for simulations of Br and FC. Surface straining rates were similar for Br and bacteria. Simulations of Br and FC concentrations were least successful for the funnels closest to the source. This could be related to the finger-like flow of the manure from the strip along the bare slopes, to the transport of Br and FC with manure colloids that became strained at the grass slope, and to the presence of micro-ponds at the grassed slope. The two-dimensional model abstraction of the actual 3D transport worked well for flux-averaged concentrations. The model developed in this work is suitable to simulate surface and subsurface transport of agricultural contaminants on hillslopes and to evaluate efficiency of grass strip buffers, especially when lateral subsurface flow is important.

Rainfall-induced release of fecal coliforms and other manure constituents: comparison and modeling.

Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl-), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl-, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents.

Effect of manure on Escherichia coli attachment to soil.

Attachment of bacteria to soil is an important component of bacterial fate and transport. Escherichia coli are commonly used as indicators of fecal contamination in the environment. Despite the fact that E. coli are derived exclusively from feces or manure, effect of the presence of manure colloids on bacteria attachment to agricultural soils was never directly studied. The objective of this work was to evaluate the magnitude of the effect of manure on E. coli attachment to soil. Escherichia coli attachment to soil was studied in batch experiments with samples of loam and sandy clay loam topsoil that were taken in Pennsylvania and Maryland. Escherichia coli cells were added to the water-manure suspensions containing 0, 20, and 40 g L(-1) of filtered liquid bovine manure, which subsequently were equilibrated with air-dry sieved soil in different soil to suspension ratios. The Langmuir isotherm equation was fitted to data. Manure dramatically affected E. coli attachment to soil. Attachment isotherms were closer to linear without manure and were strongly nonlinear in the presence of manure. The maximum E. coli attachment occurred in the absence of manure. Increasing manure content generally resulted in decreased attachment.

Runoff transport of faecal coliforms and phosphorus released from manure in grass buffer conditions.

AIMS: To test the hypothesis that faecal coliform (FC) and phosphorus (P) are transported similarly in surface runoff through the vegetative filter strip after being released from land-applied manure. METHODS AND RESULTS: The Hagerstown soil was packed into boxes that were 10 cm deep, 30 cm wide and 100, 200 or 300 cm long. Grass was grown in boxes prior experiments. Same-length boxes were placed under rainfall simulator and tilted to have with either 2% or 4% slopes. Dairy manure was broadcast on the upper 30-cm section. Rainfall was simulated and runoff samples were collected and analysed for Cl, FC and total phosphorus (TP). Mass recovery, the concentration decrease rate k, and the ratio FC : TP showed that there was a consistent relationship between FC and TP in runoff. CONCLUSION: The FC and TP transport through simulated vegetated buffer strips were highly correlated. SIGNIFICANCE AND IMPACT OF THE STUDY: As a knowledge base on the effect of the environmental parameters on P transport in vegetated buffer strips is substantially larger than for manure-borne bacteria, the observed similarity may enhance ability to assess the efficiency of the vegetated buffer strips in retention of FC currently used as indicator organisms for manure-borne pathogens.

Do microcracks decrease or increase fatigue resistance in cortical bone?

Fatigue of cortical bone produces microcracks; it has been hypothesized that these cracks are analogous to those occurring in engineered composite materials and constitute a similar mechanism for fatigue resistance. However, the numbers of these linear microcracks increase substantially with age, suggesting that they contribute to increased fracture incidence among the elderly. To test these opposing hypotheses, we fatigued 20 beams of femoral cortical bone from elderly men and women in load-controlled four point bending having initial strain ranges of 3000 or 5000 microstrain. Loading was stopped at fracture or 10(6) cycles, whichever occurred first, and microcrack density and length were measured in the loaded region and in a control region that was not loaded. We studied the dependence of fatigue life and induced microdamage on initial microdamage, cortical region, subject gender and age, and several other variables. When the effect of modulus variability was controlled, longer fatigue life was associated with higher rather than lower initial crack density, particularly in the medial cortex. The increase in crack density following fatigue loading was greater in specimens from older individuals and those initially having longer microcracks. Crack density increased as much in specimens fatigued short of the failure point as in those that fractured, and microcracks were, on average, shorter in specimens with greater numbers of resorption spaces, a measure of remodeling rate.

Rapid extraction of DNA From Escherichia coli and Cryptosporidium parvum for use in PCR.

The Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were evaluated for their ability to rapidly extract PCR-quality DNAs from Escherichia coli O157:H7 and Cryptosporidium parvum. All methods provided satisfactory DNA from E. coli, and the Xtra Amp and Instagene reagents provided satisfactory DNA from C. parvum.

Quantitative detection of Escherichia coli O157 in surface waters by using immunomagnetic electrochemiluminescence.

A protocol for the quantitative detection of Escherichia coli O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized. Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca. 1.0, indicative of a constant ECL signal per cell. The dynamic range was ca. 10(1) to 10(5) cells ml(-1) in phosphate-buffered saline and in raw water samples. The monoclonal IM beads selectively captured E. coli O157 cells in the presence of ca. 10(8) cells of a non-O157 strain of E. coli ml(-1). Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone. Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples. The linear dynamic range with SBC was ca. 10(2) to 10(5) E. coli O157 cells ml of concentrated water(-1). To validate the protocol, 10-liter surface water samples were spiked with ca. 5,000 E. coli O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations. Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for E. coli O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water. The potential sensitivity of the IM-ECL assay is ca. 25 E. coli O157 cells ml of raw water(-1), 25 cells 100 ml of 100-fold concentrated water(-1), or 1 to 2 viable cells liter(-1) with concentration and enrichment. The IM-ECL assay appears suitable for routine analysis and screening of water samples.