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D.S. Francy

Publications and source records attributed to D.S. Francy.

12 recordsLinked to original sources

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

Letters in Applied Microbiology↗

Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration

Aims: To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large-volume drinking-water samples concentrated by ultrafiltration (UF). Methods and Results: Drinking-water samples (100 l) were seeded with Bacillus anthracis, Cryptospordium parvum, Francisella tularensis, Salmonella Typhi, and Vibrio cholerae and concentrated by UF. Recoveries by traditional methods were variable between samples and between some replicates; recoveries were not determined by qPCR. Francisella tularensis and V. cholerae were detected in all 14 samples after UF, B. anthracis was detected in 13, and C. parvum was detected in 9 out of 14 samples. Numbers found by qPCR after UF were significantly or nearly related to those found by traditional methods for all organisms except for C. parvum. A qPCR assay for S. Typhi was not available. Conclusions: qPCR can be used to rapidly detect biological agents after UF as well as traditional methods, but additional work is needed to improve qPCR assays for several biological agents, determine recoveries by qPCR, and expand the study to other areas. Significance and Impact of the Study: To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples. ?? 2009 The Society for Applied Microbiology.

Journal of Applied Microbiology↗

Effects of seeding procedures and water quality on recovery of Cryptosporidium oocysts from stream water by using U.S. Environmental Protection Agency method 1623

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.

Applied and Environmental Microbiology↗

Detection of human enteric viruses in stream water with RT-PCR and cell culture

A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

Journal of Water and Health↗

Evaluation of USEPA method 1622 for detection of Cryptosporidium oocysts in stream waters

To improve surveillance for Cryptosporidium oocysts in water, the US Environmental Protection Agency developed method 1622, which consists of filtration, concentration, immunomagnetic separation, fluorescent antibody and 4, 6-diamidino-2-phenylindole (DAPI) counter-staining, and microscopic evaluation. Two filters were compared for analysis of 11 stream water samples collected throughout the United States. Replicate 10-L stream water samples (unspiked and spiked with 100–250 oocysts) were tested to evaluate matrix effects. Oocyst recoveries from the stream water samples averaged 22% (standard deviation [SD] = ±17%) with a membrane disk and 12% (SD = ±6%) with a capsule filter. Oocyst recoveries from reagent water precision and recovery samples averaged 39% (SD = ±13%) with a membrane disk and 47% (SD = ±19%) with a capsule filter. These results demonstrate that Cryptosporidium oocysts can be recovered from stream waters using method 1622, but recoveries are lower than those from reagent-grade water. This research also evaluated concentrations of indicator bacteria in the stream water samples. Because few samples were oocyst-positive, relationships between detections of oocysts and concentrations of indicator organisms could not be determined.

Journal - American Water Works Association↗

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4???,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.

Applied and Environmental Microbiology↗

Comparison of methods for determining Escherichia coli concentrations in recreational waters

Seventy water samples were collected from three Lake Erie beaches to compare recoveries of Escherichia coli (E. coli) using the USEPA-recommended method for recreational waters (mTEC) to recoveries using three alternative methods (MI, modified mTEC, and Colilert). Statistical tests showed no differences in recoveries of E. coli between MI and mTEC; however, statistically-significant differences were found between modified mTEC or Colilert and mTEC. The MI agar method provided the most similar assessment of recreational water quality to mTEC among the three alternative methods tested. The range of differences between Colilert and mTEC was widest among the three alternative methods. In a sample group with a range of values near the single-sample bathing-water standard, recoveries of E. coli were statistically lower using modified mTEC than mTEC; however, MI and Colilert compared well to mTEC in this range. Because samples were collected in a small geographic area, more work is necessary to test within-method variability of the modified mTEC, MI, and Colilert methods and to evaluate these methods as substitutes for the mTEC method in a variety of recreational waters. Copyright (C) 2000.

Water Research↗

Quality-Assurance/Quality-Control Manual for Collection and Analysis of Water-Quality Data in the Ohio District, US Geological Survey

The U.S. Geological Survey (USGS), Water Resources Division (WRD), requires that quality-assurance/quality-control (QA/QC) activities be included in any sampling and analysis program. Operational QA/QC procedures address local needs while incorporating national policies. Therefore, specific technical policies were established for all activities associated with water-quality project being done by the Ohio District. The policies described in this report provide Ohio District personnel, cooperating agencies, and others with a reference manual on QA/QC procedures that are followed in collecitng and analyzing water-quality samples and reporting water-quality information in the Ohio District. The project chief, project support staff, District Water-Quality Specialist, and District Laboratory Coordinator are all involved in planning and implementing QA/QC activities at the district level. The District Chief and other district-level managers provide oversight, and the Regional Water-Quality Specialist, Office of Water Quality (USGS headquarters), and the Branch of Quality Systems within the Office of Water Quality create national QA/QC polices and provide assistance to District personnel. In the literature, the quality of all measurement data is expressed in terms of precision, variability, bias, accuracy, completeness, representativeness, and comparability. In the Ohio District, bias and variability will be used to describe quality-control data generated from samples in the field and laboratory. Each project chief must plan for implementation and financing of QA/QC activities necessary to achieve data-quality objectives. At least 15 percent of the total project effort must be directed toward QA/QC activities. Of this total, 5-10 percent will be used for collection and analysis of quality-control samples. This is an absolute minimum, and more may be required based on project objectives. Proper techniques must be followed in the collection and processing of surface-water, ground-water, biological, precipitation, bed-sediment, bedload, suspended-sediment, and solid-phase samples. These techniques are briefly described in this report and are extensively documented. The reference documents listed in this report will be kept by the District librarian and District Water-Quality Specialist and updated regularly so that they are available to all District staff. Proper handling and documentation before, during, and after field activities are essential to ensure the integrity of the sample and to correct erroneous reporting of data results. Field sites are to be properly identified and entered into the data base before field data-collection activities begin. During field activities, field notes are to be completed and sample bottles appropriately labeled a nd stored. After field activities, all paperwork is to be completed promptly and samples transferred to the laboratory within allowable holding times. All equipment used by District personnel for the collection and processing of water-quality samples is to be properly operated, maintained, and calibrated by project personnel. This includes equipment for onsite measurement of water-quality characteristics (temperature, specific conductance, pH, dissolved oxygen, alkalinity, acidity, and turbidity) and equipment and instruments used for biological sampling. The District Water-Quality Specialist and District Laboratory Coordinator are responsible for preventive maintenance and calibration of equipment in the Ohio District laboratory. The USGS National Water Quality Laboratory in Arvada, Colo., is the primary source of analytical services for most project work done by the Ohio District. Analyses done at the Ohio District laboratory are usually those that must be completed within a few hours of sample collection. Contract laboratories or other USGS laboratories are sometimes used instead of the NWQL or the Ohio District laboratory. When a contract laboratory is used, the projec

Water-Resources Investigations Report↗

Effects of receiving-water quality and wastewater treatment on injury, survival, and regrowth of fecal-indicator bacteria and implications for assessment of recreational water quality

Bacterial injury, survival, and regrowth were investigated by use of replicate flow-through incubation chambers placed in the Cuyahoga River or Lake Erie in the greater Cleveland metropolitan area during seven 4-day field studies. The chambers contained wastewater or combined-sewer-overflow (CSO) effluents treated three ways-unchlorinated, chlorinated, and dechlorinated. At timestep intervals, the chamber contents were analyzed for concentrations of injured and healthy fecal coliforms by use of standard selective and enhanced-recovery membrane-filtration methods. Mean percent injuries and survivals were calculated from the fecal-coliform concentration data for each field study. The results of analysis of variance (ANOVA) indicated that treatment affected mean percent injury and survival, whereas site did not. In the warm-weather Lake Erie field study, but not in the warm-weather Cuyahoga River studies, the results of ANOVA indicated that dechlorination enhanced the repair of injuries and regrowth of chlorine-injured fecal coliforms on culture media over chlorination alone. The results of ANOVA on the percent injury from CSO effluent field studies indicated that dechlorination reduced the ability of organisms to recover and regrow on culture media over chlorination alone. However, because of atypical patterns of concentration increases and decreases in some CSO effluent samples, more work needs to be done before the effect of dechlorination and chlorination on reducing fecal-coliform concentrations in CSO effluents can be confirmed. The results of ANOVA on percent survivals found statistically significant differences among the three treatment methods for all but one study. Dechlorination was found to be less effective than chlorination alone in reducing the survival of fecal coliforms in wastewater effluent, but not in CSO effluent. If the concentration of fecal coliforms determined by use of the enhanced-recovery method can be predicted accurately from the concentration found by use of the standard method, then increased monitoring and expense to detect chlorine-injured organisms would be unnecessary. The results of linear regression analysis, however, indicated that the relation between enhanced-recovery and standard-method concentrations was best represented when the data were grouped by treatment. The model generated from linear regression of the unchlorinated data set provided an accurate estimate of enhanced-recovery concentrations from standard-method concentrations, whereas the models generated from the chlorinated and dechlorinated data sets did not. In addition, evaluation of fecal-coliform concentrations found in field studies in terms of Ohio recreational water-quality standards showed that concentrations obtained by standard and enhanced-recovery methods were not comparable. Sample treatment and analysis methods were found to affect the percentage of samples meeting and exceeding Ohio's bathing-water, primary-contact, and secondary-contact standards. Therefore, determining the health risk of swimming in receiving waters was often difficult without information on enhanced-recovery method concentrations and was especially difficult in waters receiving high proportions of chlorinated or dechlorinated effluents.

Water-Resources Investigations Report↗

Escherichia coli and fecal-coliform bacteria as indicators of recreational water quality

In 1986, the U.S. Environmental Protection Agency (USEPA) recommended that Escherichia coli (E. coli) be used in place of fecal-coliform bacteria in State recreational water-quality standards as an indicator of fecal contamination. This announcement followed an epidemiological study in which E. coli concentration was shown to be a better predictor of swimming-associated gastrointestinal illness than fecal-coliform concentration. Water-resource managers from Ohio have decided to collect information specific to their waters and decide whether to use E. coli or fecal-coliform bacteria as the basis for State recreational water-quality standards. If one indicator is a better predictor of recreational water quality than the other and if the relation between the two indicators is variable, then the indicator providing the most accurate measure of recreational water quality should be used in water-quality standards. Water-quality studies of the variability of concentrations of E. coli to fecal-coliform bacteria have shown that (1) concentrations of the two indicators are positively correlated, (2) E. coli to fecal-coliform ratios differ considerably from site to site, and (3) the E. coli criteria recommended by USEPA may be more difficult to meet than current (1992) fecal-coliform standards. In this study, a statistical analysis was done on concentrations of E. coli and fecal-coliform bacteria in water samples collected by two government agencies in Ohio-- the U.S. Geological Survey (USGS) and the Ohio River Valley Water Sanitation Commission (ORSANCO). Data were organized initially into five data sets for statistical analysis: (1) Cuyahoga River, (2) Olentangy River, (3) Scioto River, (4) Ohio River at Anderson Ferry, and (5) Ohio River at Cincinnati Water Works and Tanners Creek. The USGS collected the data in sets 1, 2, and 3, whereas ORSANCO collected the data in sets 4 and 5. The relation of E. coli to fecal-coliform concentration was investigated by use of linear-regression analysis and analysis of covariance. Log-transformed E. coli and fecal-coliform concentrations were highly correlated in all data sets (r-values ranged from 0.929 to 0.984). Linear regression analysis on USGS and ORSANCO data sets showed that concentration of E. coli could be predicted from fecal-coliform concentration (coefficients of determination (R2) ranged from 0.863 to 0.970). Results of analysis of covariance (ANCOVA) indicated that the predictive equations among the three USGS data sets and two ORSANCO data sets were not significantly different and that the data could be pooled into two large data sets, one for USGS data and one for ORSANCO data. However, results of ANCOVA indicated that USGS and ORSANCO data could not be pooled into one large data set. Predictions of E. coli concentrations calculated for USGS And ORSANCO regression relations, based on fecal-coliform concentrations set to equal Ohio water-quality standards, further showed the differences in E. coli to fecal-coliform relations among data sets. For USGS data, a predicted geometric mean of 176 col/100 mL (number of colonies per 100 milliliters) was greater than the current geometric-mean E. coli standard for bathing water of 126 col/100mL. In contrast, for ORSANCO data, the predicted geometric mean of 101 col/100 mL was less than the current E. coli standard. The risk of illness associated with predicted E. coli concentrations for USGS and ORSANCO data was evaluated by use of the USEPA regression equation that predicts swimming-related gastroenteritis rates from E. coli concentrations.1 The predicted geometric-mean E. coli concentrations for bathing water of 176 col/100 mL for USGS data and 101 col/100 mL for ORSANCO data would allow 9.4 and 7.1 gastrointestinal illnesses per 1,000 swimmers, respectively. This prediction compares well with the illness rate of 8 individuals per 1,000 swimmers estimated by the USEPA for an E. coli concentration of 126 col/100 mL. Therefore, the

Water-Resources Investigations Report↗

Geologic setting and water quality of selected basins in the active coal-mining areas of Ohio, 1989-91, with a summary of water quality for 1985-91

This report presents streamwater- and ground-water-quality data collected to characterize the baseline water quality for 21 drainage basins in the coal-mining region of eastern Ohio. The study area is mostly within the unglaciated part of eastern Ohio along the western edge of the Appalachian Plateaus Physiographic Province. The data collected from 1989-91 and presented in this report represent the third and final phase of a 7-year study to assess baseline water quality in Ohio's coal region during 1985-1991. During 1989-91, 246 samples from 41 streamwater sites were collected periodically from a long-term site network. Ranges and medians of measurements made at the long-term streamwater sites were following: specific conductance, 270 to 5,170 and 792 microsiemens per centimeter at 25 degrees Celsius; pH, 2.7 to 9.1 and 7.8; alkalinity, 1 to 391 and 116 mg/L (milligrams per liter). Ranges and medians of laboratory analyses of the same samples were the following: dissolved sulfate, 13 to 2,100 and 200 mg/L; dissolved aluminum, <10 to 17,000 and 300 ? /L (micrograms per liter); dissolved iron, <10 to 53,000 and 60 ? /L; and dissolved manganese, <10 to 17,000 and 295 ? /L. The ranges for concentrations of total recoverable aluminum, iron, and manganese were similar to the ranges of concentrations found for dissolved constituents. Medians of total recoverable aluminum and iron were about 10 times greater than the medians of dissolved aluminum and iron. During 1989-91, once-only sample collections were done at 45 streamwater sites in nine basins chosen for synoptic sampling. At several sites in the Middle Hocking River basin and Leading Creek basin, water had low pH and high concentrations of dissolved aluminum, iron and manganese. These water-quality characteristics are commonly associated with ace mine drainage. Throughout the entire 7-year study (1985-91), medians for most constituents at the long-term streamwater-sampling sites were fairly consistent, despite the geographic diversity of the study area. Waters from several long-term sites, including several sites in Moxahala Creek and Middle Hocking River basins, had low pH and high concentrations of several constituents, including dissolved sulfate, iron, aluminum, and manganese; this combinations characteristics is indicitive of acid drainage from surface-mining operations. At many of the streamwater sites where concentration of these constituents were high, pH values in the neutral or alkaline range were indicative of stream buffering by carbonate rock or restoration of mined lands in the drainage system. The basins with sites in this category include Yellow and Cross Creeks and Wheeling Creek basins. Water quality at other sites showed little or no effects from surface mining. Ground-water samples collected during the last phase of the study (1989-91) were mostly from unconsolidated aquifers. The waters were generally hard to very hard and calcium bicarbonate in type. During the entire 7-year study period, medians of pH in ground-water samples varied little, and most values were in the alkaline range. Except for a few sites where concentrations of dissolved sulfate exceeded 250 mg/L and concentrations of total recoverable and dissolved iron and manganese exceeded 1,000 ? /L, the quality of ground water at the wells sampled in the study area showed little effect from coal mining.

Water-Resources Investigations Report↗

Emulsification of hydrocarbons by subsurface bacteria

Biosurfactants have potential for use in enhancement of in situ biorestoration by increasing the bioavailability of contaminants. Microorganisms isolated from biostimulated, contaminated and uncontaminated zones at the site of an aviation fuel spill and hydrocarbon-degrading microorganisms isolated from sites contaminated with unleaded gasoline were examined for their abilities to emulsify petroleum hydrocarbons. Emulsifying ability was quantified by a method involving agitation and visual inspection. Biostimulated-zone microbes and hydrocarbon-degrading microorganisms were the best emulsifiers as compared to contaminated and uncontaminated zone microbes. Biostimulation (nutrient and oxygen addition) may have been the dominant factor which selected for and encouraged growth of emulsifiers; exposure to hydrocarbon was also important. Biostimulated microorganisms were better emulsifiers of aviation fuel (the contaminant hydrocarbon) than of heavier hydrocarbon to which they were not previously exposed. By measuring surface tension changes of culture broths, 11 out of 41 emulsifiers tested were identified as possible biosurfactant producers and two isolates produced large surface tension reductions indicating the high probability of biosurfactant production.

Journal of Industrial Microbiology↗