Name: concentration of virus particles from environmental water and wastewater samples using skimmed milk flocculation and ultrafiltration
Uploaded: 2023-03-17T13:20:00
Description: Watch this Scientific Journal Video about Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration at JoVE.com

This work was supported by NSERC Alliance Covid-19 Grant (Award No. 431401363, 2020-2021, Drs. Yuan and Uyaguari-D&#237;az). MUD would like to thank the University Research Grants Program (Award No. 325201). Both JF and JZA are supported by the Visual and Automated Disease Analytics (VADA) graduate training program. KY and JF both received fellowships from the Mitacs Accelerate program. MUD and his laboratory members (KY, JF, JZA) are supported by NSERC-DG (RGPIN-2022-04508) and the Research Manitoba New Investigator Operating grant (No 5385). Special thanks to the City of Winnipeg, Manitoba. This research was conducted at the University of Manitoba. We would like to acknowledge that the University of Manitoba campuses are located on the original lands of Anishinaabeg, Cree, Oji-Cree, Dakota, and Dene peoples and on the homeland of the M&#233;tis Nation.

1. Comparison of serial ultrafiltration and skimmed milk flocculation to concentrate viruses in wastewater samples
<ol>
	<li>Sample preparation
	<ol>
		<li>Collect 2 L of 24 h flow-proportional composite raw (influent) wastewater samples. Samples were collected from the three major wastewater treatment plants (WWTPs) in Winnipeg, Canada, during the summer and fall of 2020 (Table 1).</li>
		<li>Transport the samples to the laboratory in light-proof bottles in an icebox and process them ...

<ol>
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One of the critical steps in this study is the elimination of solid particles by applying a prefiltration step with 0.2 &#181;m and 0.45 &#181;m membrane filters. Considering the partitioning of viruses into solid particles, especially enveloped viruses, prefiltration can cause a significant loss in viral recovery30. While a prefiltration step for ultrafiltration methods is almost always necessary for environmental and wastewater samples to prevent ultrafiltration devices from clogging, prefiltrat...

Determining the effective concentration of microorganisms in surface and wastewater samples for microbial community analysis&#160;and&#160;epidemiology studies, is one of the important steps for monitoring and predicting the course of outbreaks in communities1,2. The COVID-19 pandemic, unfolded the importance of improving concentration methods. COVID-19 emerged in late 2019 and, as of March 2023, still poses a threat to human health, social life, and the economy. Effective surveillance and control strategies to alleviate the impacts of COVID-19 outbreaks in communities have become an important research topic, as new waves and variants of COVID-19 have been emerging in addition to the rapid transmission and spread of the virus, as well as unreported and undiagnosed asymptomatic cases3,4,5. The use of wastewater-based epidemiology for COVID-19 by civil society organizations, government agencies, and public or private utilities has been helpful in providing rapid outbreak-related information and mitigating the impacts of COVID-19 outbreaks6,7,8,9. However, the concentration of SARS-CoV-2, an enveloped RNA virus, in wastewater samples still poses challenges10. For example, one of these challenges is the partitioning of SARS-CoV-2 in wastewater solids, which may impact recovery when the solids are eliminated during concentration11. If this is the case, the focus of quantification/assessment should be on both solid and aqueous phases of environmental water samples, rather than the aqueous phase only. Furthermore, the choice of concentration method can be modified based on downstream tests and analyses. The concentration of virus particles and pathogens from environmental samples has become an urgent research topic with developments in sequencing and microbiome fields.
Various virus concentration methods have been applied in the field of virus concentration from environmental water and wastewater samples. Some commonly used methods are filtration, skimmed milk flocculation (SMF), adsorption/elution, and polyethylene glycol precipitation12-17. Among them, SMF has been considered a cheap and effective method, successfully tested, and applied for recovering viruses, including SARS-CoV-2, from wastewater and surface waters12,15,16,18. The SMF procedure is a relatively new approach that has gained increased recognition among many environmental studies as an appropriate methodology to simultaneously recover a broad array of microorganisms such as viruses, bacteria, and protozoans from all types of water samples, namely sludge, raw sewage, wastewater, and effluent samples19. When compared to other known methodologies to recover viruses from environmental samples such as ultrafiltration and glycine-alkaline elution, lyophilization-based approach, or ultracentrifugation and glycine-alkaline elution, SMF has been reported as the most efficient method with higher viral recovery and detection rates18,20. In the present study, we used Armored RNA as a test virus to assess the recovery efficiency of virus concentration methods, including tests for assessing SARS-CoV-2 recovery21,22.
Here, we tested wastewater and environmental water samples to demonstrate the utility of SMF and a sequential ultrafiltration method to concentrate microbial fractions for quantitative polymerase chain reaction (qPCR), sequence-based metagenomics, and deep-amplicon sequencing. SMF is a relatively cheaper method and optimal for a larger volume of samples compared to ultrafiltration methods. The idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 M sodium phosphate buffer with a pH 7.5</td>
			<td>Alfa Aesar</td>
			<td>J62041AP</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>0.2 &#956;m 47-mm Supor-200 membrane disc filters</td>
			<td>VWR</td>
			<td>66234</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>0.45 &#956;m 47-mm Supor-200 membrane disc filters</td>
			<td>VWR</td>
			<td>60043</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>4X TaqMan Fast Virus 1-Step Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444432</td>
			<td>Life Technologies, Carlsbad, CA, USA</td>
		</tr>
		<tr>
			<td>Armored RNA Quant IPC-1 Processing Control</td>
			<td>Asuragen</td>
			<td>49650</td>
			<td>Asuragen, Austin, TX, USA</td>
		</tr>
		<tr>
			<td>Brand A, Jumbosep Centrifugal Device, 30-kDa</td>
			<td>Pall&#160;</td>
			<td>OD030C65</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>Brand B, Microsep Advance Centrifugal Device, 30-kDa</td>
			<td>Pall</td>
			<td>MCP010C46</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 ml)&#160;</td>
			<td>Nalgene</td>
			<td>3119-0050PK</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Invitrogen</td>
			<td>18047019</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Dyna Mag-2</td>
			<td>Invitrogen</td>
			<td>12027</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>GWV High Capacity Groundwater Sampling Capsules - 0.45 &#181;m</td>
			<td>Pall</td>
			<td>12179</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 1N standard solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>AC124210025</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>MagMAX Microbiome Ultra Nucleic Acid Isolation Kit</td>
			<td>Applied biosystems</td>
			<td>A42358</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Promega</td>
			<td>P1197</td>
			<td>Promega Corporation, Fitchburg, WI, USA</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Masterflex, Cole-Parmer instrument</td>
			<td>7553-20</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>pH meter&#160;</td>
			<td>Denver instrument</td>
			<td>RK-59503-25</td>
			<td>Cole-Parmer. This product has been discontinued</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:isoamyl alcohol 25:24:1</td>
			<td>Invitrogen</td>
			<td>15593031</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>Primers and probe sets</td>
			<td>IDT</td>
			<td></td>
			<td>Integrated DNA Technologies, Inc., Coralville, IA, USA</td>
		</tr>
		<tr>
			<td>Qiagen All-prep DNA/RNA power microbiome kit</td>
			<td>Qiagen</td>
			<td></td>
			<td>Qiagen Sciences, Inc., Germantown, MD, USA</td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A34322</td>
			<td>Life Technologies, Carlsbad, CA, USA</td>
		</tr>
		<tr>
			<td>Qubit 1X dsDNA High Sensitivity (HS) assay kit</td>
			<td>Invitrogen</td>
			<td>Q33231</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer, with WiFi</td>
			<td>Invitrogen</td>
			<td>Q33238</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Qubit RNA High Sensitivity (HS) assay kit</td>
			<td>Invitrogen</td>
			<td>Q32855</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>Invitrogen</td>
			<td>EN0531</td>
			<td>Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>RNeasy PowerMicrobiome Kit</td>
			<td>Qiagen</td>
			<td>26000-50</td>
			<td>Qiagen Sciences, Inc., Germantown, MD, USA</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Difco (BD Life Sciences)</td>
			<td>DF0032173</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
		<tr>
			<td>Sodium phosphate buffer</td>
			<td>Alfa Aesar</td>
			<td></td>
			<td>Alfa Aesar, Ottawa, ON, Canada</td>
		</tr>
		<tr>
			<td>Synthetic seawater</td>
			<td>VWR&#160;</td>
			<td>RC8363-1</td>
			<td>RICCA chemical company</td>
		</tr>
		<tr>
			<td>Synthetic single-stranded DNA gBlock</td>
			<td>IDT</td>
			<td></td>
			<td>Integrated DNA Technologies, Inc., Coralville, IA, USA</td>
		</tr>
		<tr>
			<td>VacuCap 90 Vacuum Filtration Devices - 0.1 &#181;m, 90 mm, gamma-irradiated</td>
			<td>Pall</td>
			<td>4621</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>VacuCap 90 Vacuum Filtration Devices - 0.2 &#181;m, 90 mm, gamma-irradiated</td>
			<td>Pall</td>
			<td>4622</td>
			<td>Pall Corporation, Ann Arbor, MI</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>Fisher Scientific, Fair Lawn, NJ, USA</td>
		</tr>
	</tbody>
</table>

concentration of virus particles from environmental water and wastewater samples using skimmed milk flocculation and ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Evaluation of viral RNA concentration methods 
All six samples processed with UF-3k x g were positive and resulted in a 13.38% &#177; 8.14% recovery (Figure 1). Only one sample was positive when the samples were processed with UF-7.5k x g. All samples processed with SMF were positive and resulted in a 15.27% &#177; 2.65% recovery (Figure 1). The average recovery rates of UF-3K x g and SMF were significantly and co...

Watch this Scientific Journal Video about Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration at JoVE.com

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Water-based and wastewater epidemiology has emerged as an alternative method to monitor and predict the course of outbreaks in communities. The skimmed milk flocculation method represents an alternative approach to concentrate microbes. Skimmed milk flocculation method represents a versatile, cheap, and easy hands-off method to concentrate microbial fractions with no significant differences compared to ultra filtration or tangential flow filtration approaches.This approach is used for metagenomics or targeted PCR studies. Once the microbial fractions are concentrated, nucleic acid extraction can be conducted using commercial extraction kits or in-house protocol. The skimmed milk flocculation method is relatively easier for large or small volumes of water or wastewater samples.A background control with Milli-Q water should be included as part of this method. Demonstrating the procedure will be Kadir Yanac, Jhannelle Francis, and Jocelyn Zambrano-Alvarado, graduate students from Miguel Uyaguari's laboratory. To begin, collect two liters of 24-hour flow proportional composite raw wastewater samples.Transport the samples to the laboratory in light-proof bottles in an icebox and process them within 24 hours. Get the wastewater characteristics data from wastewater treatment plant technical staff. For each ultra filtration method, spike five times 10 to the fourth copies of armored RNA into 140 milliliters of each of six fresh wastewater samples collected on one date and six fresh wastewater samples on another date.Homogenize the armored RNA in the sample by stirring for 30 minutes at four degrees Celsius on a magnetic stir. For skimmed milk flocculation or SMF, prepare 1%skimmed milk solution by dissolving 0.5 grams of skimmed milk powder in 50 milliliters of synthetic seawater. Adjust the pH of SMF to 3.5 using one normal hydrochloric acid.Add five milliliters of skimmed milk solution to 500 milliliters of raw wastewater sample. Stir the sample for eight hours and allow the formed flocks to settle for another eight hours at room temperature. Remove the supernatant using a serological pipette without disturbing the settled flocks.Transfer a final volume of 50 milliliters containing the flocks to a centrifuge tube and centrifuge at 8, 000 G for 30 minutes at four degrees Celsius. Remove the supernatant and carefully scrap the pellet using a sterilized spatula. Resuspend the remaining pellet in 250 microliters of 0.2 molar sodium phosphate buffer.Transfer the scraped and resuspended pellets to the same 1.5 milliliter mini centrifuge tube. Perform viral RNA extraction from viral concentrates of wastewater samples and recovery efficiency assays using an RNA extraction kit with a 25:24:2:1 ratio of phenol to chloroform to isoamyl alcohol and beta mercaptoethanol according to the manufacturer's instructions. Finally, elute the RNA in 50 microliters of elution buffer.For RT-qPCR analysis, quantify armored RNA using tenfold dilutions of a synthetic single stranded DNA or gBlock construct. Obtain calibration curves for each RT-qPCR run. Include negative controls and run the samples in triplicate.Collect 10 liters of environmental surface water sample. Include 10 liters of deionized water for background control. Perform capsule and vacuum filtration using membrane disc filters of 0.45, 0.2, and 0.1 micron sizes to minimize noise during metagenomics sequencing.After filtration, adjust the pH of the water sample to 3.5 using one normal hydrochloric acid. Prepare a 1%pre-flocculated skimmed milk solution and adjust the pH to 3.5. Add 100 milliliters of the acidified skimmed milk solution to 10 liters of acidified environmental water sample.Stir the sample for eight hours at room temperature on a magnetic shaker and allow the flocks to sediment by gravity for an additional eight hours. After settling, carefully remove the supernatant using a vacuum pump without disturbing the flocks. Aliquot and balance the remaining flocks according to the weight in a 50 milliliter centrifuge tube and centrifuge.Remove the supernatant and dissolve the pellet in 200 microliters of 0.2 molar sodium phosphate buffer. Treat the dissolved pellets with DNase I and RNase A following the manufacturer's instructions to eliminate free DNA and RNA coprecipitated during the extraction process. After incubation, inactivate DNase I and RNase A.Extract nucleic acids from the dissolved pellet using a DNA or RNA extraction kit.Quantify the total amount of extracted DNA or RNA from the environmental surface water sample using a fluorometer. Perform qPCR analysis to target enteric viruses of interest and Illumina sequencing to identify the viral community structure. Collect two liters of water samples from protected and impacted waterways.If the sample contains considerable debris, use a cheesecloth to remove them. Add 20 milliliters of 1%skimmed milk solution to the previously pH adjusted two liter freshwater sample. Stir the sample for eight hours at room temperature and allow the flocks to sediment by gravity for an additional eight hours.After settling, carefully remove the supernatant using a peristaltic pump. Transfer the sedimented flocks to a 50 milliliter centrifuge tube and centrifuge them at 8, 000 G for 30 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellets in 200 microliters of 0.2 molar sodium phosphate buffer.Select around 0.5 grams of the flock for DNA extraction using the nucleic acid isolation kit of choice following the manufacturer's instructions. Determine the DNA concentration and purity using a fluorometer. All six samples processed with ultra filtration at 3, 000 G were positive.In contrast, only one sample was positive when the samples were processed with ultra filtration at 7, 500 G.All samples processed with SMF were positive and resulted in better recovery. The average recovery rates of 3, 000 G and SMF were significantly and consistently higher than ultra filtration at 7, 500 G.Water quality parameters in influenced samples and nucleic acids extracted from environmental water samples collected within Winnipeg across three seasons are summarized here. Samples collected from April to October 2022 from Assiniboine River water yielded the highest DNA concentration from Forested 1 and the lowest concentration was recovered from agricultural site three.It is very important to use the right concentration of skimmed milk and acidify the sample prior to use. This technique can provide a quick screening of different microbial pathogens present in environmental samples such as water and wastewater. Additional modifications can be implemented using solid matrices such as soil or sediments.

concentration-virus-particles-from-environmental-water-wastewater

Research

JoVE Journal

Environment

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ...