We gratefully acknowledge funding for this project from the USDA National Institute of Food and Agriculture grants 2009-01208 and 2009-01984.

1. Concentration of Bacteria from Large Volumes of Agricultural Water Using MMS
<ol>
	<li>MMS Preparation
	<ol>
		<li>Cut a rectangular section of 4-ply cheesecloth measuring 40 x&#160;12 cm.</li>
		<li>Fold the cheesecloth along both axes to obtain a rectangle of 20 x&#160;6 cm.</li>
		<li>Roll the cheesecloth tightly along its long axis to form a cylindrical swab of approximately 6 cm tall and 3&#160;cm in diameter.</li>
		<li>Autoclave the cheesecloth swab in aluminum foil, do not autoclave the cassette. Decontaminate the cassette by soaking it for 30 min in 10% household bleach, and deactivate the residual chlorine by soaking the cassette for 15 min in a sodium thiosulfate pentahydrate (Na2S2O3&#183;5H2O) solution (50 mg/L prepared in sterile water).</li>
		<li>Insert the swab into the MMS cassette. 
		NOTE: If the non-disposable version of the MMS cassette is used, start with a 4-ply sheet of cheesecloth measuring 80 x&#160;22 cm, fold it to 40 x&#160;11 cm and roll it to form a cylinder approximately 11 cm tall and 6 cm in diameter.</li>
		<li>Assemble the MMS.</li>
		<li>Attach vinyl tubing to spigots on both sides of the MMS, ensuring the tubing fully adheres to the spigots, and fix tubing into the peristaltic pump. Ensure that the tubing upstream of the peristaltic pump is of sufficient length for sampling.</li>
	</ol>
	</li>
	<li>Concentration of Bacteria
	<ol>
		<li>Sample at least 10 L of agricultural water by running the peristaltic pump at high speed.</li>
		<li>After sampling is complete, withdraw the peristaltic pump inlet from the water sample and continue running the pump until no effluent water is observed exiting the MMS.</li>
		<li>If a manifold is used to run several samples at the same time, collect the effluent of each MMS, and when 10 L is filtered through a single MMS close the valve for that particular MMS and continue running the pump.
		<ol>
			<li>When sampling is completed, withdraw the peristaltic pump inlet from the agricultural water, open all the valves of the manifold, and continue running the pump until no effluent water is observed exiting the MMSs.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>When concentration has been completed for all MMSs, carefully remove the vinyl tubing from the MMS spigots and cap the spigots on both sides of the MMS. Tightly capped MMS can be stored for up to 12 hr before adding enrichment media.</li>
</ol>
2. MMS Enrichment and Sample Preparation
<ol>
	<li>Enrichment
	<ol>
		<li>Unscrew the lid of the MMS, and aseptically add 20 ml of the appropriate sterile enrichment broth. Use one MMS for each selective enrichment. Alternatively, perform a non-selective enrichment to propagate all three target bacteria in the same sample. 
		NOTE: If the non-disposable version of the MMS cassette is used, aseptically remove cheesecloth filter from the cassette and place in a sterile bag before adding 225 ml of the appropriate enrichment media.
		<ol>
			<li>For enrichment of E. coli, add 20 ml of buffered peptone water (BPW) supplemented with 8 mg/L of vancomycin hydrochloride.</li>
			<li>For enrichment of Salmonella spp., add 20 ml of BPW supplemented with the Salmonella supplement (4 ml/L).</li>
			<li>For enrichment of L. monocytogenes, add 20 ml of VIDAS UP Listeria (LPT) broth.</li>
			<li>For simultaneous non-selective enrichment for all three microorganisms using a single MMS, use universal preenrichment broth (UPB).</li>
		</ol>
		</li>
		<li>Screw the lid of the MMS back on tightly. Ensure that the spigots are tightly capped to avoid spills and possible contamination during enrichment.</li>
		<li>Incubate the MMS for up to 18-24 hr in a shaking incubator at 200 rpm. Use 42 &#176;C for E. coli and Salmonella spp. selective enrichments. Use 30 &#176;C for L. monocytogenes selective enrichment, and use 37 &#176;C for non-selective enrichment.</li>
	</ol>
	</li>
	<li>Sample Preparation
	<ol>
		<li>When incubation has completed, remove the MMS from the incubator, uncap one of the spigots, and gently dispense about 0.5 ml into a 1.5 ml microcentrifuge tube. Alternatively, unscrew the MMS and pipette out 0.5 ml of overnight enrichment. 
		NOTE: If the not-disposable version of the MMS cassette is used, pipette 0.5 ml of enrichment from the bag.</li>
		<li>For colorimetric analysis of E. coli selective enrichment, or for the analysis of the non-selective enrichments, sonicate 0.5 ml of enrichment at 5 W, 22 kHz for 20 sec using a probe sonicator.</li>
	</ol>
	</li>
</ol>
3. Preparation and Embedding of Colorimetric Substrates in &#181;PADs
<ol>
	<li>Prepare enough &#181;PADs for each sample and target tested. To detect E. coli prepare CPRG and X-Gluc &#181;PADs, for Salmonella spp. detection prepare MC &#181;PADs, and for L. monocytogenes detection prepare X-InP &#181;PADs.
	<ol>
		<li>Prepare HEPES buffer [0.1 M HEPES with 0.1% Bovine Serum Albumin (BSA)] for the colorimetric substrates and adjust the pH of the buffer according to the substrate that is going to be used: pH 7.5 for preparation of CPRG or X-Gluc, pH 9 for MC, and pH 7 for X-InP.</li>
		<li>Prepare stock solutions of the substrates in the pH adjusted HEPES buffer at concentrations of 3 mM (CPRG), 62.5 mM (X-Gluc), 15 mM (MC), 100 mM (X-InP). Protect the stock solutions from light.</li>
		<li>Place the &#181;PADs inside a sterile Petri dish, then imbed the &#181;PAD microspot with 24 &#181;l of each substrate stock solution.</li>
	</ol>
	</li>
</ol>
4. Detection and Data Analysis
<ol>
	<li>Detection
	<ol>
		<li>Add 6 &#181;l of the enriched sample to each appropriate &#181;PAD microspot contained in a Petri dish.</li>
		<li>Place the cover back on the Petri dish and seal it with Parafilm.</li>
		<li>Incubate the sample with the &#181;PAD at 37 &#176;C for up to 3 hr to allow for color development and drying of the microspots.</li>
	</ol>
	</li>
	<li>Visual Examination of the Results
	<ol>
		<li>Perform detection by simple visual inspection of the color changes of the &#181;PADs. 
		NOTE: Typically, strong reactions that produce definitive color changes are expected following 18-24 hr of enrichment, which should obviate the need for further clarification and should be considered positive.</li>
		<li>Determine the presence of an E. coli positive sample by observing microspots imbedded with CPRG change from yellow to red-violet and microspots imbedded with X-Gluc change from colorless to blue-green.</li>
		<li>Determine the presence of a L. monocytogenes positive sample by observing microspots imbedded with X-InP change from colorless to indigo.</li>
		<li>Determine the presence of a Salmonella spp. positive sample by observing microspots imbedded with MC change from colorless to purple-mauve.</li>
	</ol>
	</li>
	<li>Software Aided Data Analysis 
	NOTE: Conducted to clearly determine whether weak reactions are positive or negative and to enable a semi-quantitative analysis of the results.
	<ol>
		<li>Allow the &#181;PADs to fully dry.</li>
		<li>Scan the &#181;PAD using a flat-bed scanner.</li>
		<li>Use ImageJ software to process the image.
		<ol>
			<li>&#34;Open&#8221; scanned image to be analyzed in ImageJ located under the &#8220;File&#8221; tab on the main menu (or press &#8220;Clt+O&#8221;).</li>
			<li>Invert the image selecting &#8220;Invert&#8221; located under the &#8220;Edit&#8221; tab on the main menu (or press &#8220;Clt+Shift+I&#8221;).</li>
			<li>Use the &#8220;Analyze&#8221; tab on the main menu and select &#8220;Set Measurements&#8230;&#8221; to select the reported measurements analyzed.</li>
			<li>Make sure that the &#8220;Mean grey intensity,&#8221; &#8220;Limit to threshold,&#8221; and &#8220;Display label&#8221; boxes are checked, and press &#8220;OK.&#8221; This sets how the software analyzes each area selected and how it reports it.</li>
			<li>Use the &#8220;Oval&#8221; tool to draw a circle that outlines the area that you want to measure within.</li>
			<li>Under the &#8220;Analyze&#8221; tab select &#8220;Measure,&#8221; (or press &#8220;Clt+M&#8221;). The software will measure the average grey intensity within the area defined and report it in a pop-up screen that can be copied and pasted to excel for further analysis. 
			NOTE: For more accurate analyses, at least two technical replicates of each sample should be performed.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidance for industry: Guide to minimize microbial food safety hazards for fresh fruits and vegetables. , (1998).</li><li>Bisha, B., P&#233;rez-M&#233;ndez, A., Danyluk, M. D., Goodridge, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Modified+Moore+swabs+and+continuous+flow+centrifugation+for+concentration+of+Salmonella+and+Escherichia+coli+O157:H7+from+large+volumes+of+water).">Evaluation of Modified Moore swabs and continuous flow centrifugation for concentration of Salmonella and Escherichia coli O157:H7 from large volumes of water).</a> J Food Prot. 74, 1934-1937 (2011).</li><li>Sbodio, A., Maeda, S., Lopez-Velasco, G., Suslow, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+Moore+swab+optimization+and+validation+in+capturing+E.+coli+O157:H7+and+Salmonella+enterica+in+large+volume+field+samples+of+irrigation+water.">Modified Moore swab optimization and validation in capturing E. coli O157:H7 and Salmonella enterica in large volume field samples of irrigation water.</a> Food Res Int. 51, 654-662 (2013).</li><li>McEgan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Salmonella+spp.+from+large+volumes+of+water+by+modified+Moore+swabs+and+tangential+flow+filtration.">Detection of Salmonella spp. from large volumes of water by modified Moore swabs and tangential flow filtration.</a> Lett Appl Microbiol. 56, 88-94 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solicitation Number: FDA-SS-1116253. , (2013).</li><li>Jokerst, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+paper-based+analytical+device+for+colorimetric+detection+of+select+foodborne+pathogens.">Development of a paper-based analytical device for colorimetric detection of select foodborne pathogens.</a> Analytical Chemistry. 84, 2900-2907 (1021).</li><li>Martinez, A. W., Phillips, S. T., Butte, M. J., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+paper+as+a+platform+for+inexpensive,+low-volume,+portable+bioassays.">Patterned paper as a platform for inexpensive, low-volume, portable bioassays.</a> Angew Chem Int Edit. 46, 1318-1320 (2007).</li><li>Martinez, A. W., Phillips, S. T., Wiley, B. J., Gupta, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLASH:+A+rapid+method+for+prototyping+paper-based+microfluidic+devices.">FLASH: A rapid method for prototyping paper-based microfluidic devices.</a> Lab on a Chip. 8, 2146-2150 (1039).</li><li>Abe, K., Suzuki, K., Citterio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet-printed+microfluidic+multianalyte+chemical+sensing+paper.">Inkjet-printed microfluidic multianalyte chemical sensing paper.</a> Analytical Chemistry. 80, 6928-6934 (1021).</li><li>Cheng, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millimeter-scale+contact+printing+of+aqueous+solutions+using+a+stamp+made+out+of+paper+and+tape.">Millimeter-scale contact printing of aqueous solutions using a stamp made out of paper and tape.</a> Lab on a Chip. 10, 3201-3205 (1039).</li><li>Lu, Y., Shi, W. W., Jiang, L., Qin, J. H., Lin, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+paper-based+microfluidics+with+wax+for+low-cost,+portable+bioassay.">Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay.</a> Electrophoresis. 30, 1497-1500 (2009).</li><li>Charles, S., Henry, L. D. G., Jana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jokerst+Rapid+detection+of+pathogens+using+paper+devices.">Jokerst Rapid detection of pathogens using paper devices.</a> US patent. , (2012).</li><li>Bisha, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T10-06+Colorimetric+paper-based+detection+of+Salmonella+spp.+and+Escherichia+coli+from+artificially+contaminated+irrigation+river+water.">T10-06 Colorimetric paper-based detection of Salmonella spp. and Escherichia coli from artificially contaminated irrigation river water.</a> , (2012).</li></ol>

The authors have nothing to disclose.

This protocol describes an integrated method for detecting E. coli, Salmonella spp., and L. monocytogenes in agricultural water. Here, MMS concentration of bacteria from large volumes (10 L) of agricultural water, is coupled with bacterial enrichment, and bacterial-indicative colorimetric detection using &#181;PADs. The MMS procedure can cope with high particulate content in the water samples while concentrating the bacteria 10-fold, is robust and simple enough for field applications by minimal...

It is important that foodborne disease agents are detected rapidly and preferably in field-based settings in order to reduce the burden of foodborne disease. Common strategies to detect foodborne bacterial pathogens include biochemical profiling, selective and differential culturing, immunological isolation and detection, and molecular detection. However, these methods are hampered by sporadic contamination, small sample sizes tested, the often low concentrations of the foodborne pathogenic bacteria, require long processing times, and/or are not applicable for field settings. Further, compounds in many food matrices are inhibitory to detection and diagnostic applications. In order to improve the likelihood of microbial detection, the United States Food and Drug Administration has suggested that testing agricultural water (such as wash water and irrigation water) which either comes in contact with a large surface area of fresh produce or serves as a vehicle for produce contamination is a viable alternative to direct testing of food1. Even so, the often low natural pathogen-burden coupled with the dilution effect of the representative agricultural water sample makes sample preparation methods for pathogen concentration essential. Such a method would require sampling large volumes of water (&#8805;10 L), adequate pathogen-concentration, and compatibility with downstream detection strategies.
Modified Moore swabs (MMS) are inexpensive, simple, and rugged devices used for concentrating bacteria from large volumes (&#8805;10 L) of water2-4. The MMS consists of a plastic cassette filled with gauze, which serves as a coarse filter for large volumes of water pumped through the cassette using a peristaltic pump. The MMS is a non-discriminatory method of bacterial concentration (&#8805;10 fold concentration) that captures organic and inorganic particulate material including microorganisms in processed liquid samples. It is likely that the excellent efficacy of concentration of target microorganisms by the MMS can be explained by the fact that microorganisms are expected to be attached to the silt-clay fraction or organic micro-aggregates of the suspended solids3. The rugged design of the MMS allows for overcoming most shortcomings associated with other filtration methods for capture and concentration of bacteria from water, such as clogging of filters, inability to process large volumes, filter samples with high turbidity, and high costs. For these reasons, the FDA is recommending that MMS&#8217;s be incorporated into official procedures for environmental and produce-related sample collection procedures5.
Here, a method is described for the concentration, enrichment, and detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from agricultural waters. A MMS is used for concentration of bacteria, and also serves as a vessel for selective or non-selective bacterial enrichment. Bacterial detection is achieved biochemically using paper-based analytical devices (&#181;PADs)6. &#181;PADs can be manufactured as fluidic networks or spot tests using a variety of methods including photolithography, inkjet printing, stamping, and wax printing7-11. Examples of fluidic designs can be dendritic channel patterns where the sample is deposited in the center and subsequently flows to distal reservoirs or single channel patterns in which the sample or substrate are pulled from the outer reservoirs of the channel by capillary action into the center12. For this protocol, we have chosen to employ for 7-mm-diameter wax-paper spot arrays imbedded with chromogenic substrates that can be processed by enzymes indicative of the microorganisms tested here: Chlorophenol red &#946;-D-galactopyranoside (CPRG) and 5-bromo-4-chloro-3-indolyl &#946;-D-glucuronide (X-Gluc) for detection of &#946;-galactosidase and &#946;-glucuronidase produced by E. coli; 5-bromo-6-chloro-3-indolyl caprylate (magenta caprylate) for the detection of C8-esterase produced by Salmonella spp.; and 5-bromo-4-chloro-3-indolyl-myo-inositol phosphate (X-InP) for detection of phosphatidylinositol-specific phospholipase C (PI-PLC) produced by L. monocytogenes6. Thus, the presence of a particular bacterium can be observed visually without the need for complex equipment or data interpretation. The specificity and sensitivity of the enzyme-based colorimetric &#181;PAD detection of these specific target bacteria has been previously explored6. In addition, the sensitivity of the integrated concentration-detection method for these target bacteria was evaluated by spiking of large volumes of water with pre-determined levels of microorganisms (unpublished data and Bisha&#160;et al.13).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Agricultural water</td>
			<td></td>
			<td></td>
			<td>Irrigation water, produce wash water, well water, etc.</td>
		</tr>
		<tr>
			<td>Vinyl tubing</td>
			<td>Wilmar</td>
			<td>BN-CVT1005</td>
			<td>&#160;1/4&#34; inner diameter,&#160; 3/8&#34; outer diameter, available at:&#160; http://www.wilmar.com</td>
		</tr>
		<tr>
			<td>Modified Moore Swab cartridge&#160;</td>
			<td>Lumiere Diagnostics</td>
			<td></td>
			<td>11 &#189; cm in length and 4 &#189; cm in width, available at:&#160; http://www.lumierediagnostics.com.&#160; Alternativelly, a non-disposable version of the cartridge can be used (refer to the text)</td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td>Chesapeake Wiper &#38; Supply, Inc.</td>
			<td>CC90</td>
			<td>Grade #90, 44 &#215; 36 weave, available at:&#160; www.raglady.com</td>
		</tr>
		<tr>
			<td>Household Bleach</td>
			<td>Various</td>
			<td></td>
			<td>Sodium hypochlorite concentration approx. 6%</td>
		</tr>
		<tr>
			<td>Sodium thiosulphate 5-hydrate</td>
			<td>Mallinckrodt Baker Inc</td>
			<td>8100-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Manifold</td>
			<td>Built in-house</td>
			<td></td>
			<td>Optional, device can be constructed from PVC pipes and appropriate fittings</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Micron Meters</td>
			<td>RPP1300</td>
			<td>Available at:&#160; http://www.micronmeters.com</td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Various</td>
			<td></td>
			<td>Disposable, 10ml</td>
		</tr>
		<tr>
			<td>Universal preenrichment broth</td>
			<td>Difco</td>
			<td>223510</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffered peptone water</td>
			<td>Difco</td>
			<td>218105</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella supplement</td>
			<td>Biom&#233;rieux Industry</td>
			<td>42650</td>
			<td>http://www.biomerieux-usa.com</td>
		</tr>
		<tr>
			<td>VIDAS UP Listeria (LPT) Broth</td>
			<td>Biom&#233;rieux Industry</td>
			<td>410848</td>
			<td>http://www.biomerieux-usa.com</td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td>Sigma-Aldrich</td>
			<td>861987</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Pipet-Aid</td>
			<td>Various</td>
			<td></td>
			<td>Drummond DP-110 used here</td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Various</td>
			<td></td>
			<td>Excella E25, New Brunswick Scientific used here</td>
		</tr>
		<tr>
			<td>Micropipette&#160;</td>
			<td>Various</td>
			<td></td>
			<td>10 &#956;l, 1 ml</td>
		</tr>
		<tr>
			<td>Micropipette tips</td>
			<td>Various</td>
			<td></td>
			<td>Barrier, 10 &#956;l, 1 ml</td>
		</tr>
		<tr>
			<td>1.5 microcentrifuge tubes</td>
			<td>Various</td>
			<td></td>
			<td>RNase- and DNase- free</td>
		</tr>
		<tr>
			<td>Probe sonicator</td>
			<td>Q Sonica LLC</td>
			<td>XL-2000 series</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;PADs</td>
			<td>Avant&#160;</td>
			<td></td>
			<td>Wax printed 7 mm diameter circles, with 4 pt line thickness. Contact Dr. Charles Henry for additional information</td>
		</tr>
		<tr>
			<td>HEPES [N-(2-Hydroxyethyl)piperazine-N&#8242;-2-ethanesulfonic acid]</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8022</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorophenol red-galactopyranoside (CPRG)</td>
			<td>Sigma-Aldrich</td>
			<td>59767</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-glucuronide (X-Gluc)</td>
			<td>Sigma-Aldrich</td>
			<td>B8174</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-6-chloro-3 indolylcaprylate (magenta caprylate)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>53451</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-myo-inositol phosphate (X-InP)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>38896</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene 100mm by 15 mm</td>
			<td>Various</td>
			<td></td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Flat bed scanner</td>
			<td>Various</td>
			<td></td>
			<td>Xerox USB scanner</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

colorimetric paper-based detection of escherichia coli, salmonella spp., and listeria monocytogenes from large volumes of agricultural water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

As described in this protocol, concentration of bacteria using the MMS (Figure 1) can be performed within approximately 15-20 min. The MMS in constructed from acrylonitrile butadiene styrene (ABS) in two separate components; a lid and a cartridge both with an integrated spigot assembly into which a cylindrical cheesecloth swab is inserted (Figure 1A). Both components are then screwed together forming the MMS (Figure 1B). MMS-based processing is driven by a battery-powere...

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Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

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14.3K Views.  University of Wyoming. A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

Video: Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

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 ...

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod. We provide methodology for how to conduct field surveys to characterize the morphology and develop stage-storage curves for a vernal pond. Additionally, we provide methodology for monitoring the water level, temperature, pH, oxidation-reduction potential, dissolved oxygen, and electrical conductivity of water in a vernal pond, as well as monitoring rainfall data. This information can be used to better quantify the ecosystem services that vernal ponds provide and the impacts of anthropogenic activities on their ability to provide these services.

Understanding the ecosystem services and processes provided by vernal ponds and the impacts of anthropogenic activities on their ability to provide these services requires intensive hydrologic monitoring. This sampling protocol using in-situ monitoring equipment was developed to understand the impact of anthropogenic activities on water levels and quality.

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...