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. Decontamina...

<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>

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...

Watch this Scientific Journal Video about Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water at JoVE.com

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 visualizações.  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.