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

1. Concentração de bactérias a partir de grandes volumes de água agrícolas que utilizam MMS <ol><li> Preparação MMS <ol><li> Corte uma seção retangular de 4-ply gaze medindo 40 x 12 cm. </li><li> Dobre o pano ao longo de ambos os eixos para obter um retângulo de 20 x 6 cm. </li><li> Enrole a gaze firmemente ao longo do seu eixo longitudinal de modo a formar uma haste cilíndrica de cerca de 6 cm de altura e 3 cm de diâmetro. </li><li> Autoclave o cotonete gaze em papel alumínio, não autoclave cassete. Descontaminar a cassete por imersão durante 30 minutos em 10% de água sanitária, e desactivar o cloro residual por imersão a cassete durante 15 minutos em um pentahidrato de tiossulfato de sódio (Na 2 S 2 O 3 · 5H 2 O) de solução (50 mg / L preparadas em água estéril). </li><li> Insira o cotonete na cassete MMS. NOTA: Se a versão não-descartável da cassete MMS é usado, comece com uma folha de 4-ply de gaze menta 80 x 22 cm, dobre-o para 40 x 11 cm e enrole para formar um cilindro de aproximadamente 11 cm de altura e 6 cm de diâmetro. </li><li> Monte o MMS. </li><li> Anexar tubo de vinil de espigões em ambos os lados do MMS, garantindo a tubagem adere completamente aos espigões, e corrigir tubo na bomba peristáltica. Certifique-se de que a tubagem a montante da bomba peristáltica é de comprimento suficiente para amostragem. </li></ol></li><li> A concentração de bactérias <ol><li> Amostra de pelo menos 10 litros de água agrícola, executando a bomba peristáltica em alta velocidade. </li><li> Após a amostragem for concluída, retirar a entrada da bomba peristáltica a partir da amostra de água e continuar a executar a bomba até que nenhuma água efluente é observado sair do MMS. </li><li> Se um colector é usado para executar várias amostras ao mesmo tempo, recolher o efluente de cada MMS, e quando é 10 L é filtrada através de um único MMS fechar a válvula para que o MMS particulares e continuar a funcionar a bomba. <ol><li> Quando a amostragem is concluído, retire a entrada da bomba peristáltica da água na agricultura, abrir todas as válvulas do colector, e continuar a executar a bomba até que nenhuma água efluente é observado sair do MMS. </li></ol></li></ol></li><li> Quando a concentração tenha sido concluída para todas as mensagens MMS, remova cuidadosamente o tubo de vinil a partir das torneiras MMS e tampar as torneiras em ambos os lados do MMS. MMS bem fechados pode ser armazenado por até 12 horas antes de adicionar mídia de enriquecimento. </li></ol> 2. MMS Enriquecimento e Preparação de Amostras <ol><li> Enriquecimento <ol><li> Soltar a tampa do MMS, e adicionar assepticamente 20 ml de caldo de enriquecimento estéril apropriado. Use um MMS para cada enriquecimento seletivo. Alternativamente, realizar um enriquecimento não selectivo para propagar as três bactérias alvo na mesma amostra. NOTA: Se a versão não-descartável da cassete MMS é usado, de forma asséptica remover filtro de pano da cassete e coloque em um saco estéril antes de anúncioding 225 ml da mídia enriquecimento adequado. <ol><li> Para o enriquecimento de E. coli, adicionar 20 ml de água de peptona tamponada (BPW) suplementado com 8 mg / L de cloridrato de vancomicina. </li><li> Para o enriquecimento de Salmonella spp., Adicionar 20 ml de BPW suplementado com o suplemento de Salmonella (4 ml / l). </li><li> Para o enriquecimento de L. monocytogenes, adicione 20 ml de VIDAS UP Listeria (LPT) caldo. </li><li> Para enriquecimento não seletivo simultânea para todos os três microrganismos utilizando um único MMS, use caldo preenrichment universal (UPB). </li></ol></li><li> Feche a tampa do MMS para trás com força. Certifique-se de que as torneiras estão bem tampada, para evitar derrames e possível contaminação durante o enriquecimento. </li><li> Incubar as MMS para até 18-24 horas numa incubadora com agitação a 200 rpm. Utilizar 42 ° C durante E. coli e Salmonella spp. enriquecimentos seletivos. Use 30 ° C para L. monocytogenes enriquecimento seletivo, e usar 37 &amp; #176; C para o enriquecimento não-seletivo. </li></ol></li><li> Preparação de Amostras <ol><li> Quando incubação estiver concluída, remova as MMS da incubadora, destampar uma das torneiras, e gentilmente dispensar cerca de 0,5 ml em um tubo de microcentrífuga de 1,5 ml. Alternativamente, desapertar os MMS e pipeta fora 0,5 ml de enriquecimento durante a noite. NOTA: Se a versão não-descartável da cassete MMS é usado, pipetar 0,5 ml de enriquecimento do saco. </li><li> Para a análise colorimétrica de E. coli de enriquecimento selectivo, ou para a análise dos enriquecimentos não selectivos, sonicar 0,5 ml de enriquecimento a 5 W, 22 kHz, durante 20 segundos usando um sonicador de sonda. </li></ol></li></ol> 3. Preparação e incorporação de substratos colorimétricos em μPADs <ol><li> Prepare μPADs suficientes para cada amostra e alvo testado. Para detectar E. coli preparar CPRG e X-Glic μPADs, por Salmonella spp. detecção preparar μPADs MC, e por L. monocytdetecção ogenes preparar μPADs X-InP. <ol><li> Preparar tampão HEPES [0,1 M de HEPES com 0,1% de albumina de soro bovino (BSA)] para os substratos colorimétricos e ajustar o pH do tampão de acordo com o substrato que vai ser usado: pH 7,5 para a preparação de CPRG ou X-Gluc, pH 9 para MC, e pH 7 para X-InP. </li><li> Preparar soluções stock dos substratos em que o pH ajustado a concentrações de tampão de HEPES de 3 mM (CPRG), 62,5 mM de (X-Gluc), 15 mM de (MC), 100 mM de (X-InP). Proteger as soluções estoque da luz. </li><li> Coloque os μPADs dentro de uma placa de Petri estéril, em seguida, encaixar o microspot μPAD com 24 mL de cada solução de substrato. </li></ol></li></ol> 4. Detecção e Análise de Dados <ol><li> Detecção <ol><li> Adicionar 6 ml da amostra enriquecida para cada microspot μPAD apropriado contido em uma placa de Petri. </li><li> Coloque a tampa de volta na placa de Petri e selá-lo com Parafilm. </li><li> Incubar a amostracom a μPAD a 37 ° C durante até 3 horas para permitir o desenvolvimento da cor e secagem dos microspots. </li></ol></li><li> Exame visual dos resultados <ol><li> Realizar a detecção por simples inspeção visual das mudanças de cor das μPADs. NOTA: Normalmente, fortes reações que produzem mudanças de cor definitivos são esperados com a 18-24 horas de enriquecimento, o que deve evitar a necessidade de mais esclarecimentos e deve ser considerado positivo. </li><li> Determinar a presença de uma E. coli amostra positiva, observando microspots encaixados com a mudança CPRG do amarelo ao vermelho-violeta e microspots encaixados com X-Gluc mudança de incolor a azul-esverdeada. </li><li> Determinar a presença de um L. monocytogenes amostra positiva, observando microspots encaixados com X-InP mudança de incolor a índigo. </li><li> Determinar a presença de uma Salmonella spp. amostra positiva por microspots observando encaixados com MC mudança de colorless ao roxo-lilás. </li></ol></li><li> Software Aided Análise de Dados NOTA: Conduzido para determinar claramente se reacções fracas são positivos ou negativos e permitir uma análise semi-quantitativa dos resultados. <ol><li> Permitir que as μPADs para secar completamente. </li><li> Digitalizar o μPAD usando um scanner plano. </li><li> Use o software ImageJ para processar a imagem. <ol><li> &quot;Abrir&quot; imagem digitalizada para ser analisado em ImageJ localizado na guia &quot;Arquivo&quot; no menu principal (ou pressione &quot;Clt + O&quot;). </li><li> Inverta a imagem selecionando &quot;Inverter&quot;, localizada na aba &quot;Editar&quot; no menu principal (ou pressione &quot;Clt + Shift + I&quot;). </li><li> Use a guia &quot;Analisar&quot; no menu principal e selecione &quot;Definir Medidas ...&quot; para selecionar as medições relatados analisados. </li><li> Certifique-se de que a &quot;intensidade de cinza média&quot;, &quot;Limite de limiar&quot;, e &quot;caixas rótulo de exibição&quot; sãomarcada, e pressione &quot;OK&quot;. Isso define a forma como o software analisa cada área selecionada e como ela relata ele. </li><li> Use a ferramenta &quot;Oval&quot; para desenhar um círculo que define a área que você deseja medir dentro. </li><li> Sob a guia &quot;Analisar&quot;, selecione &quot;Measure&quot;, (ou pressione &quot;Clt + M&quot;). O software irá medir a intensidade cinza médio dentro da área definida e relatá-lo em uma tela pop-up que pode ser copiado e colado para se destacar para análise posterior. NOTA: Para uma análise mais precisa, pelo menos, duas repetições técnicos de cada amostra deve ser realizada. </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.

Este protocolo descreve um método para a detecção de E. integrado coli, Salmonella spp., e L. monocytogenes em água agrícola. Aqui, a concentração de MMS de bactérias a partir de grandes volumes (10 L) de água na agricultura, é acoplado com o enriquecimento bacteriana, e detecção colorimétrica de bactérias indicativo usando μPADs. O procedimento MMS pode lidar com elevado teor de partículas nas amostras de água enquanto se concentra a bactérias de 10 vezes, é robusta e simpl...

É importante que os agentes de doenças de origem alimentar são detectados rapidamente e de preferência em ambientes baseados em campo, a fim de reduzir a carga de doenças transmitidas por alimentos. As estratégias comuns para detectar bactérias patogênicas transmitidas por alimentos incluem perfis bioquímicos, cultura seletivo e diferencial, isolamento e detecção imunológica e detecção molecular. No entanto, estes métodos são prejudicados pela contaminação esporádica, pequenas amostras testadas, as baixas concentrações muitas vezes das bactérias patogênicas transmitidas por alimentos, requerem longos tempos de processamento e / ou não são aplicáveis ​​para as definições de campo. Além disso, os compostos em diversas matrizes alimentares são inibitórios para aplicações de diagnóstico e detecção. A fim de melhorar a probabilidade de detecção microbiana, os Estados Unidos Food and Drug Administration tem sugerido que o teste da água na agricultura (como água de lavagem e água de irrigação), que quer entrar em contato com uma grande superfície de produtos frescos ou serve como um veículo para pcontaminação roduce é uma alternativa viável para o teste direto de alimentos 1. Mesmo assim, a freqüência baixa patógeno-peso natural, juntamente com o efeito de diluição da amostra de água agrícola representante torna os métodos de preparação de amostras para a concentração do patógeno essencial. Tal método poderia exigir a amostragem de grandes volumes de água (≥ 10 L), adequada patógeno-concentração, e compatibilidade com estratégias de detecção a jusante. Cotonetes Moore Modificados (MMS) são dispositivos de baixo custo, simples e robustos utilizados para concentrar bactérias de grandes volumes (≥ 10 L) de água 2-4. O MMS é composta de uma cassete de plástico cheio com gaze, que serve como um filtro grosseiro para grandes volumes de água bombeados através da caixa, utilizando uma bomba peristáltica. O MMS é um método não-discriminatório de concentração bacteriana (≥ concentração 10 vezes) que captura material particulado orgânico e inorgânico, incluindo microrganismos em li processadoamostras quid. É provável que a excelente eficiência de concentração de microrganismos alvo por o MMS pode ser explicado pelo fato de que os microorganismos devem ser ligados à fracção-argilo ou micro-agregados orgânicos dos sólidos em suspensão 3. O design robusto do MMS permite superar a maioria dos defeitos associados a outros métodos de filtragem para a captura e concentração de bactérias da água, como o entupimento dos filtros, a incapacidade de processar grandes volumes, as amostras de filtro com alta turbidez, e custos elevados. Por estas razões, o FDA está recomendando que da MMS ser incorporados procedimentos oficiais para procedimentos de coleta de amostras ambientais e relacionadas com a produzir 5. Aqui, é descrito um método para a concentração, o enriquecimento, e a detecção de Escherichia coli, Salmonella spp., E Listeria monocytogenes de águas agrícolas. A MMS é usado para concentração de bactériaeria, e também serve como um recipiente para o enriquecimento selectivo de bactérias ou não selectiva. Detecção bacteriana é conseguido bioquimicamente usando dispositivos analíticos baseados em papel (μPADs) 6. μPADs pode ser fabricado como redes fluídicas ou testes local usando uma variedade de métodos, incluindo fotolitografia, impressão a jato de tinta, estampagem, e cera de impressão 7-11. Exemplos de modelos de fluidos podem ser os padrões de canal dendríticas em que a amostra é depositada no centro e, subsequentemente, os fluxos de reservatórios distais ou padrões de canal único, no qual a amostra ou o substrato são puxados a partir dos reservatórios exteriores do canal, por acção capilar até ao centro 12. Para este protocolo, optou-se por empregar para 7 mm de diâmetro com papel de cera matrizes ponto encaixados com substratos cromogênicos que podem ser processados ​​por enzimas indicativas dos microrganismos testados aqui: Clorofenol vermelho β-D-galactopyranoside (CPRG) e 5 - bromo-4-cloro-3-indolil β-D-glucuronido (X-Gluc)para detecção de β-galactosidase e β-glucuronidase produzidas por E. coli; Caprilato de 5-bromo-6-cloro-3-indolilo (magenta caprilato) para a detecção de C8-esterase produzidas por Salmonella spp.; e-5-bromo-4-cloro-3-indolil-fosfato de mio-inositol (X-InP) para a detecção específica de fosfatidilinositol-fosfolipase C (PLC-PI) produzida por L. monocytogenes 6. Assim, a presença de uma bactéria em particular pode ser observado visualmente, sem a necessidade de equipamento complexo ou a interpretação de dados. A especificidade e sensibilidade da detecção colorimétrica μPAD à base de enzimas destas bactérias alvo específicas tem sido explorada anteriormente 6. Além disso, a sensibilidade do método de detecção de concentração integrada para estas bactérias alvo foi avaliada por cravação de grandes volumes de água com níveis de microorganismos (dados não publicados e Bisha et al. 13) pré-determinados.

<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

Este protocolo descreve a detecção colorimétrica rápida de Escherichia coli, Salmonella spp., E Listeria monocytogenes, a partir de grandes volumes (10 L) de águas agrícolas. Aqui, a água é filtrada através de estéril modificação Moore Cotonetes (MMS), que consistem de um filtro de gaze simples fechado em um cartucho de plástico, para concentrar as bactérias. Após a filtração, enriquecimentos não selectivos ou selectivos para as bactérias alvo são realizadas no MMS. Para a detecção colorimétrica da bactéria alvo, os enriquecimentos são então ensaiadas usando os dispositivos de análise à base de papel (μPADs) incorporados com substratos bactérias indicativos. Cada substrato reage com enzimas bacterianas alvo indicativo, gerando produtos coloridos que podem ser detectados visualmente (detecção qualitativa) na μPAD. Alternativamente, as imagens digitais das μPADs reagiram podem ser gerados com a digitalização comum ou dispositivos fotográficos e analisados ​​usando o software ImageJ, almugido para interpretação mais objetiva e padronizada de resultados. Embora os procedimentos de rastreio bioquímicos são concebidos para identificar os agentes patogénicos bacterianos acima mencionados, em alguns casos, enzimas produzidas por bactérias do fundo ou a degradação dos substratos colorimétricos podem produzir um falso positivo. Portanto, é necessária a confirmação através de um diagnóstico mais discriminatória. No entanto, esta plataforma de concentração e detecção de bactérias é barato, sensível (0,1 CFU limite de detecção / ml), de fácil execução e rápida (concentração, enriquecimento e detecção são realizados dentro de aproximadamente 24 horas), justificando o seu uso como um método de triagem inicial para a qualidade microbiológica da água na agricultura.

Tal como descrito no presente protocolo, a concentração de bactérias que usam o MMS (Figura 1) pode ser realizada dentro de aproximadamente 15-20 min. Os MMS no construídos a partir de acrilonitrila-butadieno-estireno (ABS) em dois componentes separados; uma tampa e um cartucho de ambos com um espigão de montagem integrado no qual uma haste de gaze cilíndrica é inserida (Figura 1A). Os dois componentes são então aparafusado em conjunto, formando o MMS (Figura 1B).</stro...

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.

Papel colorimétrico à base de Detecção de<em&gt; Escherichia coli</em&gt;,<em&gt; Salmonella</em&gt; Spp. E<em&gt; Listeria monocytogenes</em&gt; A partir de grandes volumes de água Agrícola

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

colorimetric-paper-based-detection-escherichia-coli-salmonella-spp

Research

JoVE Journal

Environment

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

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.

Vídeo: 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.

Determinados pelo Laboratório Fósforo Flux do Lago Sedimentos como medida de Fósforo Interno Carregando

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

Ambiente

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.

Medição do fluxo de gases com efeito de Solos Agrícolas Usando Chambers estáticos

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 Método 1615. Medição de Enterovirus e Norovirus Ocorrência na água por cultura e RT-qPCR. Parte III. Detecção de vírus por 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 ...

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.

Um ensaio de PCR Duplex Digital para a simultânea Quantificação da<em&gt; Enterococcus spp.</em&gt; Ea Human Fecal-associado HF183 marcador no 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.

Caracterização e aplicação de amostradores passivos de monitorização de pesticidas em água

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.

Monitoramento Contínuo de Nutrientes e Sedimentos em Bacias Agrícolas

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.

Medição e mapeamento de padrões de erosão e deposição relacionadas às concentrações de carbonato de solo sob gestão agrícola

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.

Capturando o fluxo ponderado água e partículas em suspensa de canais agrícolas durante eventos de drenagem

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.

Contínua hidrológicos e monitoramento da qualidade do Vernal lagoas de água

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.

Específicos e detecção precisa do Using PCR convencional em amostras de tecido de folhas cítricas cítricas Greening patógeno Candidatus liberibacter spp.

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