La financiación de este trabajo fue proporcionado por un premio del Fondo de crecer Iowa Valores de BFBS.

1. Superficie de muestreo con cinta adhesiva estéril <ol><li> Seleccione una cinta a utilizar para el muestreo. Disponibles en el comercio Hongos-Tape o póngase en contacto-Es cintas de muestreo son estériles y envasadas especialmente para la facilidad de uso. Sin embargo, hemos encontrado que la transparencia (ópticamente transparente) cinta de la oficina de genéricos también se puede utilizar. </li><li> Use un marcador permanente para dibujar 1 cm 2 plazas en el lado no pegajosa de una pieza de 10 cm de cinta adhesiva (una plantilla de papel se puede utilizar). Esto servirá como una guía visual para destacar que parte de la cinta se ha utilizado para probar la superficie del alimento o del medio ambiente. </li><li> Formar una &quot;C&quot; en forma de lazo de cinta, con el lado adhesivo hacia la superficie a muestrear. Para ello, mantenga los extremos pegajosos con el pulgar y el dedo medio y la posición del dedo índice contra el cuadrado dibujado en la espalda (no pegajosa lado) de la cinta (Figura 1). </li><li> Coloque la cinta en la superficie para tomar muestras y presionar suavemente el área marcada sobre la superficie. Sin soltar los bordes de la cinta, utilice el dedo índice para asegurarse de que el lado pegajoso de la cinta llega en pleno contacto con la superficie de la muestra, evitando las burbujas. </li><li> El uso de un movimiento, incluso, lentamente tire de la cinta fuera de la muestra, de extraer físicamente unidas a la superficie, los microbios. Fije la cinta de células con carga, lado pegajoso hacia arriba, sobre un portaobjetos de vidrio con cinta transparente genérico de oficina. Asegúrese de que la tensión adecuada / estiramiento de la cinta para que un piso, no arrugada superficie se crea. Esto ayudará a minimizar los problemas con la deformación / curling de la cinta durante el calentamiento posterior durante la hibridación. </li></ol> 2. En fase sólida de enriquecimiento y Miniculture superficie del líquido <ol><li> En fase sólida de enriquecimiento se realiza mediante la colocación de la cinta boca abajo en la xilosa-lisina-Tergitol 4 (XLT-4) placas de agar, para que las células de la muestra se ponen en contacto directo con la superficie del agar. La plantilla parte / muestra el contacto de la cinta se coloca al ras con la superficie del agar y un extremo de la cinta está ligeramente adherida a la pared lateral de la placa de Petri para facilitar una fácil extracción de la cinta de la superficie del agar después del enriquecimiento. </li><li> Las placas son invertidas (para evitar la condensación) y se incubaron a 35 - 37 ° C para permitir el crecimiento suficiente de Salmonella spp. La duración del período de incubación necesario para enriquecer a las células a un nivel detectable dependerá de los niveles iniciales de contaminación por Salmonella. Hemos observado la formación de microcolonias excelente a partir de inóculos iniciales de baja después de las 8 h de enriquecimiento. </li><li> Tras el periodo de enriquecimiento deseado, abra la placa de agar. La cinta mantendrá su adherencia durante la incubación. Presione suavemente la cinta contra el agar usando el dedo índice para asegurar la recuperación máxima de microcolonias formado en la interfaz de cinta-agar. Sujete la cinta desde el borde pegado a la pared de la cápsula de Petri y retírela con un lento, incluso movimiento. Montar la cinta de células cargadas, el lado pegajoso arriba, sobre un portaobjetos como se describe en el punto 1.5. Proceder a la &quot;fijación y deshidratación&quot; un paso adelante. </li><li> Para miniculture superficie del líquido, comience por la fijación de la cinta utilizada para el muestreo sobre un portaobjetos como se describe en el punto 1.5. A continuación, cubrir la parte de plantilla / muestra el contacto de la cinta con una cámara de silicona no estéril de perfusión (Coverwell, Grace Bio-Labs, Inc.), formando una cámara sellada, cuyo fondo está compuesto por células de la cinta cargada, mirando hacia arriba. Firmemente pero con cuidado de prensa de la cámara en su lugar para asegurar un sello hermético. </li><li> El uso de un gel flexible de carga punta de la pipeta, transferir ≤ 500 l de caldo de tripticasa de soja u otro medio de enriquecimiento necesario a través de uno de los puertos de entrada de la cámara pequeña. Sello de los dos puertos con cinta transparente para evitar que la oficina de diapositivas evaporación e incubar a 35 -37 ° C, según sea necesario para el enriquecimiento suficiente. A pesar de todas las operaciones deben realizarse de acuerdo a buenas prácticas de muestreo y la manipulación, la esterilidad del puerto cinta aislante o cámaras de perfusión no es necesario durante miniculture superficie del líquido. La combinación de FISH y citometría de flujo permitirá una clara discriminación de Salmonella spp. a partir de bacterias que no son objeto de que puedan estar presentes, con la mayor contribución de la flora no objetivo espera que provenga de la propia muestra. Proceder a la &quot;fijación y deshidratación&quot; un paso adelante. </li></ol> 3. La fijación y deshidratación <ol><li> Para el muestreo de superficie directa o para el enriquecimiento de la fase sólida de Salmonella spp. en XLT-4 agar, lleve a cabo en la cinta de fijación de células durante 30 min a 25 ° C, cubriendo el área de contacto de la muestra con 500 l 10% de formol tamponado neutro. </li><li> Deseche fijador en un recipiente hermético (bajo una campana de productos químicos para minimizar la exposición a irritantes / vapores tóxicos). </li><li> Deshidratar en una serie de etanol (50%, 80% y 95%, 300 l / min 3 cada concentración). Proceder a la &quot;hibridación&quot; un paso adelante. </li><li> Para la fijación de 500 l de muestra de líquido miniculture superficies, cámaras de perfusión son abiertos, flexibles y un gel de carga punta de la pipeta se utiliza para extraer la enrichate. Pipeteo rápido arriba y hacia abajo se utiliza para ayudar a asegurar la eliminación efectiva de los restantes obligados cinta de las células. </li><li> A continuación, todo el volumen de 500 l miniculture se transfiere a un tubo de microcentrífuga y centrifuga a 2.000 xg (5 min). Se desecha el sobrenadante, el pellet se agita vigorosamente durante 30 a 60 s, luego resuspendido en un volumen igual de 10% de formol tamponado neutro. Se fijan las células durante 30 min a 25 ° C. </li><li> Fijador A continuación, se retira y la muestra se resuspendió en tampón de celda de almacenamiento, de la siguiente manera. La muestra se centrifuga a 2.000 xg (5 min, 25 ° C) se desecha el sobrenadante, el pellet se agita vigorosamente durante 30 - 60 s, la volvieron a suspender en un volumen igual de 50%, no desnaturalizado, etanol 50% de fosfato solución salina tamponada. Las células fijadas se pueden almacenar por tiempo indefinido (año) a -20 ° C. Nota: Cuando se cambian los líquidos (por ejemplo, al introducir un buffer de fijador o diferentes), es importante para volver a suspender completamente (con agitación) el sedimento celular en una mínima parte (~ 10 a 20 l) del sistema de &quot;salida&quot; líquido . Esto ayudará a prevenir el agrupamiento y asegurar que las suspensiones de células individuales, incluso se obtienen. Proceder a la &quot;hibridación&quot; un paso adelante. </li></ol> 4. Hibridación <ol><li> Prepare la hibridación / tampón de lavado con el comercial grado biología molecular de soluciones indica en la Tabla de Materiales. Final de las concentraciones de componentes de amortiguación son 0,7 M NaCl, 0,1 M TRIS [pH 8.0], 10 mM EDTA, 0,1% dodecilsulfato de sodio, hasta alcanzar el volumen final deseado con molecular calidad del agua y se filtra con una jeringa de 0,22 micras o filtro de la taza. El mismo tampón, sin la adición de sondas (ver más abajo) se utiliza como un tampón de lavado. Precaliente la hibridación y tampones de lavado a 55 º C. </li><li> Añadir fluorescente marcada con Sal3 (Nordentoft et al, 1997;. 5&#39;-AAT CAC TTC CAC TAC GTG-3 &#39;) y Salm-63 (Kutter et al, 2006;. 5&#39;-GAC TCG ACT TTC TCC AGC-3&#39; ) sondas de oligonucleótidos (Tabla de Materiales) de tampón de hibridación precalentada. La concentración total de la sonda de 5 ng l -1 se utiliza (por ejemplo, 2,5 ng l -1 de cada sonda, Bisha y Brehm-Stecher, 2009a). </li><li> Para las muestras de directo-a-cinta y sólida fase de enriquecimiento, cubrir el área de contacto de la muestra de plantilla de la cinta con 300 L de tampón de hibridación que contiene el cóctel de hibridación de la sonda y atado de cintas de las células en una cámara húmeda, la incubación sellada ajustado a 55 ° C. Si una incubadora de contacto directo, como el instrumento Foso de diapositivas (Tabla de Materiales) se utiliza, las muestras se hibridó durante 15 min y&gt; 20 diapositivas se pueden procesar de forma simultánea. Si un horno de hibridación giratorio al estilo, como el instrumento Bambino (Tabla de Materiales) se utiliza, las diapositivas se colocan en tubos de 50 ml de centrífuga de polipropileno para la hibridación, una diapositiva por tubo. Debido a la transferencia de calor no es directa, estas muestras se hibridó durante períodos más largos (hasta 30 min). </li><li> Tras la hibridación, las diapositivas se eliminan y la sonda que contienen superposición de tampón de hibridación se descarta. Portaobjetos se sea breve y suave enjuagarse con buffer de lavado precalentado pipeteando un pequeño volumen de tampón sobre un portaobjetos inclinado (3 enjuagues de 300 cada l), o el agua de manera formal (hasta 30 min), ya sea con una capa de tampón de lavado precalentado o inmersión en un tubo de polipropileno de centrífuga de 50 ml que contiene el buffer de lavado precalentado. En nuestra experiencia, a pesar de un lavado formales proporcionará los mejores resultados (menos neblina de la sonda no unido), un simple enjuague es adecuado para la detección inequívoca de Salmonella spp. A continuación, los portaobjetos se secaron al aire antes de pasar a la &quot;detección&quot; un paso adelante. </li><li> La hibridación de muestras de la superficie líquida miniculture se realiza girando por las muestras (recién fija o se mantiene en el buffer de almacenamiento a -20 ° C) durante 5 min a 2.000 xg seguido por agitación vigorosa de la pastilla y resuspensión en buffer de 100 l de hibridación precalentada que contiene el cóctel de la sonda. Las muestras se hibridó a 55 ° C durante 30 minutos en un bloque de calor o de otra estación de incubación adecuado (Tabla de Materiales), seguido por la adición de 500 l buffer de lavado precalentado. Al igual que con la cinta con destino las muestras, estas muestras pueden ser formalmente lavado de hasta 30 minutos con incubación a 55 ° C en el tampón de lavado, con agitación intermitente. Por otra parte, las muestras pueden ser completamente vortex después de la adición de 500 l buffer de lavado precalentado, seguido de la cosecha inmediata para el análisis (abajo). </li><li> En preparación para la detección de Salmonella spp. mediante citometría de flujo, líquido muestras miniculture superficie se centrifuga a 2.000 g durante 5 min, se desecha el sobrenadante y las muestras se resuspenden en 300 l temperatura ambiente (~ 25 ° C) PBS. Si las muestras requieren transporte a una instalación a distancia, o si el retraso se espera entre la hibridación y el análisis, las muestras se pueden refrigerar o mantenidos en hielo antes de analisis. Por otra parte, las muestras podrán ser transferidos a buffer de almacenamiento de células (una mezcla de 50:50 de la PBS y etanol absoluto) y se mantiene a -20 ° C hasta una semana sin pérdidas apreciables en la sonda confiere fluorescencia (Bisha y Brehm-Stecher, 2009b) . </li></ol> 5. Detección <ol><li> Para la detección en la cinta de Salmonella spp. a través de microscopía de fluorescencia, directo a cinta o sólidas muestras de enriquecimiento fase se superpone con aproximadamente 10 l Vectashield H-1200 montaje de soporte de la nuclear de contraste 4 &#39;,6-diamidino-2-phenylindole (DAPI), montado con una hoja de cubierta, después se incuban en la oscuridad durante 10 min. </li><li> Aceite de inmersión se coloca en la hoja de la cubierta, y las muestras se analizan utilizando un objetivo de gran aumento (63x o 100x). El filtro de DAPI se utiliza para transportar la muestra en el foco, el microscopio se cambia entonces con el filtro correspondiente (verde o rojo, dependiendo del medio de contraste utilizado para las sondas de la etiqueta final) y las células de Salmonella se anotó visualmente de acuerdo con su fluorescencia (Figuras 2 y 3). </li><li> Para la detección por citometría de líquido muestras de la superficie miniculture, diversos instrumentos se pueden utilizar, dependiendo de la disponibilidad local. En nuestro laboratorio, PBS-suspendido muestras se transfieren a 5 ml de tubos redondos de muestreo inferior (BD Falcon) y se examinaron con un citómetro de flujo FACSCanto con excitación a 647 nm (Tabla de Materiales). Para las muestras enriquecidas que contienen un alto número de bacterias totales, la &quot;baja tasa de flujo&quot; de ajuste (10 min l -1) se utiliza y 5.000-50.000 eventos se recogen. Los datos se analizaron mediante FlowJo software (versión 8.8.6, Árbol Star, Inc., Ashland, Oregón) u otro software de análisis adecuado (Figura 4, los grupos A y B). </li></ol> 6. Resultados representante <img src="/files/ftp_upload/2308/2308fig1.jpg" alt="Figura 1" /> Figura 1. El uso de cinta adhesiva para el muestreo de Salmonella spp. de la superficie de un tomate contaminado artificialmente. <img src="/files/ftp_upload/2308/2308fig2.jpg" alt="Figura 2" /> Figura 2. Los resultados típicos de muestreo directo a cinta y de detección de peces de Salmonella Typhimurium ATCC 14028 de la superficie de un tomate (100 objetivo de aceite X). Un cóctel de dos sondas de Texas Red marcado con sondas (Sal3/Salm-63) se utilizó para etiquetar estas células. <img src="/files/ftp_upload/2308/2308fig3.jpg" alt="Figura 3" /> Figura 3. Microcolonias de Salmonella Typhimurium ATCC 14028 forman en la superficie de la xilosa lisina Tergitol-4 (XLT-4) agar después de 8 h en cinta de enriquecimiento a 37 ° C. El inóculo inicial se tomaron muestras de la superficie de un tomate contaminado artificialmente. En fase sólida de enriquecimiento aumenta el número de células disponibles para la detección y también mejora el contenido celular rRNA. <img src="/files/ftp_upload/2308/2308fig4.jpg" alt="Figura 4" /> Figura 4. El uso de cinta adhesiva para recoger muestras de S. enterica serovar Typhimurium en la superficie de un tomate contaminadas artificialmente, seguido por el análisis directo mediante FISH y citometría de flujo (panel A) o después de 5 h no selectivo de enriquecimiento líquido miniculture superficie en una cámara de perfusión llena con 500 l de caldo de soja tripticasa (panel B ).

<ol>
	<li>Almeida, C., Azevedo, N. F., Fernandes, R. M., Keevil, C. W., Vieira, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+method+using+a+peptide+nucleic+acid+probe+for+the+identification+of+Salmonella+spp.+in+a+broad+spectrum+of+samples.">Fluorescence in situ hybridization method using a peptide nucleic acid probe for the identification of Salmonella spp. in a broad spectrum of samples.</a> Appl. Environ. Microbiol. 76, 4476-4485 (2010).</li><li>Barnetson, R. S., Milne, L. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+sampling+for+Candida+with+adhesive+tape.">Skin sampling for Candida with adhesive tape.</a> Br. J. Dermatol. 88, 487-491 (1973).</li><li>Bisha, B., Brehm-Stecher, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+adhesive-tape-based+sampling+of+tomato+surfaces+combined+with+rapid+fluorescence+in+situ+hybridization+for+Salmonella+detection.">Simple adhesive-tape-based sampling of tomato surfaces combined with rapid fluorescence in situ hybridization for Salmonella detection.</a> Appl. Environ. Microbiol. 75, 1450-1455 .</li><li>Bisha, B., Brehm-Stecher, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-through+imaging+cytometry+for+characterization+of+Salmonella+subpopulations+in+alfalfa+sprouts,+a+microbiologically+complex+food+system.">Flow-through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a microbiologically complex food system.</a> Biotechnol. J. 4, 880-887 (2009).</li><li>Edwards, R. W., Hartman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+collecting+fungus+specimens+from+infected+surfaces.">A simple technique for collecting fungus specimens from infected surfaces.</a> Lloydia. 15, 39-39 (1952).</li><li>Evancho, G. M., Sveum, W. H., Moberg, L. J., Frank, J. F., Pouch Downes, F., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+monitoring+of+the+food+processing+environment.">Microbiological monitoring of the food processing environment.</a> Compendium of Methods for the Microbiological Examination of Foods. , (2001).</li><li>Fung, D. Y. C., Lee, C. Y., Kastner, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+tape+method+for+estimating+microbial+load+on+meat+surfaces.">Adhesive tape method for estimating microbial load on meat surfaces.</a> J. Food. Prot. 43, 295-297 (1980).</li><li>Kutter, S., Hartmann, A., Schmid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+barley+(Hordeum+vulgare)+with+Salmonella+enterica+and+Listeria+spp.">Colonization of barley (Hordeum vulgare) with Salmonella enterica and Listeria spp.</a> FEMS Microbiol. Ecol. 56, 262-271 (2006).</li><li>La Cono, V., Urz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+applied+on+samples+taken+with+adhesive+tape+strips.">Fluorescent in situ hybridization applied on samples taken with adhesive tape strips.</a> J. Microbiol. Meth. 55, 65-71 (2003).</li><li>Lakshmanan, C., Schaffner, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+controlling+microbiological+contamination+of+beverage+dispensers+in+university+foodservice+operations.">Understanding and controlling microbiological contamination of beverage dispensers in university foodservice operations.</a> Food Prot. Trends. 26, 27-31 (2005).</li><li>Langvad, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+qualitative+and+quantitative+study+of+the+fungal+flora+of+leaves.">A simple and rapid method for qualitative and quantitative study of the fungal flora of leaves.</a> Can. J. Microbiol. 26, 666-670 (1980).</li><li>Nordentoft, S., Christensen, H., Wegener, H. C. <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+a+fluorescence-labelled+oligonucleotide+probe+targeting+23S+rRNA+for+in+situ+detection+of+Salmonella+serovars+in+paraffin-embedded+tissue+sections+and+their+rapid+identification+in+bacterial+smears.">Evaluation of a fluorescence-labelled oligonucleotide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears.</a> J. Clin. Microbiol. 35, 2642-2648 (1997).</li></ol>

Métodos sencillos y rápidos para la detección de patógenos en las superficies de los productos pueden ayudar a mitigar las enfermedades transmitidas por los alimentos, proporcionando información oportuna y procesable. Adhesivo a base de cinta de métodos de muestreo se han utilizado en el medio ambiente, clínica y microbiología de los alimentos desde la década de 1950 e implican apremiantes de &quot;Scotch&quot; al estilo de la cinta a la superficie para la eliminación de microorganismos, seguido por el examen ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Fungi-Tape sampling tape</td>
<td>&nbsp;</td>
<td>Scientific Device Laboratory, Des Plaines, IL</td>
<td>745</td>
<td>http://www.scientificdevice.com/</td>
</tr>
<tr>
<td>Con-Tact-It sampling tape</td>
<td>&nbsp;</td>
<td>Birko Corporation, Denver, CO</td>
<td>&nbsp;</td>
<td>http://www.birkocorp.com/</td>
</tr>
<tr>
<td>Clear office tape, generic</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Should be optically clear, have low intrinsic fluorescence</td>
</tr>
<tr>
<td>Food surface</td>
<td>&nbsp;</td>
<td>Local grocery</td>
<td>&nbsp;</td>
<td>Tomatoes (red tomatoes on the vine, not waxed or oiled) used here</td>
</tr>
<tr>
<td>Trypticase Soy Broth</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>211768</td>
<td>For non-selective liquid surface miniculture enrichment</td>
</tr>
<tr>
<td>Xylose-lysine-Tergitol 4 agar base</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>223420</td>
<td>For Salmonella-selective agar (XLT-4)</td>
</tr>
<tr>
<td>Xylose-lysine-Tergitol 4 agar supplement</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>235310</td>
<td>For Salmonella-selective agar (XLT-4)</td>
</tr>
<tr>
<td>Formalin solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>HT5011</td>
<td>10% solution, neutral, buffered (cell fixative)</td>
</tr>
<tr>
<td>Absolute ethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>E7023</td>
<td>Molecular biology grade (pre-hybridization dehydration)</td>
</tr>
<tr>
<td>1.5 ml microcentrifuge tubes</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>RNase- and DNase-free</td>
</tr>
<tr>
<td>Microscope slides and cover slips</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific, Waltham, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>S5150</td>
<td>Molecular biology grade, 5M solution (hybridization buffer component)</td>
</tr>
<tr>
<td>Tris-EDTA buffer solution (100X concentrate)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>T9285</td>
<td>1M Tris [pH 8.0], 0.1M EDTA (hybridization buffer component)</td>
</tr>
<tr>
<td>Sodium dodecyl sulfate solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>L4522</td>
<td>10% solution in 18 megohm water (hybridization buffer component)</td>
</tr>
<tr>
<td>Sal3 and Salm-63 oligonucleotide probes</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies, Coralville, IA</td>
<td>&nbsp;</td>
<td>5&#x2019;-labeled with 6-carboxyfluorescein (FAM) or Texas Red (for microscopy) or Cy5 (for cytometry), HPLC-purified</td>
</tr>
<tr>
<td>Variable speed microcentrifuge</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Use rotor diameter to calculate RPM needed for RCF values described in protocol</td>
</tr>
<tr>
<td>CoverWell perfusion chamber </td>
<td>&nbsp;</td>
<td>Grace Bio-Labs Inc., Bend, OR</td>
<td>PC1R-2.0</td>
<td>Non-sterile</td>
</tr>
<tr>
<td>Gel loading pipette tips (FS MultiFlex)</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific, Waltham, MA</td>
<td>05-408-151</td>
<td>Long, thin tips for easy access to small sampling ports and maneuverability within chamber</td>
</tr>
<tr>
<td>Aluminum heat block or precision-controlled heating station</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Eppendorf Thermomixer R dry block heating and cooling shaker used here</td>
</tr>
<tr>
<td>Bambino mini hybridization oven</td>
<td>&nbsp;</td>
<td>Boekel Scientific, Feasterville, PA</td>
<td>Model 230300</td>
<td>Slides are placed in 50 ml polypropylene centrifuge tubes for hybridization, heat transfer not direct</td>
</tr>
<tr>
<td>Slide Moat slide hybridizer</td>
<td>&nbsp;</td>
<td>Boekel Scientific, Feasterville, PA</td>
<td>Model 240000</td>
<td>Provides rapid, direct transmission of heat through glass slide</td>
</tr>
<tr>
<td>Vectashield H-1200 mounting medium with 4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Vector Laboratories, Inc., Burlingame, CA</td>
<td>H-1200</td>
<td>Minimizes quenching of fluorescence during microscopy, provides DAPI counterstain</td>
</tr>
<tr>
<td>Fluorescence microscope</td>
<td>&nbsp;</td>
<td>Various suppliers </td>
<td>&nbsp;</td>
<td>Leitz Laborlux S used here</td>
</tr>
<tr>
<td>Digital camera</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Canon PowerShot A640 camera used here</td>
</tr>
<tr>
<td>Image acquisition software</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Axiovision software v. 4.6 (Carl Zeiss) used</td>
</tr>
<tr>
<td>Adobe Photoshop</td>
<td>&nbsp;</td>
<td>Adobe Inc.</td>
<td>&nbsp;</td>
<td>For minimal processing of images (overlay of images taken in different channels)</td>
</tr>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>FACSCanto flow cytometer (BD Biosciences, San Jose, CA) with red (647 nm) excitation used</td>
</tr>
<tr>
<td>Flow cytometry analysis software</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>FlowJo software v. 8.7.1 (Tree Star, Inc.) used</td>
</tr>		</tbody>
		</table>
		</div>

combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of salmonella on fresh produce

Este protocolo describe un método sencillo para que el adhesivo, la cinta basada en el muestreo de tomate y otras superficies de los productos frescos, seguido por fluorescencia en cinta de hibridación in situ (FISH) para un rápido cultura independiente de la detección de Salmonella spp. Cintas cargadas de células también puede ser colocado boca abajo en agar selectivo para la fase sólida de enriquecimiento antes de la detección. Por otra parte, bajo volumen de enriquecimiento líquido (miniculture superficie del líquido) se puede realizar en la superficie de la cinta en el no selectivo caldo, seguidos por el pescado y el análisis mediante citometría de flujo. Para comenzar, cinta adhesiva estéril se pone en contacto con productos frescos, se aplica presión leve, y de retirar la cinta, de extraer físicamente los microbios presentes en estas superficies. Las cintas están montadas pegajoso hacia arriba en portaobjetos de vidrio y las células de la muestra se fijan con formol al 10% (30 min) y deshidratados mediante una serie gradual de etanol (50, 80, y 95%, 3 min cada concentración). A continuación, las células cargadas las cintas se han manchado con un tampón que contiene un cóctel de ADN de Salmonella objetivo de la sonda y se hibridaron durante 15 - 30 min a 55 ° C, seguido de una breve aclaración en un tampón de lavado para eliminar la sonda no unido. Adherente, PESCADO marcado células se contrastados con el ADN tinte 4 &#39;,6-diamidino-2-phenylindole (DAPI) y los resultados se ven usando la microscopía de fluorescencia. Para la fase sólida de enriquecimiento, las células cargadas las cintas se colocan boca abajo sobre una superficie de agar selectivo adecuado y se incuban para permitir el crecimiento in situ de microcolonias Salmonella, seguidos por el pescado y la microscopia como se describió anteriormente. Para miniculture superficie del líquido, las células cargadas las cintas se colocan lado pegajoso hacia arriba y una cámara de perfusión de silicona se aplica para que la cinta y la forma del portaobjetos del microscopio la parte inferior de una cámara hermética en la que un pequeño volumen (≤ 500 l) de tripticasa de soja caldo (TSB) se introduce. Los puertos de entrada están cerrados y las cámaras se incuban a 35 - 37 ° C, permitiendo que el crecimiento basado en la amplificación de extraer la cinta-microbios. Después de la incubación, los puertos de entrada son sin sellar, las células se separan y se mezclan con la espalda vigorosa y pipeteo adelante, cosechadas por centrifugación y se fija en 10% neutros formalina tamponada. Finalmente, las muestras se hibridan y se examina a través de citometría de flujo para revelar la presencia de Salmonella spp. Como se describe aquí, nuestra &quot;cinta-FISH&quot; enfoque puede proporcionar un muestreo simple y rápida y la detección de Salmonella en las superficies de tomate. También hemos utilizado este enfoque para el muestreo de otros tipos de productos frescos, como la espinaca y chiles jalapeños.

Watch this Scientific Journal Video about Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce at JoVE.com

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Este protocolo describe un simple adhesivo cinta enfoque basado en el muestreo de tomate y otras superficies de los productos frescos, seguido por la rápida detección de células enteras de<em&gt; Salmonella</em&gt; Mediante fluorescencia<em&gt; In situ</em&gt; Hibridación (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

combination-adhesive-tape-based-sampling-fluorescence-situ

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

12.9K vistas.  Colorado State University. Este protocolo describe un simple adhesivo cinta enfoque basado en el muestreo de tomate y otras superficies de los productos frescos, seguido por la rápida detección de células enteras de Salmonella Mediante fluorescencia In situ Hibridación (FISH).

Video: Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Flujo de ácido nucleico cerrado Citometría de fluorescencia In situ</emHibridación> (LNA flujo-FISH): un método para la detección de bacterias pequeñas de ARN

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Investigación

Inmunología e Infección

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Hibridación<em&gt; In situ</em&gt; De las glándulas salivales, los ovarios y embriones de los mosquitos vectores

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescente<em&gt; In situ</em&gt; La hibridación en los cromosomas mitóticos de mosquitos

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

Fluorescencia<em&gt; In situ</em&gt; Las hibridaciones (FISH) para la localización de los virus y las bacterias endosimbióticas en plantas e insectos tejidos

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Método de muestreo hisopo para la Detección de norovirus humano en superficies

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

La neuraminidasa Ensayo de inhibición basado en la fluorescencia para evaluar la susceptibilidad de los virus de la influenza a El Inhibidor de la neuraminidasa clase de antivirales

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Muestreo no invasivo de revestimiento mucoso líquido para la cuantificación In Vivo de la vía aérea superior mediador inmune niveles

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una plataforma de alto rendimiento para la detección de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Fluorescencia cuantitativa en hibridación situ (FISH) e inmunofluorescencia (IF) de productos genéticos específicos en células infectadas por KSHV

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...