Los fondos para el desarrollo de este protocolo fue proporcionada por el Consorcio Sureste de Pensilvania para la Educación Superior y el Departamento de Ciencias Cabrini Colegio. Financiamiento y apoyo adicional vino de Arcadia University y la Universidad Immaculata. Agradecemos especialmente a la doctora Karen Snetselaar en la Universidad de San José para tomar amablemente las imágenes de microscopía electrónica de nuestros fagos aislados. Apoyo adicional fue proporcionado por los cazadores del Instituto Médico Howard Hughes de la Alianza de Educación Científica de fagos que subieron Genómica y programa (SEA-FAGOS) Evolutiva Ciencia.

1. Preparación de las células para el aislamiento de fagos Arthobacter <ul><li> Arthrobacter Cultura sp. KY3901 colonias estrías en una Luria Bertaini (LB) placa de agar se incubaron a 30 ° C durante 2-3 días. Escoja una colonia y utilizar un asa estéril para añadirlo a 250 ml de caldo LB en un matraz de cultivo desconcertado e incubar en una incubadora de agitación a 225 rpm a 30 ° C. </li><li> Deje aproximadamente 24 h de crecimiento para obtener células en fase exponencial finales / temprana estacionarias para experimentos de infección del fago. Supervisar el estado de crecimiento bacteriano de cerca para evitar que las células entren en el medio y tardío estado de crecimiento estacionario. NOTA: El crecimiento celular debe consistir en una densidad óptica OD 600 entre 0,5-aproximadamente 0.7 para los procedimientos de aislamiento y purificación de fagos / pasos descritos a continuación. </li></ul> 2. Recopilación de fagos de muestras de suelo <ol><li> Reúna 200-400 g de suelo desde la ubicación deseada. Nota: Puesto que las bacterias en la Arthrobagénero CTER son bacterias comunes del suelo y que los fagos que infectan a ellos se pueden encontrar en y son omnipresentes en la mayoría de los tipos de suelo. </li><li> Añadir al menos 400 ml de tampón de fago (PB) [NaCl 68 mM, 10 mM MgSO 4, 10 mM Tris-CL (pH 7,5)] para el suelo y mezclar en un matraz lo suficientemente grande como para que al menos 200 ml de PB es en el sobrenadante y es capaz de ser extraído. </li><li> Mezcle por agitación o agitación suave hasta que se suspendió el suelo y permitir que el sedimento del suelo se asiente, típicamente 30 min. </li><li> Pase el &quot;extracto de tierra&quot; a través de papel de filtro por filtración por gravedad para eliminar los restos de sedimentos del suelo. </li><li> Añadir polvo de LB y mezclar para obtener una solución al 2% (4 g LB en polvo en 200 ml de extracto de suelo). </li><li> Pasar la solución a través de un filtro de 0,22 micras por filtración a vacío para obtener un filtrado estéril. Si es posible, continúe con el paso 2.7 inmediatamente. Si es necesario, la tienda filtra muestras a 4 ° C durante un máximo de una semana antes de continuar, sin embargo, el número de partículas de fagos viablesse reducirá. </li><li> Alícuota de múltiples porciones de 50 ml de la mezcla de extracto de LB / suelo esterilizada por filtración en individuales 250 ml esterilizados agitando los matraces de cultivo utilizando técnicas asépticas con deflectores. Añadir M CaCl solución estéril 2,0 2 a las concentraciones finales deseadas (0-50 mm) desde diferentes fagos Arthrobacter crecen óptimamente en diferentes CaCl 2 condiciones. NOTA: Nos encontramos con que la mayoría de los fagos Arthrobacter identificados por nosotros están dentro del rango de 1 mM-2,5 mM CaCl2. Sin embargo, varios de los fagos aislados crecieron por nosotros exclusivamente a 0 mM CaCl 2 .o a muy altas concentraciones de CaCl 2. Proceda inmediatamente al paso 3.1. </li></ol> Aislamiento 3. Fago <ol><li> Añadir 1 a 2,5 ml de cultivo de bacterias estacionaria tardía fase exponencial / temprano para cada uno de los matraces. Para una DO 600 0,5-0,7, utilice 1 ml de células. Para un OD 600 mayor que un diámetro exterior de 0,7 utilizar 2,5 ml delas células. NOTA: Las células en fase de crecimiento exponencial típicamente producen más fagos y los títulos más altos que las células en fase estacionaria del crecimiento. </li><li> Agite matraces a 250 rpm a 30 ° C durante aproximadamente 24 horas en un incubador con agitación. </li><li> Después de un período de incubación de 24 hr eliminar matraces de enriquecimiento de la incubadora de agitación y diluir las muestras de enriquecimiento de diez veces en tampón de fago. </li><li> Establecer tubos de cultivo con 0,5 ml de cultivo de bacterias estacionaria tardía fase exponencial / temprano y añadir diversas cantidades del cultivo de enriquecimiento (5 l a 500 l de una dilución 10 -1 en PB) a los tubos de cultivo. NOTA: Es recomendable probar una amplia gama de concentraciones ya que cada muestra de suelo genera diferentes títulos de fagos después del enriquecimiento. </li><li> Añadir CaCl 2 a tubos de cultivo para hacer una concentración final igual a la concentración presente en el matraz de enriquecimiento inicial. Utilice una gama de diferentes concentraciones de CaCl 2 para seleccionar para muchos fagos wi-ésima variable CaCl 2 dependencias. NOTA: La mayoría de los fagos serán aislados dentro de la gama de CaCl2 de 1 a 2,5 mM pero hay fagos aislados más óptima en cualquier lugar 0-50 mM CaCl 2 concentraciones. Debido a esto lo mejor es comprobar una serie de CaCl 2 concentraciones de maximizar el número de fagos únicos Arthrobacter aisladas. </li><li> Mezclar 4,5 ml de agar superior LB en el tubo de cultivo y verter la mezcla sobre una placa de agar LB. Agitar suavemente para distribuir a través de la placa de manera uniforme y permitir agar superior solidificar durante aproximadamente 15 min. NOTA: Top agar se puede preparar en grandes lotes (250 ml) y agar superior adicional se puede almacenar a temperatura ambiente y se reutiliza para experimentos posteriores durante al menos dos semanas. Aunque existen diferentes fórmulas utilizadas para hacer subir Agar, hacemos subir Agar utilizando 7 g / l de agar mezclado con LB. Top agar Colocar en un baño de agua a 60 ° C para conservar el estado líquido durante el curso de un experimento. </li><li> Invertir tél placa e incubar a temperatura deseada (temperatura a 30 ° C) O / N a 48 h. Vary estas condiciones para optimizar los fagos presentes. La mayoría fagos aislados provienen de placas se incubaron a 30 ° C en 1-2,5 mM CaCl concentración 2 después de un período de incubación de 24 horas. </li></ol> 4. Los fagos Purificación <ol><li> Después de una verificación de incubación de 24 h para la formación de placa en placas. Continuar la incubación durante un máximo de 72 horas si no hay placas son evidentes o para determinar si van a aparecer placas adicionales. Si es necesario, las placas, almacenarlas con placas putativos para hasta una semana a 4 ° C antes de continuar. NOTA: No es raro encontrar una variedad de diferentes morfologías de placa en un plato. Encontrar placas después de 3 días de incubación es posible pero poco frecuente. </li><li> Purificar fagos deseados de placas claramente aislados utilizando el método de la placa racha. Toque una placa con un palo de madera estéril. Racha de la placa mediante la ejecución de un palo de madera a través de una llama, allowing el palo para enfriar brevemente, y luego frotando el palo en la placa de agar en el movimiento suave como se muestra en la Figura 1. Repita este usando un palo limpio para cada pasada. </li><li> Como alternativa, utilice el ensayo de valoración placa tradicional para aislar placas de fagos individuales. El ensayo de valoración de placa requiere el uso de más reactivos tales como agar LB y agar superior LB y materiales tales como placas de Petri para el aislamiento de placas de fagos. Hemos tenido un gran éxito con el método de la placa racha pero es técnicamente más fáciles de aislar placas individuales utilizando el ensayo de valoración de la placa. </li><li> Una vez que la placa de agar LB se raya al menos tres veces, marcar el área con la concentración más baja de partículas de fago putativo y aplique suavemente 0,5 ml de Arthrobacter y 4,5 ml de agar superior LB fundido (que contiene la misma concentración de cloruro de calcio como la placa matriz ) y deje que se distribuye uniformemente en toda la placa. </li><li> Invierta la placa e incubar O / N. Repita la placa rachase aísla técnica para al menos tres iteraciones para garantizar una especie de fagos puros. Placas se almacenan a 4 ° C durante hasta una semana, según sea necesario entre rayas y sin pérdida significativa de la viabilidad del fago. </li><li> Una vez que las placas deseados se han cultivado y aislado en la placa racha final, tocar un pozo aislado placa con una punta de la micropipeta y volver a suspender en 100 l PB. En serie diluir esta solución 10 0 a la dilución 10 -8 haciendo pasar 20 l de la muestra en 180 l de PB fresco. </li><li> Añadir 10 l de cada dilución a 0,5 ml de Arthrobacter crecido en un tubo de cultivo de 5 a 10 min a TA y la placa mediante la mezcla de las bacterias infectadas con 4,5 ml de agar superior LB fundido que contiene la misma concentración de CaCl 2 usado para aislar el fago de la muestra original del suelo. Guarde todas las diluciones hechas a 4 ° C hasta el día siguiente. </li><li> Determinar la dilución en serie fago necesaria para hacer un patrón web utilizando el título de la placa tradicionaldecir. El propósito del ensayo es el título de la placa para determinar el título del fago se necesita para obtener una placa patrón de web. Identificar un patrón placa web como la mayoría desprovisto de bacterias, pero que contengan restos de bacterias que aún no han sido lisadas por los fagos. Añadir 5 ml de la cultura Arthrobacter estacionaria exponencial / principios del XX a un matraz de cultivo de 50 ml estéril. <ol><li> Añadir 100 l de la dilución (que dio un patrón de web a la cultura Arthrobacter) en el matraz de cultivo y permitir que los fagos para infectar las células de las bacterias de 5 a 10 min a TA. Mezclar con agar superior LB fundido que contiene la misma concentración de CaCl 2 tal como se utiliza para identificar inicialmente el fago y la placa 5 ml de esta mezcla sobre 10 placas de LB fresco. Dejar solidificar e incubar las placas invertidas a 30 ° C. </li></ol></li><li> Al día siguiente, inundar las placas modelo web con 5 ml de PB y tienda de O / N a 4 ° C o 4 horas a temperatura ambiente. </li><li> Filtrar el lisado de fago a través de un filtro de jeringa de 0,22 micrasy almacenar a 4 ° C. Utilice esta fago final para experimentos adicionales, tales como imágenes de microscopía electrónica de partículas de fago, fago aislamiento de ADN genómico para el análisis con enzimas de restricción y secuenciación de ADN genómico utilizando procedimientos estándar. Para el almacenamiento a largo plazo de la población de fagos, añadir una cantidad igual del lisado del fago a glicerol estéril en frascos pequeños para la acción de archivado a -80 ° C. NOTA: Para los que no experimentado con estas técnicas de fagos, un gran recurso es el sitio web en línea www.phagesdb.org accesible. </li></ol>

<ol>
	<li>Shapir, N., Mongodin, E. F., Sadowsky, M. J., Daugherty, S. C., Nelson, K. E., Wackett, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Catabolic+Pathways:+Genomic+Insights+into+Microbial+s-Triazine+Metabolism.">Evolution of Catabolic Pathways: Genomic Insights into Microbial s-Triazine Metabolism.</a> J Bacteriol. 189 (3), 674-682 (2007).</li><li>Qingyan, L., Ying, L., Xikun, Z., Baoli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Atrazine+Degrading+Arthrobacter+sp.+AD26+and+Use+of+this+Strain+in+Bioremediation+of+Contaminated+Soil.">Isolation and Characterization of Atrazine Degrading Arthrobacter sp. AD26 and Use of this Strain in Bioremediation of Contaminated Soil.</a> J Environ Sci. 20, 1226-1230 (2008).</li><li>Wang, J., Zhu, L., Liu, A., Ma, T., Wang, Q., Xie, H., Wang, J., Jiang, T., Zhao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+an+Arthrobacter+sp.+Strain+HB-5+that+Transforms+Atrazine.">Isolation and characterization of an Arthrobacter sp. Strain HB-5 that Transforms Atrazine.</a> Environ Geochem Hlth. 33, 259-266 (2011).</li><li>Mages, I. S., Frodl, R., Bernard, K. A., Funke, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identities+of+Arthrobacter+spp.+And+Arthrobacter-Like+Bacteria+Encountered+in+Human+Clinical+Specimens.">Identities of Arthrobacter spp. And Arthrobacter-Like Bacteria Encountered in Human Clinical Specimens.</a> J Clin Microbiol. 46 (9), 2980-2986 (2008).</li><li>Brown, D. R., Holt, J. G., Pattee, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Arthrobacter+Bacteriophages+and+Their+Application+to+Phage+Typing+of+Soil+Arthrobacters.">Isolation and Characterization of Arthrobacter Bacteriophages and Their Application to Phage Typing of Soil Arthrobacters.</a> Appl Environ Microbiol. 35 (1), 185-191 (1978).</li><li>Petrovski, S., Seviour, R. J., Tillett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Gordonia+and+Nocardia+Stabilized+Foam+Formation+by+Using+Bacteriophage+GTE7.">Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7.</a> Appl Environ Microbiol. 77 (21), 7864-7867 (2011).</li><li>Le Marrec, C., Moreau, S., Loury, S., Blanco, C., Trautwetter, a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Characterization+of+Site-specific+Integration+Functions+of+phi+AAU2+infecting+&#8220;Arthrobacter+aureus&#8221;+C70.">Genetic Characterization of Site-specific Integration Functions of phi AAU2 infecting &#8220;Arthrobacter aureus&#8221; C70.</a> J Bacteriology. 178 (7), 1996-2004 (1996).</li><li>Casida, L. E., Liu, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthrobacter+globiformis+and+Its+Bacteriophage+in+Soil.">Arthrobacter globiformis and Its Bacteriophage in Soil.</a> J Appl Microbiol. 28 (6), 951-959 (1974).</li><li>Einck, K. H., Pattee, P. A., Holt, J. G., Hagedorn, C., Miller, J. A., Berryhill, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+a+Bacteriophage+of+Arthrobacter+globiformis.">Isolation and Characterization of a Bacteriophage of Arthrobacter globiformis.</a> J Virol. 12 (5), 1031-1033 (1973).</li><li>Twest, R., Kropinski, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+Enrichment+from+Soil+and+Water.">Bacteriophage Enrichment from Soil and Water.</a> Methods Mol Bio. 501, 15-21 (2009).</li><li>Fullner, K. J., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacteriophage+L5+Infection+of+Mycobacterium+bovis+BCG:+Implications+for+Phage+Genetics+in+the+Slow-growing+Mycobacteria.">Mycobacteriophage L5 Infection of Mycobacterium bovis BCG: Implications for Phage Genetics in the Slow-growing Mycobacteria.</a> Mol Microbiol. 26 (4), 755-766 (1997).</li><li>Robb, S. M., Woods, D. R., Robb, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Growth+Characteristics+on+Stationary+Phase+Achromobacter+cells.">Phage Growth Characteristics on Stationary Phase Achromobacter cells.</a> J Gen Virol. 41 (2), 265-272 (1978).</li><li>Bolger-Munro, M., Cheung, K., Fang, A., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T4+Bacteriophage+Average+Burst+Size+Varies+with+Escherichia+coli+B23+Cell+Culture+Age.">T4 Bacteriophage Average Burst Size Varies with Escherichia coli B23 Cell Culture Age.</a> Journal of Experimental Microbiology and Immunology. 17, 115-119 (2013).</li></ol>

Autores declaran no tener intereses financieros en competencia.

A pesar de muchos intentos anteriores para aislar fagos capaces de infectar ordenadores Arthrobacter, tuvimos poco éxito utilizando procedimientos de enriquecimiento estándar. El método generalizado de enriquecimiento bacteriana desarrollado y adaptado por van Twest y Kropinski 10 para enriquecer fagos a partir de muestras ambientales sigue siendo la base para la mayoría de procedimientos de enriquecimiento. La evidencia de estudios previos sugieren que los métodos de siembra directa han produci...

La ubicuidad de las especies Arthrobacter en entornos de suelo ofrece un gran número y diversidad de fagos capaces de ser aislado a partir de esta especie de bacteria huésped. Miembros bacterianas de la familia Acintobacteriaceae son más notables por sus vías catabólicas de degradar compuestos recalcitrantes como la atrazina y varios otros pesticidas y herbicidas 1,2,3. Aunque la mayoría de investigaciones se ha hecho uso de cepas ambientales de Arthrobacter, aislados clínicos de este género se encuentran en la sangre, la orina, los ojos y muchas otras fuentes humanas que muestran toda la heterogeneidad filogenético 4. Si bien no es un lugar amplio cuerpo de investigación sobre bacterias Arthrobacter, sólo unos pocos estudios informan sobre los fagos capaces de infectar a los miembros de este diverso género. Curiosamente, sin embargo, el trabajo realizado previamente en Arthrobacter fagos toques sobre varios temas clave diferenciadas, como la tipificación de suelo &lt;em&gt; especies Arthrobacter 5, usos industriales con el fin de reducir la espuma perjudicial en las plantas de tratamiento de lodos activados 6, y el trabajo destacando recombinación específica de sitio y los genes de la integrasa 7. Varios protocolos técnica de enriquecimiento se han empleado para generar pura fago aísla en especies de Arthrobacter. Los primeros procedimientos incluyen incubaciones de suelo con agentes tóxicos adicionales como sales de nicotina durante períodos de más de un año 8 dando lugar a los fagos capaces de infectar sólo A. globiformis. Los estudios realizados utilizando suelo percola con productos orgánicos lábiles parecían producir fagos detectables a través de técnicas de ensayo de placa, omitiendo largos períodos de incubación 8. Curiosamente, sin embargo, se utilizó una técnica parecida a la siembra directa en el pasado dando lugar a varios fagos mientras que todavía tiene una baja tasa de éxito particular por los investigadores 5, citando estudios anteriores con bajas tasas de éxito <sarriba&gt; 8. En general, las técnicas de aislamiento utilizados en el pasado eran destaca por tener poca eficacia en la práctica a pesar de género Arthrobacter que representan el suelo aeróbico más común aislar en la naturaleza 4,9,, Van Twest y Kropinski 10 presentes métodos de enriquecimiento para el aislamiento de fagos a partir de agua y el suelo adaptado de técnicas anteriores utilizadas para enriquecer aislamientos bacterianos ambientales, pero estas técnicas de enriquecimiento resultó ineficaz en el aislamiento de los fagos Arthrobacter. El propósito del método descrito aquí es mostrar &quot;prueba de concepto&quot; que los métodos de enriquecimiento primeros pueden ser adaptados para aislar de manera consistente y eficaz fagos Arthrobacter, la superación de dificultades técnicas anteriores asociadas con el aislamiento de fagos de este género bacteriano.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>LB Broth powder</td>
			<td>Fisher</td>
			<td>BP9722-2</td>
			<td>It&#39;s best to order these in bulk.</td>
		</tr>
		<tr>
			<td>Granulated Agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>It&#39;s best to order these in bulk.</td>
		</tr>
		<tr>
			<td>0.22 um syringe filters</td>
			<td>Fisher</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>.22 um buchner filters</td>
			<td>Fisher</td>
			<td align="right">430320</td>
			<td>More than 50 mL of liquid can be obtained by carefully swapping the receiving tube.</td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher</td>
			<td align="right">76002844</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher</td>
			<td align="right">76002845</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-676-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman qualitative filter paper</td>
			<td>Fisher</td>
			<td>1001-824</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized enrichment technique for the isolation of arthrobacter bacteriophage species from soil sample isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

Para demostrar la reproducibilidad de la técnica de enriquecimiento mejorado para fagos Arthrobacter, se utilizaron 30 muestras de suelo diferentes en diferentes momentos y lugares durante la primavera y el verano de 2014. De estos 30 muestras de suelo fagos Arthrobacter únicas se obtuvieron a partir de 22 muestras de suelo recogidas mediante este enriquecimiento procedimiento. El procedimiento de enriquecimiento estándar produjo fagos únicos de 3 de las mismas muestras de suelo. Las muestras de en...

Watch this Scientific Journal Video about An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates at JoVE.com

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Una técnica de enriquecimiento optimizado para el aislamiento de<em&gt; Arthrobacter</em&gt; Bacteriófago Especies de Aísla Muestra de Suelo

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

an-optimized-enrichment-technique-for-isolation-arthrobacter

Research

JoVE Journal

Environment

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

19.3K vistas.  University of the Sciences. We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Video: An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Diseño y aplicación de un continuo<sup&gt; 13</sup&gt; C y<sup&gt; 15</sup&gt; N Cámara Etiquetado de Uniforme o Diferencial, metabólico y estructural, Planta de isótopos de etiquetado

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Investigación

Medio ambiente

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

Diseño y Construcción de un Centro de Investigación La escorrentía urbana

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Muestreo de suelos y aislamiento de nematodos entomopatógenos (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

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

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

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

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

A CO<sub&gt; 2</sub&gt; Fondo gradiente de concentración para las pruebas de CO<sub&gt; 2</sub&gt; Enriquecimiento y suelo Efectos sobre la Función de los Ecosistemas del Prado

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

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Aislamiento y análisis de comunidades microbianas en el suelo, rizosfera y raíces en los experimentos de pastos perennes

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Un protocolo de Rhizobox optimizada para visualizar el crecimiento de las raíces y la capacidad de respuesta a nutrientes localizadas

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Uso de Caenorhabditis elegans para el estudio de los efectos trans y multigeneracionales de los tóxicos

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

Diseño de microparplots y preparación de muestras de plantas y suelos para 15 análisisde nitrógeno

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Electrostatic Method to Remove Particulate Organic Matter from Soil

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Removing recently deposited and incompletely decomposed plant material from soil samples reduces the influence of temporary seasonal inputs on soil organic carbon measurements. Attraction to an electrostatically charged surface can be used to quickly remove a substantial amount of particulate organic matter.

Método electrostático para eliminar la materia orgánica particulada del suelo

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of ...

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Estimación del contenido de celulosa cristalina de biomasa vegetal utilizando el método Updegraff

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...