O financiamento para o desenvolvimento deste protocolo foi fornecido pela Pennsylvania Consortium Sudeste do Ensino Superior e do Departamento de Ciência Cabrini College. Financiamento e apoio adicional veio da Universidade de Arcadia e Universidade Imaculada. Agradecemos especialmente ao Dr. Karen Snetselaar em St. Joseph Universidade para gentilmente tomando as imagens microscópicas de elétrons dos nossos fagos isoladas. Apoio adicional foi fornecido pelos Hunters Instituto Médico Howard Hughes Educação Ciência Alliance fagos Avançando Genomics e Evolutiva Ciência programa (SEA-fagos).

1. Preparação de células Arthobacter para Fago Isolation <ul><li> Cultura de Arthrobacter sp. KY3901 colónias riscadas em um Bertaini Luria (LB) agar placa incubada a 30 ° C durante 2-3 dias. Escolha uma colónia e usar uma ansa estéril para adicioná-lo a 250 ml de caldo LB num frasco de cultura com chicanas e incubar numa incubadora com agitação a 225 rpm a 30 ° C. </li><li> Reserve em torno de 24 horas de crescimento para obter células estacionárias exponenciais / início de final de fase para experimentos de infecção fago. Monitorar o estado de crescimento bacteriano de perto para evitar que as células de entrar no meio para o final estado estacionário de crescimento. NOTA: O crescimento celular deve consistir de uma densidade óptica OD 600 entre 0,5-.0.7 para os procedimentos de isolamento e purificação de fago / passos descritos abaixo. </li></ul> 2. Recolha de Fago de amostras de solo <ol><li> Reúna 200-400 g de solo do local desejado. Nota: Uma vez que as bactérias no ArthrobaCTER género são bactérias comuns do solo e que os fagos que eles infectam pode ser encontrado em e são difundidas na maior parte dos tipos de solo. </li><li> Adicionar, pelo menos, 400 ml de tampão de fagos (PB) [NaCl 68 mM, 10 mM de MgSO 4, 10 mM de Tris-Cl (pH 7,5)] para o solo e misturar num frasco grande o suficiente para que, pelo menos, 200 ml de PB é no sobrenadante e é capaz de ser extraído. </li><li> Misture por agitação ou rodando suavemente até que o solo está suspenso e permitir que os sedimentos do solo para resolver, tipicamente 30 min. </li><li> Passe o &quot;extrato de solo&quot; por papel de filtro por filtração por gravidade para remover os restos de sedimentos do solo. </li><li> Adicionar LB pó e misturar para fazer uma solução a 2% (4 g LB pó em 200 ml de extracto de solo). </li><li> Filtrar a solução através de um filtro de 0,22 um por filtração em vácuo para se obter um filtrado estéril. Se possível, continue no passo 2.7 imediatamente. Caso seja necessário, armazenar as amostras filtradas a 4 ° C durante até uma semana antes de continuar, no entanto, o número de partículas fágicas viáveisserá reduzido. </li><li> Múltipla alíquota de 50 ml da mistura de extrato de LB / solo esterilizado por filtração em individuais de 250 ml esterilizadas tremendo frascos de cultura perplexos usando técnicas assépticas. Adicionar estéril M de CaCl2 solução 2,0 para as concentrações finais pretendidas (0-50 mM) uma vez que diferentes fagos de Arthrobacter crescem optimamente sob diferentes condições de CaCl 2. NOTA: Descobrimos que a maioria dos fagos Arthrobacter identificados por nós estão dentro da gama de 1 mM-2,5 mM de CaCl2. No entanto, vários dos fagos isolados cresceram nos exclusivamente a 0 mM CaCl2 .ou a concentrações muito elevadas de CaCl 2. Prossiga imediatamente para a etapa 3.1. </li></ol> 3. Fago Isolation <ol><li> Adicionar 1 a 2,5 ml de exponencial tardia / estacionária antecipada bactérias em fase de cultura para cada um dos frascos. Para uma DO600 0,5-0,7, utilizar 1 ml de células. Para uma DO600 superior a uma DO de 0,7 2,5 ml de usarcélulas. NOTA: As células em fase de crescimento exponencial normalmente produzem mais fagos e títulos mais elevados do que as células em crescimento fase estacionária. </li><li> Frascos de agitação a 250 rpm a 30 ° C durante aproximadamente 24 horas numa incubadora com agitação. </li><li> Após um período de incubação de 24 h remover frascos de enriquecimento da incubadora com agitação e diluir as amostras de enriquecimento de dez vezes em tampão de fago. </li><li> Configure tubos de cultura com 0,5 ml de tarde exponencial / início de bactérias em fase estacionária cultura e adicionar várias quantidades de a cultura de enriquecimento (5 mL a 500 mL de uma diluição 10 -1 em PB) para os tubos de cultura. NOTA: É aconselhável testar uma ampla gama de concentrações uma vez que cada amostra de solo gera diferentes títulos de fagos após enriquecimento. </li><li> Adicionar CaCl2 para tubos de cultura para obter uma concentração final igual à concentração presente no frasco de enriquecimento inicial. Use uma variedade de diferentes concentrações de CaCl 2 para selecionar para muitos fagos wom variando CaCl 2 dependências. NOTA: A maioria dos fagos será isolado dentro do CaCl2 gama de 1-2,5 mM mas há fagos isolados mais de forma ideal em qualquer lugar 0-50 mM de CaCl2 concentrações. Por isso, o melhor é verificar uma série de CaCl 2 concentrações de maximizar o número de Arthrobacter fagos únicas isoladas. </li><li> Misturar 4,5 ml de LB agar superior no tubo de cultura e despeje a mistura em uma placa de ágar LB. Agitar suavemente o frasco para distribuir uniformemente ao longo da placa e para permitir agar de topo solidificar durante aproximadamente 15 minutos. NOTA: Topo de agar podem ser preparadas em lotes maiores (250 ml) e agar de topo extra pode ser armazenado à temperatura ambiente e reutilizado para as experiências subsequentes, durante pelo menos duas semanas. Embora existam diferentes fórmulas utilizadas para fazer agar top, fazemos top agar usando 7g / L de agar misturado com LB. Ponto de agar de topo num banho de água a 60 ° C para manter o estado líquido durante o decurso de uma experiência. </li><li> Inverter tele placa e incubar a temperatura desejada (temperatura de 30 ° C) O / N a 48 h. Alterar estas condições para otimizar os fagos presentes. Mais fagos isolados provenientes de placas incubadas a 30 ° C a 1-2,5 mM de CaCl 2 a concentração após um período de incubação de 24 horas. </li></ol> 4. Fago Purificação <ol><li> Após uma incubação de 24 horas de verificação para a formação de placas em placas. Continue incubação por até 72 horas, se não há placas são evidentes ou para determinar se as placas adicionais aparecerão. Se necessário, as placas de loja com placas putativas de até uma semana a 4 ° C antes de prosseguir. NOTA: Não é raro encontrar uma variedade de diferentes morfologias de placa em um prato. Encontrar placas após 3 dias de incubação, é possível, mas raro. </li><li> Purificar fagos desejados de placas claramente isolado utilizando o método raia placa. Toque em uma placa com uma vara de madeira estéril. Streak a placa executando uma vara de madeira através de uma chama, allowing o pau para esfriar brevemente, e, em seguida, esfregando o pau na placa de agar no movimento suave, como mostrado na Figura 1. Repita esta usando uma vara limpa para cada passagem. </li><li> Em alternativa, usar o ensaio de título de placa tradicional para isolar placas de fagos individuais. O ensaio de titulação da placa requer a utilização de mais reagentes, tais como agar LB e agar de topo LB e materiais, tais como placas de petri para o isolamento do fago de placa. Temos sido muito bem sucedida usando o método raia placa, mas é tecnicamente mais fácil isolar placas individuais utilizando o ensaio título placa. </li><li> Uma vez que a placa de agar LB é riscada pelo menos três vezes, marcar a área com a menor concentração de partículas fágicas putativas e suavemente aplicar 0,5 ml de Arthrobacter e 4,5 ml de agar de topo LB fundida (contendo a mesma concentração de cloreto de cálcio, tal como a placa-mãe ) e deixe-a espalhar uniformemente ao longo da placa. </li><li> Inverter e incubar placa O / N. Repita a placa raiatécnica para, pelo menos, três iterações para garantir uma espécie de fagos puros é isolado. Armazenar as placas a 4 ° C durante até uma semana, conforme necessário entre estrias, sem perda significativa da viabilidade do fago. </li><li> Uma vez que as placas desejadas tenham sido cultivadas e isolado na placa raia final, toque em uma placa bem isolado com uma ponta de micropipeta e suspender novamente em 100 PB ul. Serialmente dilua esta solução a 10 0 para a diluição de 10 -8, passando 20 ul da amostra em 180 ul de PB fresco. </li><li> Adicionam-se 10 ul de cada diluição de 0,5 ml de Arthrobacter crescido num tubo de cultura para 5 a 10 minutos à temperatura ambiente e placa misturando as bactérias infectadas com 4,5 ml de agar de topo LB fundida contendo a mesma concentração de CaCl2 utilizada para isolar o fago a partir da amostra original do solo. Salve todas as diluições feitas a 4 ° C até o dia seguinte. </li><li> Determinar a diluição em série fago necessária para fazer um teste padrão da web usando o título de placa tradicional comodizer. O objectivo do ensaio de titulação de placa é determinar o título de fago necessária para obter uma placa padrão de teia. Identificar uma placa padrão de teia como na maior parte isenta de bactérias, mas que contém vestígios de bactérias que ainda não tenham sido submetidas a lise por fagos. Adicionar 5 ml de tarde exponencial / início de cultura Arthrobacter estacionário a um frasco estéril cultura de 50 ml. <ol><li> Adicionar 100 ul da diluição (que deu um padrão de teia para a cultura de Arthrobacter) no frasco de cultura e permitir que os fagos para infectar as células de bactérias por 5 a 10 min a RT. Misturar com fundido de topo LB agar contendo a mesma concentração de CaCl 2 como inicialmente utilizados para identificar o fago e placa 5 ml desta mistura sobre 10 placas de LB fresco. Deixa-se solidificar e incubar as placas invertidas a 30 ° C. </li></ol></li><li> No dia seguinte, inundar as placas em forma de rede com 5 ml de PB e armazenamento de O / N a 4 ° C ou 4 horas à temperatura ambiente. </li><li> Filtra-se o lisado de fago através de um filtro de seringa de 0,22? Me armazenar a 4 ° C. Use este inventário final fago para experiências adicionais, tais como imagens de microscopia electrónica de partículas de fago, o fago de isolamento de ADN genómico para análise de enzimas de restrição e sequenciação de DNA genómico usando procedimentos padrão. Para armazenamento a longo prazo do stock de fagos, adicionar uma quantidade igual de lisato de fago de glicerol estéril em frascos pequenos para arquivamento estoque a -80 ° C. NOTA: Para aqueles que não experimentaram com estas técnicas de fagos, um grande recurso é o site www.phagesdb.org acessível online. </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>

Os autores declaram não há interesses financeiros concorrentes.

Apesar de muitas tentativas anteriores para isolar fagos capazes de infectar hospedeiros Arthrobacter, tivemos pouco sucesso utilizando procedimentos de enriquecimento padrão. O método de enriquecimento bacteriana generalizada desenvolvido e adaptado por van Twest e Kropinski 10 para enriquecer os fagos a partir de amostras ambientais continuam a base para a maioria dos procedimentos de enriquecimento. Evidências de estudos anteriores sugerem que métodos de plaqueamento direto produziram placas d...

A onipresença de espécies Arthrobacter em ambientes de solo oferece um vasto número e diversidade de fagos capazes de serem isolados nessa espécie de bactérias hospedeiras. Bacterianas membros da família Acintobacteriaceae são o mais notável para suas vias catabólicas de degradar compostos recalcitrantes como atrazina e vários outros pesticidas e herbicidas 1,2,3. Embora a maioria das pesquisas tem sido feito utilizando estirpes ambientais de Arthrobacter, isolados clínicos deste gênero é encontrado no sangue, urina, olhos, e muitas outras fontes humanas todos exibindo heterogeneidade filogenética 4. Enquanto não há um corpo bastante extensa pesquisa sobre bactérias Arthrobacter, apenas alguns estudos relatam sobre os fagos capazes de infectar membros deste gênero diverso. Curiosamente, porém, o trabalho feito anteriormente em Arthrobacter fagos toques sobre diversos temas distintos-chave, tais como a digitação de solo &lt;em&gt; espécies Arthrobacter 5, usos industriais, com o objetivo de reduzir espuma deletéria em estações de tratamento de lamas activadas 6, e destacando o trabalho de recombinação sítio específico e genes integrase 7. Vários protocolos técnica de enriquecimento têm sido empregadas para gerar pura fago isolados em espécies Arthrobacter. Procedimentos iniciais incluem incubações de solo com agentes tóxicos adicionais como sais de nicotina por períodos de mais de um ano 8 dando origem a fagos capazes de infectar apenas A. globiformis. Estudos feitos usando solo percolados com orgânicos instáveis ​​apareceu para produzir fagos detectáveis ​​através de técnicas de ensaio de placa, omitindo períodos de incubação longos 8. Curiosamente, porém, uma técnica parecida com semeadura direta foi utilizado no passado dando origem a vários fagos enquanto continua a ter uma baixa taxa de sucesso nomeadamente pelos investigadores 5, citando estudos anteriores com baixas taxas de sucesso <saté&gt; 8. No geral, as técnicas de isolamento usados ​​no passado eram notável por ter pouca eficácia na prática, apesar das género Arthrobacter representativas do solo aeróbico mais comum isolar na natureza 4,9,, Van Twest e Kropinski 10 atuais métodos de enriquecimento para isolamento de fagos a partir da água e do solo adaptado a partir de técnicas anteriores utilizadas para enriquecer isolados bacterianos ambientais, mas estas técnicas de enriquecimento se mostrou ineficiente para isolar fagos Arthrobacter. O objectivo do método descrito aqui é mostrar &quot;prova de conceito&quot; que os métodos de enriquecimento precoces podem ser adaptados de forma consistente e eficaz isolar fagos Arthrobacter, ultrapassar os desafios técnicos anteriores, associados com o isolamento de fagos a partir deste 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 demonstrar reprodutibilidade da melhoria técnica de enriquecimento de fagos Arthrobacter, 30 amostras de solo diferentes foram usados ​​em diferentes momentos e locais durante a primavera eo verão de 2014. Destes 30 amostras de solo fagos Arthrobacter originais foram obtidos a partir de 22 de amostras de solo coletadas usando este enriquecimento procedimento. O processo de enriquecimento de fagos padrão originou únicas de 3 das mesmas amostras de solo. As amostras de enriquecimento podem t...

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.

Uma técnica de enriquecimento Aperfeiçoado para o isolamento de<em&gt; Arthrobacter</em&gt; Bacteriophage Espécie de amostra de solo Isolados

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

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

19.4K visualizações.  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.

Vídeo: 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.

Projeto e operação de uma contínua<sup&gt; 13</sup&gt; C e<sup&gt; 15</sup&gt; N Labeling Câmara de uniforme ou Diferencial, metabólica e estrutural, Fábrica de Marcação por Isótopo

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

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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.

Projeto e Construção de um Mecanismo de Pesquisa escoamento urbano

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.

Amostragem do solo e isolamento de nematóides entomopatogênicos (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; Facility Concentração Gradiente para Teste CO<sub&gt; 2</sub&gt; Enriquecimento do solo e efeitos sobre a função da pastagem de Ecossistemas

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

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Protocolo para Microplásticos Amostragem na superfície do mar e análise de amostras

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

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.

Isolamento e análise de comunidades microbianas na rizosfera, solo e raízes em experiências de grama perene

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.

Um protocolo otimizado de Rhizobox para visualizar o crescimento da raiz e receptividade aos 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 ...

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.

Projeto de microtrat e preparação de amostras de plantas e solo para 15análises de nitrogênio

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 eletrostático para remover matéria orgânica particulada do solo

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.

Estimativa do Teor de Celulose Cristalina de Biomassa Vegetal utilizando o 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 ...