资助这项协议的发展是由东南宾夕法尼亚财团高等教育和卡布里尼学院科学系提供。额外的资金和支持来自阿卡迪亚大学和Immaculata大学。我们特别感谢莫文蔚Snetselaar博士在圣约瑟夫大学好心参加我们的孤立的噬菌体电子显微图像。额外的支持是由霍华德·休斯医学研究所科学教育联盟噬菌体猎人推进基因组学和进化科学（SEA-噬菌体）计划提供的。

1.制备Arthobacter细胞对噬菌体分离<ul><li>培养节杆菌。KY3901菌落划线上的Luria Bertaini（LB）琼脂平板上培养，在30℃下进行2-3天。选择一个菌落并使用无菌环到它在225转添加250ml的LB肉汤中在带挡板的培养瓶中，并培育在振荡培养箱中于30℃。 </li><li>允许约24小时的生长，得到迟指数/早期静止期细胞噬菌体感染实验。监测细菌生长的状态密切以防止细胞进入到中间后期静止生长状态。 注意:细胞生长应0.5-.0.7对于下面描述的噬菌体的分离和纯化步骤/步骤之间包括一个光密度OD 600。 </li></ul>从土壤样品2.收集噬菌体<ol><li>收集200-400克土壤所需的位置。注:由于细菌在ArthrobaCTER属是常见的土壤细菌，他们和他们感染的噬菌体中可以找到并普遍存在于大多数类型的土壤。 </li><li>添加到土壤至少400毫升噬菌体缓冲液（PB）[68 mM氯化钠，的10mM 硫酸镁 ，10毫摩尔Tris-CL（pH为7.5）]，并混合在一个足够大的烧瓶，使至少200毫升的PB是在上清液中，并能够被提取。 </li><li>轻轻搅拌或涡旋直至土壤悬浮混合，并且允许在土壤沉积物沉降，一般为30分钟。 </li><li>通过重力过滤通过"土壤提取物"，通过过滤纸，除去泥沙土屑。 </li><li>添加LB粉末并混合，使2％的溶液（4克的LB粉末在200毫升土壤提取物）。 </li><li>通过真空过滤通过0.22微米过滤的溶液，得到的无菌滤液。如果可能的话，请继续，立即步骤2.7。可行的噬菌体颗粒，如果需要的话，店内过滤样品在4℃长达一个星期前进行，然而，数会降低。 </li><li>多个等分试样（每份50ml）的过滤灭菌的LB /土壤提取物的混合物的成单个250毫升灭菌摇使用无菌技术挡板的培养瓶中。因为不同的节杆菌噬菌体不同的CaCl 2的条件下生长最佳添加无菌的2.0M的CaCl 2溶液至最终所需的浓度（0-50毫米）。 注意:我们发现，多数确定我们的节杆菌噬菌体的是为1mM-2.5mM的氯化钙2的范围内。然而，一些孤立的噬菌体的增长我们专门在0毫米氯化钙2。或者在非常高的氯化钙浓度。立即进行步骤3.1。 </li></ol> 3.噬菌体隔离<ol><li>加12.5毫升晚指数/早期稳定期细菌培养物到每个烧瓶。对于OD 600从0.5-0.7，使用1毫升细胞。对的OD 600高于0.7的OD使用将2.5ml细胞。 注:指数生长期细胞通常产生更多的噬菌体和更高的滴度比细胞静止期增长。 </li><li>在振荡培养箱摇瓶中以250rpm在30℃下大约24小时。 </li><li>经过24小时的潜伏期从摇床删除富集瓶和稀释的浓缩样品十倍噬菌体缓冲。 </li><li>建立培养管0.5下旬指数/早期固定相细菌培养毫升，加入不同量的富集培养（5微升至500微升10 -1稀释PB），以文化管。 注意:最好是测试各种浓度的，因为每个土样富集后会产​​生不同的噬菌体滴度。 </li><li>加入氯化钙至培养管，使最终浓度等于存在于原始富集烧瓶浓度。使用一定范围的不同的CaCl 2浓度的选择用于许多噬菌体瓦特第i个不同的氯化钙的依赖。 注:大多数噬菌体将在氯化钙范围1-2.5毫米之内孤立的，而是有噬菌体中分离最优化的任何地方从0-50毫米氯化钙2浓度。因为这一点，最好是检查范围的CaCl 2浓度最大化的独特分离节杆菌的噬菌体的数目。 </li><li>混合4.5毫升的LB顶层琼脂中培养管，将混合物倾到的LB琼脂板上。轻轻摇动整个板均匀地分布，并允许上层琼脂凝固约15分钟。 注:顶部琼脂可以在较大的批量（250毫升）和附加的顶层琼脂制备可以存储在RT并再用于随后的实验中，至少有两个星期。虽然有用来使顶层琼脂不同的公式，我们使上层琼脂使用7克/ L琼脂的LB中与混合在60℃的水浴代替顶层琼脂的实验过程中保持液态。 </li><li>反转吨他板和孵化在所需温度（温度为30°C）O / N为48小时。改变这些条件进行优化为存在的噬菌体。最孤立的噬菌体来自一个24小时的潜伏期后，培养在30℃1-2.5毫米氯化钙2浓度板。 </li></ol> 4.净化噬菌体<ol><li>经过24小时培养检查平板上斑块的形成。继续温育长达72小时，如果没有斑块是显而易见的，或以确定是否附加的噬菌斑出现。如果需要的话，店里板的假定斑块长达一个星期在4℃之前，诉讼的。 注:这是不难发现各种不同的斑块形态的一个板块。孵化后3天发现斑块是可能的，但很少见。 </li><li>净化使用条纹斑点法明确孤立斑块预期的噬菌体。触摸牌匾用无菌木棍。连胜斑块通过火焰运行木棍，allowin克棒简单冷却，然后擦在琼脂平板在柔和的运动的棒， 如图1。使用清洁棒对各遍重复此。 </li><li>另外，使用传统的牌匾滴度检测，以分离单个噬菌斑。噬菌斑测定法滴度确实需要使用多种试剂，如LB琼脂和LB顶层琼脂和材料如培养皿噬菌体斑块隔离。我们已经非常成功使用条纹菌斑的方法，但它在技术上是容易分离使用噬菌斑的滴度测定个体斑块。 </li><li>一旦在LB琼脂平板上划线接种是在至少三次，马克与推定的噬菌体颗粒的最低浓度的区域，并轻轻申请0.5毫升节杆菌和4.5毫升的LB熔融顶层琼脂（含有氯化钙的相同浓度的父板），并允许它均匀地分布在整个板。 </li><li>反转，并培育板O / N。重复连胜牌匾技术至少三次迭代，以确保一个纯噬菌体物种是分离的。商店板在4℃下进行长达一周需要条纹之间不显著损失噬菌体生存能力。 </li><li>一旦需要的斑块已经发展壮大，孤立的最终连胜板，触摸一体以及孤立的牌匾用枪头，重新悬浮于100微升PB。串联通过使20微升样品放入180微升新鲜稀释的PB这10 0的溶液出到10 -8稀释。 </li><li>在培养管中进行5加10微升每种稀释液以0.5 节杆菌生长毫升至室温10分钟，板通过混合感染的细菌具有4.5的含氯化钙的相同浓度用于分离噬菌体熔融的LB上层琼脂毫升从原来的土样。保存发在4°C，直到第二天的所有稀释。 </li><li>确定需要做出采用传统的牌匾滴度为web模式的噬菌体连续稀释说了。菌斑的滴度测定的目的是确定需要获得一个web图案板的噬菌体效价。识别网页模板的大多是缺乏细菌，但包含尚未通过裂解噬菌体细菌的残余。加入5 ml后期指数/早期固定节杆菌培养到无菌的50ml培养瓶中。 <ol><li>添加100微升的稀释液（即给一个web图案的节杆菌培养物）中培养瓶，并允许噬菌体感染的细菌细胞进行5至10分钟，在RT。混合使用作为用于最初鉴定噬菌体和板5毫升该混合物到10新鲜LB平板含有氯化钙的相同浓度的熔融的LB琼脂顶部。允许固化并孵育倒置平板，于30℃。 </li></ol></li><li>第二天，洪水幅图案板用5ml PB和商店O / N的，在4℃或4小时，在室温。 </li><li>通过0.22微米的注射器过滤器过滤该噬菌体裂解物并存储在4℃。使用此最终噬菌体股票为另外的实验，如噬菌体颗粒的电子显微镜图像，噬菌体基因组DNA分离为使用标准程序DNA的限制酶分析和基因组测序。对于长期储存的噬菌体股票，噬菌体裂解液等量的小药瓶股票归档于-80℃加入到无菌甘油。 注:对于那些没有经历过这些噬菌体技术，一个巨大的资源是在线访问www.phagesdb.org网站。 </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>

作者宣称没有竞争的财务权益。

尽管许多以前曾试图孤立能够感染主机节杆菌噬菌体的，我们有一点成功使用标准的浓缩过程。细菌富集开发并适于通过面包车Twest和Kropinski 10从环境样品富集的噬菌体的广义方法仍是依据对大多数富集过程。从以往的研究证据表明，直接电镀的方法都对节杆菌菌株的检测斑块尽管有隔离5的成功率极低。与直接镀法，噬菌体是从土壤样品中提取噬菌体缓冲和过滤，以...

节杆菌种在土壤环境中无处不在提供能够从这种寄主细菌被噬菌体分离出一个庞大的数量和多样性。在Acintobacteriaceae家族的细菌成员是最显着的降低顽固的化合物，如莠去津和其他各种杀虫剂和除草剂1,2,3他们的代谢途径。虽然大多数研究已经完成使用的节杆菌菌株环境，本属临床分离株血，尿，眼睛，和其他许多人来源的所有显示系统发育不均匀4找到。 虽然有相当广泛的对节杆菌的细菌研究机构，只有少数研究能够感染这种多样化属成员的噬菌体报告。但有趣的是，在此前节杆菌所做的工作噬菌体几个关键的不同的主题接触，如土壤类型&lt;EM&gt;节杆菌物种5，工业用途以减少有害的泡沫在活性污泥处理厂6和工作突出位点特异性重组和整合基因7的目的。 各种富集技术协议已被利用来产生纯的噬菌体分离物在节杆菌物种。早期的程序包括土壤添加了有毒物质尼古丁一样的盐每年超过8引起的时期孵化到能够只感染A的噬菌体球形节杆菌 。研究使用的土壤渗出完成与不稳定的有机物经出现斑块检测技术，生产检测噬菌体，省略了漫长的潜伏期8。但有趣的是，一个类似的技术直接电镀用在过去的几个噬菌体引起同时还具有一个显着低成功率的调查5，理由是低成功率过去的研究<s达&gt; 8。 总体而言，在过去所用的分离技术是值得注意的具有尽管节杆菌属的代表最常见的有氧土壤在实践中很少功效隔离性质的4,9，，范Twest和Kropinski 10本富集方法用于从水和土壤中分离的噬菌体改编自用于充实环境的细菌菌株，但这些富集技术的早期技术被证明效率低下的隔离节杆菌噬菌体。这里所描述的方法的目的是要表明"概念验证"，早期的富集方法可以适于一致地和有效地隔离节杆菌噬菌体，克服了与来自该细菌属中分离的噬菌体相关的先前的技 ​​术挑战。

<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.

为了证明对节杆菌噬菌体改进富集技术的再现，2014年这些独特的节杆菌噬菌体从采集的土壤样品22使用这种浓缩获得30土壤样品的春季和夏季期间，30个不同的土壤样品用于在不同的时间和地点流程。标准的富集过程产生独特的噬菌体3相同的土壤样品。所述富集的样品可具有非常高的噬菌体滴度需要初始稀释以分离斑块或相对低的噬菌体滴度要求富集的节杆菌的较大体积成功?...

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.

一种优化富集技术的隔离<em&gt;节杆菌</em&gt;噬菌体种从土壤样品隔离

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

19.4K 次观看.  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.

视频: 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.

设计一个连续和操作<sup&gt; 13</sup&gt; C和<sup&gt; 15</sup&gt;Ñ贴标商会统一或差分，代谢和结构，植物同位素标记

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

科研

环境科学

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.

设计一个城市径流研究设施的建设

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.

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

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.

一个外线<sub&gt; 2</sub&gt;浓度梯度用于测试CO<sub&gt; 2</sub&gt;浓度与土壤对草地生态系统功能的影响

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

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

通过他们的足迹识别个体:合模猎豹

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

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.

协议塑料微粒采样的海面和样品分析

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.

多年生草试验中土壤、根际和根系微生物群落的分离与分析

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.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

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.

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

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.

微图设计及植物和土壤样品制备，进行15次氮气分析

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. ...