本议定书适用于木质和非木质基材，概述，以及烤箱或风干的材料。通读协议第一，但是，在设置。有几点提出，可能适用于您的研究，需要规划这些点（下划线）。另外，还要注意有两个琼脂块，偶尔使用的方法，一个英国标准838和其他国际木材保护研究组（IRG - WP）英勇勋章（1978年）提交的文件。类似方法838，与修改，主要的缩影设计和控制的琼脂培养基，但再次，这两种方法往往是避免由于历史中的水分控制木块问题，造成缺氧性和多变性。尼古拉（1973年）中可以找到一个良好的审查，这些测试方法包括琼脂块的设计，其中包括838标准的讨论。 1）准备的缩影这些试验的缩影高1厘米，比一般培养皿（更深），上面的木块，增加头部空间。他们都充满了一个温和的琼脂和确切数额，以营养控制绝对的金额，除了其浓度，并远离（&gt; 3毫米）的盖子，保持木块。在这种情况下石膏测试使用的琼脂是一个低钙型一个琼脂;但是，我们显示使用布莱克斯利的介质，在ATCC建议保持测试隔离Serpula lacrymans（Wulfen:薯条）中等代表结果施罗特应变EMPA 65 （ ATCC 32750）。 这样的设计使植物组织离琼脂接触和远离盘盖和墙壁。木质纤维基板可变润湿是在琼脂块测试的变异的主要来源。润湿增加水分含量，创建缺氧，抑制甚至停止好氧生物降解。它还创建一个任何人学习棕色和白腐真菌负责木材分解氧化机制的问题。凝结板盖是一个问题，如果水滴形式和潮湿的基材。同样，木材和其他组织将"芯"从琼脂水接触时，导致水分含量在80％以上（干重为基础）和制止好氧降解迅速。组织必须远离这​​些水源，使丝状真菌的发现，连接和控制基板内水分。 <ol><li>制造足够的琼脂培养基，以填补20毫升培养基，每个培养皿板所需的数量。我们使用五个复制（N = 5），每次治疗，电源使用过去的结果的分析确定。 </li><li>对于钙和铁的A型琼脂中，我们添加15克，琼脂500毫升容量瓶中含有约400毫升去离子水1.0 克NH 4 NO 3，1.0克新一代的KH 2 PO 4，0.25 克硫酸镁4 x修正7H 2 O和1.0克葡萄糖。的混合物，使用库存的解决方案中添加微量营养素。我们添加50μL的H 3 BO 4（0.057克/ 100毫升）和硫酸锌（0.031克/ 100毫升）。我们添加MnCl 2（0.036克/ 100毫升），硫酸铜4（0.039克/ 100毫升）各50μL，（NH 4）6个月7 O 24（0.018克/ 100毫升）。一旦添加营养素，填补容量瓶中，以500毫升线。 </li><li>一个典型的钙此外，在这种情况下，将0.05克氯化钙2 × 2H 2 O / 500毫升。在我们的例子中，我们使用琼脂钙作为一种治疗，用终浓度为5毫米。我们计数器增加离子强度和氯此外，通过添加5 mM氯化钠其他缩影。同样，我们使用这个示范铁自由的媒体，但在铁是其中包含的情况下，我们的FESO 4 0.112克2毫升去离子水混合，并添加后立即震荡（股票）50μL这种新鲜的解决方案。控制养分增加您在任何媒体上，这是明智的测试高压灭菌前pH值。酸性或碱性添加剂（如FECL 3）会影响凝固。 </li><li>媒体传送到容量瓶中，并在121 ° C和16磅20分钟高压灭菌介质。我们不超过500毫升卷（每1000毫升容量瓶中），以避免琼脂凝固之前，我们可以管理所有的测试板。 （注:它是明智的，在使用其他媒体，尤其是最小的基底盐添加的营养琼脂，首先验证你的测试木耳将增长时，高离子强度可以抑制生长，甚至杀死你的测试隔离。） </li><li>使用便携式移液器援助和10毫升的无菌聚苯乙烯移液器转移无菌琼脂在生物安全柜，一旦瓶清凉。让媒体凉爽接触是重要的，这里，以尽量减少结露。另外，栈板高，因为他们浇，最大限度地减少自由水的盖子。虽然凝结在正常培养的滋扰，在这里它是一个重大的问题，如果DROplets形式和湿木。水分含量超过80％（干重）（MC），将创建在组织缺氧，限制衰减好氧菌，并增加可变性。计算管委会如下: MC * = [（新鲜重量x干重）/干重] × 100 *（MC可超过100％ - 这可能似乎是一种奇怪的方式来计算MC，但为标准） <ol class="ualpha"><li>替代的缩影:如果植物组织的测试必须预先碾磨，过筛（如玉米秸秆的秸秆和树叶，一起），有两个菜，我们使用的选项。分培养皿板提供2个或4个部分，可以从车厢粉排除和琼脂。凝固的琼脂也可以被切割和粉末离开房间的一部分拆下来，但必须小心保持平等的琼脂卷，其余的如果要控制养分利用率。 </li></ol></li><li>慷慨削减塑料网格板面，采用彻底清洗，并添加无菌蒸压电网。我们使用的产品，天沟的保护我们的Mesh（35 × 50毫米，2毫米厚），的，并使用扫描电子显微镜显示后肥皂和清水洗干净的表面，并表现出缺乏真菌渗透。我们曾用玻璃纤维过滤器和直接铺设块菌丝发达，由于排汗问题的成败参半。你会得到衰减，但您的变异系数（CV）将高，治疗比较统计学薄弱。我们用玻璃棒以前，但块容易滑出棒时推挤，离开接触琼脂块。一个很好的选择是削减完整的圆圈，以适应板，切割一个边缘容纳接种。在一般情况下，削减电网，以适应你自己的设置，但确保他们在培养皿奠定完全平坦。 </li></ol> 2）准备"阻止"基板这些协议已发展为实木，但其他植物组织的适应性。质量损失衰减退化的丝状真菌在木材进展情况的衡量标准。因此，我们的方法是使用烤箱前和后衰变，以确定质量损失的干重。然而，对于任何生物能源的研究，侧重于植物组织化学，许多人发现，空气干燥组织是可取的。我们在这里展示您的琼脂块培养，使用烘干的起始原料准备协议，但空气干燥过程，而不是粉固体物质的替代信息。 <ol><li>对于本演示，我们使用南方松（SYP）。青少年暑期活动是市售的木材占绝大多数木材在美国的住宅使用它可以是任何四个松树种之一。用于非处理的木材和无结块从一个单一的RIP（纵向切割）切割的沿晶（19 × 19毫米）。这最大限度地减少了木材的化学变异。这个长度切成19毫米的 3块。我们使用土壤块试验规模，并削减在单个会话在表上的许多块看到。 </li><li> 19毫米的 3块在琼脂块的缩影进一步沿晶劈成两半，用凿子和锤子，锯。这使得一个粗糙的块边缘面对的塑料网，并顺利标签的顶部边缘。标签块用铅笔。如果你不能标注您的基板，一定要设计一个系统，以保持样品。削减足够的块，以满足你的治疗（再次，我们使用N = 5％治疗），以及非接种控制，将服务作为污染监测和基线数据样本，在平行空调。 </li><li>烘干的材料，将样品在对流烤箱在100 ° C下48小时。如果空气干燥的材料是必需的，条件的样本分庭或具有恒定温度和湿度的房间。我们用相对湿度65％和20 ° C，和我们平时数在10-14天空调，视物。 </li><li>前衡量你的样本，以确定初始炉干或鲜重。从烤箱的样品，只需将样品干燥器中冷却和权衡。 <ol class="ualpha"><li>替代空气干燥:空气干燥材料，采取五个样本（N = 5;牺牲这些将不会在微观世界中使用），重量新鲜，烘箱干燥以上，干燥后重新权衡。计算水分修正系数为如下两个街区: MC修正系数=干重/鲜重 例如:2.46 g（干）/ 2.68克（鲜）= 0.918，因此，3.0克新鲜块是2.75克烘干 平均5个样本，以获取平均校正因子。然后，权衡所有您风干块。 MC的平均校正因子乘以每个重量来确定初始炉干重。 </li></ol></li><li>高压灭菌器标记的样品在121 ° C和16磅为1小时，或与较大的样本。紧紧箔，以尽量减少润湿。 <ol class="ualpha"><li> Alternati已经灭菌:伽玛射线可以用来杀菌，如果有的话。我们已经测试云杉和榉木以前温度对半纤维素损失，并没有发现，随着没有实力损失或颜色的变化。然而，这可能不同的衬底和你的喜好/需求，和γ射线是一种成熟的替代。 </li></ol></li><li>对于本演示，我们正在测试固体石膏的作用（1％，FESO 4与纯）松（SYP）样本退化。这些都使得精确的表面面积和体积的光碟，除了它们的浓度控制在其可用性。这些蒸压分别在接种步骤添加。我们需要一个每块盘，我们加入了两个块，让每培养皿两个收成。 </li></ol> 3）接种和标签接种琼脂块的缩影，是以上的接种块土罐费时。对于我们来说，我们指望每次取3分的接种。含有琼脂的培养皿中，这点网，木材，任何外源性的营养源（在这里，石膏颗粒）此外，和真菌。的时间盖子打开，入内参观的人数增加，因为有污染的机会。也有几种通常在这一阶段，这些都是最好的耦合与文本的视频覆盖的关键失误。观看视频。 <ol><li>在无菌的生物安全柜，组装空琼脂菜，真菌接种（使用2周文化），网格，植物基板（木），外源性材料，sharpie，封口膜带，和传输工具的源盘。我们火焰消毒用70％的乙醇，并利用接种和网格块和钳一个传输工具。应剪去封口膜用剃刀边缘，以避免任何刻痕。在封口膜裂口导致休息时，密封性好，这将争相样品，并要求重新进入。 </li><li>火焰传输工具钳或之前加入以下（按照这个顺序）: <ol class="ualpha"><li>塑料网。 </li><li>木基板。我们的演示，我们将添加两个木块沿网格的长度，无论是从相同的初始块被分裂了，所以我们可以对数据从前期和后期的收成。这些块被添加并排，粮食年底面临真菌接种源点（木截面）。 </li><li>外源性材料。在这里，我们正在测试的衰变石膏由丝状真菌的作用。因此，我们要遇到这些石膏光盘才到达木木耳，因此每个接种点和块之间的网格上添加一个石膏光盘。这意味着增加2％的缩影光盘。不要让光盘和木材之间的联系，但让他们关闭。 </li><li>真菌接种。我们用直径7毫米，从20毫升培养基中生长2周文化的插头＃4软木螟。这将有助于控制接种量。对于非接种的控制，最好是从无菌板添加一个插件。我们通常的琼脂中添加这些插头，而不是在网格上。不要让接种和外源性底物或木材之间的联系，但同样，放在并拢。每块有一个接种插件。 注意:这是明智的，开始之前，以确保将有至少3毫米的头部空间，将有至少3毫米的距离，木块和盖墙壁之间，聚集在琼脂培养皿中的这些内容，和你的塑料网尺寸轻松容纳您的基板。 </li></ol></li><li>封口膜菜，密封，保持火焰燃烧酒精和镊子准备。保持在一个平稳的连续运动的板块水平和包装封口膜。如果封口膜断裂或如果内容争抢，除去封口膜，重新输入和组装的内容，并再试一次。 </li><li>标签的菜盖使用耐酒精的标志，标签直接的块数超过各自的基板之上。我们还提请各界石膏光盘。作为木衰变，你可能会失去对承印物上的标签的阅读能力。重要的是要保持菜的位置。另外，不要低估为一种真菌降解或殖民无数其他类型的材料，包括金属的能力。 </li><li>如果您对您的网格组合细腻，仔细传输板的孵化器。 ，我们只有一次，撞毁板斌对一门堵塞，并重新组装板。把你的时间，并绘制你的路线。它是一个时间的问题，所以要小心这一次。 </li></ol> 4）培育和收获孵育板可满足您的木耳，但应保持在生物孵化器如果可能的话，以避免由于温度波动的冷凝。时间序列的收成是在无菌条件下完成的，最后收获的除外。如果这些中间收成完成后生长承印物是重要的，处理板更容易，因为菌丝交叉链接基板。 <ol><li>对于我们的演示，我们孵育板在20 ° C，并在黑暗中。在5周的收获点，我们将删除整个治疗很多，喷用70％乙醇每一个底部和顶部，并用火烧钳去除材料内的生物安全柜。 </li><li>拍摄之前销毁琼脂平板上，侧重于任何形态或黑色素照片。 </li><li>删除您的需求需要处理的块。用手指滚远多余的菌丝（丁腈手套），但要小心，不要失去任何腐朽的材料。我们通常在烘箱中干燥铝块重达平底锅，以确定质量损失，使用新鲜和干重的控制，监测过度排汗。我们也标签和跟踪任何有明确积水深褐色块，虽然这应该是最小的这种做法。质量损失数据，有助于衡量衰减进步，它是重要的数据元素浓度克重量的基础上上，后来被计算。还记得那百分比数据必须进行统计归。我们计算如下: ％质量损失= [（初始体重最终重量）/初始体重] × 100 <ol class="ualpha"><li>替代空气干燥:如果您需要与您的目标生物治疗材料的进一步空气干燥，你应该从第2.3节中使用的调节机制，以整修。如果你必须确定质量损失，将需要的水分含量的措施，解决办法之一，是分割成一个大和小部分块（或粉末）。称，但只烘箱干燥的一小部分。计算水分含量转换系数，如上，然后将其应用到总鲜重的小+大的部分（块总鲜重）。特别是，如果前预处理材料与一种真菌，糖化等工序，质量损失，将帮助您与质量平衡的决心后，帐户由真菌消耗碳水化合物。 </li></ol></li><li>在高压灭菌袋销毁的文化，但节省塑料支撑网和在今后的实验中使用任何可以回收的其他组件。 </li></ol> 5）对结果的解释木质纤维组织通常会在琼脂块设计衰减慢，但在这一点上，你应该有，即使在中等衰变水平相对较低的变异。你也应该有中度（20-50％），在控制组织不高水分。 <ol><li>木材中的水分含量（干重计算）不暴露真菌通常是温和的，在这个演示中约40％。这是上面的松树（一般为24％左右）的纤维饱和点（FSP），这意味着有一些多余的水木质纤维次生壁内的约束的自由水。这也意味着，有可能发生一些排汗。 100％的相对湿度应仍不允许木材MC超过了FSP。你的设计可实现对基板和网状选择不同的结果。关键是干重的基础上，保持水分含量低于85％左右。 </li><li> ％在木材质量损失在这些琼脂块的缩影退化通常是30％左右，后16周最真菌。在这种情况下Serpula lacrymans，对于选择的真菌，真菌会在这段时间内实现全降解，&gt; 60％的质量损失。 S. lacrymans是一个褐腐菌，消除小木质素，因此在本试验中62％的质量损失是指衰减是附近或过去完成。白腐真菌去除木质素，取决于品种，以及为底物的质量损失。 </li><li>小心记录菌丝形态。菌丝形态在这个演示中，本质上没有显示任何一个治疗的成功或失败，但黑化色彩是很重要的。它使我们能够与石膏中的杂质铁的作用研究等，如低（2000年），原生材料进行了测试，发现这个"生锈"着色。低等人 （2000年），钙和铁的作用进行了辩论，在这里，我们看到的铁，而不是钙增强衰减，以及这些相同的铁锈色的菌丝。 </li><li>如果你想在这个降级的木材测量浓度的任何东西，最好是正常化的质量损失。其重量的基础上的集中，例如，如果钙既不是进口或出口从木材腐朽的50％，将出现零时间增加一倍，达到最终的收获。这是因为现在1克粉末两次木材体积比在零时间。木耳消耗50％的矩阵，降低密度。规范化任何给定的浓度（如毫摩尔/克），以弥补质量损失如下: 归浓度=毫摩尔/ GX [1 - （％质量损失/ 100）] 例如:10毫摩尔/克钙在木材降解20％，比7毫摩尔/克，在零时刻。 问:衰变过程中钙含量的增加？ 10毫摩尔/克是一个明显的3毫摩尔/克的增加，增加了43％，但oNE克粉末退化木材密度较低，现在量也较大。规范化是比较平等的木材量的初始和最终的钙含量。 10 × [1 ×（20％/ 100）] = 8毫摩尔/克正常化 答:是的，CA确实增加了，但14％，而不是43％。 （数据也可以表示每毫摩尔/ 厘米 3，木材体积，乘以由密度。 </li></ol><img src="/files/ftp_upload/2283/2283fig1.jpg" alt="图1" /> 图1。琼脂块的缩影，成立孵化前，在这个示范。 <img src="/files/ftp_upload/2283/2283fig2.jpg" alt="图2" /> 图2。Serpula lacrymans殖民休息塑料网，提升上述琼脂接触块的松木块。这菌丝木材和外源性营养物质/元素的来源之间的重要连接，并在琼脂或固体材料的控制这些外源性的物质来源，是琼脂块设计的一个关键优势，与土壤块设计。 <img src="/files/ftp_upload/2283/2283fig3.jpg" alt="图3" /> 图3％重量损失，作为衡量一个5周和15周后，在琼脂块的缩影松的孵化Serpula lacrymans木材腐烂程度。治疗无（无钙），5毫米氯化钙2琼脂（2氯化钙），&gt; 99％纯度的石膏（硫酸钙），或1％的铁修订石膏。保护方差分析手段比较Tukey的测试，使用α= 0.05。对于每一个收获，在同一封信酒吧没有显着差异。误差棒=标准偏差。在林（2010）出版。 <img src="/files/ftp_upload/2283/2283fig4.jpg" alt="图4" /> 图4架空图片（一）所有五个衰减的第15周时重复相同的褐腐病试验木耳，Serpula lacrymans，以及（二）拆除和烘干块。注意的黑色素缺乏钙免费治疗，泛黄的纯钙治疗，锈的铁条的治疗外观。治疗标记，如图3，在控制比较（二）未接种块。 <img src="/files/ftp_upload/2283/2283fig5.jpg" alt="图5" /> 图5。架空图片从不同的使用铁浓度较高的介质，布莱克斯利的麦芽琼脂的试验。注意与图4相比，观察黑色素的损失。删除和权衡块没有表现出对减肥的治疗效果。这是作为一个示范的这些块试验的外源性成分的影响。这些影响不会在土壤块JAR设计可测试，这些外源性的投入是太难以控制。

<ol>
	<li>ASTM D1413-07. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+test+method+for+testing+wood+preservatives+by+laboratory+soil-block+cultures.+.">Standard test method for testing wood preservatives by laboratory soil-block cultures. .</a> Annual Book of ASTM Standards. , 185-192 (2007).</li><li>Bravery, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A miniaturized wood block for the rapid evaluation of wood preservative fungicides. , (1978).</li><li>Low, G. A., Young, M. E., Martin, P., Palfreyman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+relationship+between+the+dry+rot+fungus+Serpula+lacrymans+and+selected+forms+of+masonry.">Assessing the relationship between the dry rot fungus Serpula lacrymans and selected forms of masonry.</a> Int. Biodeterior. Biodegrad. 46, 141-150 (2000).</li><li>Nicolas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+I+(One/1)+-+Degradation+and+Protection+of+Wood+(Syracuse+Wood+Science+Series+#5).">Volume I (One/1) - Degradation and Protection of Wood (Syracuse Wood Science Series #5).</a> Wood Deterioration and Its Prevention by Preservative Treatments. , (1973).</li><li>Schilling, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+calcium-based+materials+and+iron+impurities+on+wood+degradation+by+the+brown+rot+fungus+Serpula+lacrymans.">Effects of calcium-based materials and iron impurities on wood degradation by the brown rot fungus Serpula lacrymans.</a> Holzforschung. 64, 93-99 (2010).</li></ol>

使用我们的琼脂块的设置（图1）Serpula lacrymans增长与石膏表面和成木块（图2）直接接触，导致60％以上的减肥控制褐色腐烂的松木块（图3 ）。这很容易满足ASTM标准的目标，&gt; 50％的衰减，和16周的平均变异系数 （五）在衰变时0.055。这个数据是发表在7林女士。同样，其他真菌比在土壤块将需要更长的的琼脂块孵化。作为参考，我们已经成功地运行在过去几个实验类似的设计与多种其他真菌物种，通常使用扫描电子显微镜与电子谱仪（SEM - EDS）和电感耦合等离子体光谱仪（ICP - OES的），以验证外生连接基板和8,9成木钙和其它元素的易位。 除了向低变化，有两个结果，我们的琼脂块的设计揭示其他。首先，有治疗效果（图3），我们的钙增加，无论是琼脂氯化钙或纯石膏抑制这种真菌的衰变。这是一个有用的结果，因为别人有理论的基础上与现实世界像砂浆和抹灰材料的研究，钙提高衰减。相反，我们看到衰变率回升，除铁，石膏，表明铁，而不是钙，是关键。第二个有用的结果，然而，没有量化，但而不是颜色的菌丝体（图4）。有外部菌丝重大和明显的黑化处理间的差异，研究人员先前已指出"生锈"黑化，当遇到这种真菌建材（如低等， 2000）。在我们最近的研究中，我们发现使用高铁中（图5），这些影响都将丢失。总的来说，它表明这种真菌利用铁，钙，这些材料和早先的研究，与"生锈"的菌丝体报道，观察铁的作用，而不是钙的效果。 总的来说，这些结果和治疗效果，使用高铁琼脂缺乏控制，琼脂块的设计可以提供，尤其是在替代土壤块的方法，研究员，是非常强烈的示范。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>4014</td>
<td>25 x 150 mm</td>
</tr>
<tr>
<td>Agar, Type A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4550</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium nitrate, NH4NO3</td>
<td>&nbsp;</td>
<td>Millinckrodt</td>
<td>3436-12</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate, KH2PO4</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3246-01</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate 7-hydrate, MgSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>230391</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>Dextrose</td>
</tr>
<tr>
<td>Boric acid, H3BO4</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>2549-04</td>
<td>&nbsp;</td>
<tr>
<td>Zinc sulfate 7-hydrate, ZnSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>8880-12</td>
<td>&nbsp;</td>
<tr>
<td>Manganous chloride 4-hydrate, MnCl2&bull;4H2O</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>2540-04</td>
<td>&nbsp;</td>
<tr>
<td>Copper(II) sulfate 5-hydrate, CuSO4&bull;5H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>209198</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium heptamolybdate 4-hydrate, (NH4)6Mo7O24&bull;4H2O</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431346</td>
<td>&nbsp;</td>
<tr>
<td>Calcium chloride dihydrate, CaCl2&bull;2H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>4160-12</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride, NaCl</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>7581-12</td>
<td>&nbsp;</td>
<tr>
<td>Ferrous sulfate 7-hydrate, FeSO4&bull;7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt</td>
<td>5056-12</td>
<td>&nbsp;</td>
<tr>
<td>Pipet-aid</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>4-000-110</td>
<td>Cordless 
EtOH the surface</td>
</tr>
<tr>
<td>10 ml sterile polystyrene pipette</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>Gutter Guard</td>
<td>&nbsp;</td>
<td>Thermwell Products Co.</td>
<td>VX620</td>
<td>Pre-scrubbed with soap 
Hardware store</td>
</tr>
<tr>
<td>Calcium sulfate hemihydrate, CaSO4&bull;0.5H2O</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>385355000</td>
<td>&nbsp;</td>
<tr>
<td>#4 cork borer</td>
<td>&nbsp;</td>
<td>Boekel</td>
<td>1601</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm "M"</td>
<td>&nbsp;</td>
<td>Pechiney</td>
<td>PM-996</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

agar-block microcosms for controlled plant tissue decomposition by aerobic fungi

研究真菌生物降解木质纤维素的植物组织的两个主要方法是开发木材防腐剂测试（块土;琼脂块）。这是广为接受的土壤块缩影收益率更高的衰减率，更少的水分问题，研究之间的变异较低，防腐剂的毒性较高的阈值。土壤块测试，因此更利用的技术，并已通过美国测试和材料协会（ASTM），（方法D 1413年至1407年）的标准化。土壤块的设计也有缺点，但是，使用本地变量的土壤来源和限制了营养素外部（外生）的控制，以腐烂组织。这些弊端已经成为一个问题，在采用此方法等，日益流行的研究目的。这些现代化的目标包括:有辱人格的lignocellulosics生物能源的研究，试验的共同代谢的有毒物质的生物修复，沿菌丝网络评估氧化机制，跟踪易位元素。土壤块不适合在这些应用中足够的控制。精致的琼脂块的方法是必要的。 在这里，我们使用的褐腐病木材降解菌Serpula lacrymans的降解木在琼脂块的缩影，采用深低钙琼脂培养皿中。我们测试在时间序列的衰减作用外源性石膏，证明效用和预期的变化。块空调从一个单一的板RIP（纵向削减），称重，高压灭菌，并介绍上盖塑料网无菌。真菌接种在每个块面，石膏外源性接口添加。无菌操作，直到最后的破坏性收获的收成。这些微观世界的设计，以避免与琼脂培养皿墙块接触。凝结在板盆满钵满，孵化过程中的最小化。最后，接种/石膏/木间距最小化，但没有允许接触。这些技术方面的琼脂块的设计也最常见的原因失败和研究之间的变异的主要来源。视频有益的出版物，因此在这种情况下，我们展示了低可变性，高品质的结果。

Watch this Scientific Journal Video about Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi at JoVE.com

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

本视频演示了一个受控环境中的方法来研究好氧菌降解木质纤维素的植物组织。 ，控制养分来源和水分的能力是一个关键优势的琼脂块的缩影，但是这种方法往往产生成败参半。我们解决关键缺陷产生重现性好，低变异的结果。

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

agar-block-microcosms-for-controlled-plant-tissue-decomposition

Nanyang Technological University

Research

JoVE Journal

Biology

15.0K 次观看.  University of Minnesota. 本视频演示了一个受控环境中的方法来研究好氧菌降解木质纤维素的植物组织。 ，控制养分来源和水分的能力是一个关键优势的琼脂块的缩影，但是这种方法往往产生成败参半。我们解决关键缺陷产生重现性好，低变异的结果。

视频: Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

测试使用微量注射蚜虫病毒传播的生理障碍

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

科研

生物学

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

酸性α-膨大诱导测量植物细胞壁扩展（蠕变）

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

链接捕食风险，草食动物生理应激和植物凋落物的微生物分解

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

从食虫植物组织分离存活的多细胞腺的，猪笼草</i

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

小鼠中自噬激活有氧运动

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...