斯蒂芬·阿尔德曼博士，博士大卫叶和Erin马克里表示诚挚的感谢与显微镜的帮助。通过赠款资助这项研究是从的NIFA特色作物研究计划，USDA得离谱SREP格兰特奖2009-02484号。梅根·莱昂哈德约瑟夫·麦科勒姆，凯文·金洛克，BRIANA Kinash玛丽亚McSharry和妮可做人提供了优良的技术援助和周到的讨论。

此过程需要至少几个月孵化BDM片土壤中，还有几个个月的时间顺序琼脂平板上和琼脂，肉汤评估已有化学定义殖民化和降解的生物测定。个人的方法列出的顺序，他们将被执行。 1。 BDM薄膜在土壤中孵化纳入BDM片土壤条件下，模仿下，他们将有望降低。收购400克（干重当量）居民的土壤和三明治一个10厘米×10厘米的BDM一个13×13厘米的尼龙网眼袋（250微米）内的土壤。关闭用尼龙线。孵化的时间是在实验者的自由裁量权。监视器和/或微生物的活动（ 例如，土壤温度，养分和水分）在整个潜伏期酌情定期修改的参数相关。 2。媒体的制备和试剂为了避免无意中进入媒体和文化管引入营养源，使用试剂级化学品，新购买的培养管，I型超（ 如 NanoPure）水，混合媒体和严格的水洗玻璃器皿。避免交叉污染，试剂 - 最好是使用一组专用的试剂及玻璃器皿用于此目的。请注意，这是可能的，可以分离真菌的生长发育受到抑制由下面列出的试剂中的一种成分。如果发生这种情况，可能需要一些优化。 <ol><li>媒体之前，洗所有必要的玻璃器皿实验室洗涤剂，然后酸洗（ 例如：10％盐酸浸泡过夜）。在蒸馏水中冲洗一个至少12倍，两次在超纯水。用干净的铝箔覆盖所有的玻璃器皿排除粉尘。戴上手套，避免接触的内部和边缘的船舶排除皮肤油指纹。为了避免任何肥皂居民的会费和促进定植，培养真菌在新购买的（未使用），冲洗和蒸压玻璃文化管子及管盖的玻璃划伤。 </li><li> 马铃薯葡萄糖琼脂 （PDA）用于真菌从土壤中进行初始分离，为建立单个孤立的菌落接种物和生成用于长期存储。根据制造商的说明准备媒体，添加氯霉素（10毫克/毫升原液在EtOH）至终浓度为30微克/毫升，高压灭菌后（冷却至50℃时），以排除细菌。这种介质现在被称为作为PDA叶绿素30。 </li><li> 真菌的最小介质 （FMM）与潜在的真菌塑料降解菌接种BDMS了坚实的基础。除了从琼脂（当使用时，固化介质），它是无碳。约800毫升去离子H 2 O中，添加6.0克纳米3，0.52克氯化钾， 硫酸镁 0.52克·7H2Ø，1.52ĝKH 2 PO 4，和1毫升Hutner的痕量元素的股票解决方案（硫酸锌2.2 ​​克4·7H 2Ø，1.1ĝħ3 BO 3，0.5ĝMNCL 2·4H 2 O，0.5ĝ硫酸亚铁4·7H 2 O，氯化钴0.16克2·5H 2 O，0.16克硫酸铜4·5H 2 O，0.11克（NH 4）6MO 7 O 24·4H 2 O，5.0克Na 4 EDTA在100毫升蒸馏H 2 O 33 34 6.5用NaOH调整pH值，并把对于液体生物测定体积至1 L，过滤灭菌FMM，以避免盐的沉淀。如果所需的固体培养基中，加16克琼脂，高压釜中。当半固体琼脂需要，减少琼脂8克/升。当冷却至50°C，添加氯霉素至终浓度为30微克/毫升，此介质称为为FMM叶绿素30，另外，作为对照，以确保真菌菌株生长在这种媒介，FMM应该是马德以上，但每公升含10克葡萄糖。这就是所谓的葡萄糖35（GMM）的基本培养基中。 </li><li> 36 磷酸盐缓冲盐水 （PBS）中用于暂停在初始的连续稀释的土壤暴露BDMS土壤颗粒和微生物细胞。为了使PBS中，加入8克氯化钠，氯化钾0.2克，1.44克Na 2 HPO 4，0.24克KH 2 PO 4 800毫升蒸馏水中。溶解盐，用盐酸调节pH值至7.4。使总体积为1升超纯水。填写廉洁文化管9.5毫升和4.5毫升PBS每管。高压灭菌。 </li><li> 30％（体积/体积）甘油用于暂停在储存过程中在-80℃下冷冻保存的细菌和酵母细胞，芽孢，真菌菌丝这种超纯水解决方案。高压灭菌。 </li><li> 0.01％（体积/体积）的Triton X-100的用于暂停真菌孢子的洗涤剂作为一种无毒的润湿剂为疏水性孢子。将100微升的Triton X-100在1升的超纯水。高压灭菌。 </li><li> 0.1M磷酸钠缓冲液（pH7.2）作为溶剂戊二醛在SEM固定期间。 68.4毫升，1M的Na 2 HPO 4和31.6毫升的1 M的NaH 2 PO 4混合，并把总体积为1升，用超纯水进行36。 </li></ol> 3。生物活性材料的制备：BDM薄膜表面去污<ol><li>因为残存的污染物，如纸张和胶带切割用具是潜在的碳来源，使用新购买的剪刀或新鲜的刀片，用于切割和无机表面。戴上手套，以避免污染皮肤油脂BDM片。 BDM片切割成小方块（4.25×4.25厘米）。此大小广场融入了标准的100×15毫米的Petri板。 </li><li>使用紫外线杀菌灯（发光波长为253.7纳米）净化BDM电影。这种灯是经常发现在一个标准的生物安全柜。确保当UV光操作，没有到生物安全柜可能污染的BDM膜的外部空气的流量。 </li><li>罩内用70％乙醇，让鼓风机运行15分钟，冲洗空气的潮湿的表面。然后，关闭窗扇和净化罩表面采用UV 2小时。 </li><li>将净化罩，行的BDM片。允许紫外光照射2小时的电影。 </li><li>使用清洁，消毒钳翻转BDM电影。前排和后排的工作时间缩至最短，手和手臂正上方新净化BDM表面。当翻转BDMS，这里的新的灭菌的一端朝下，在一个干净的区域，暴露在紫外线下，但以前没有接触侧片与非灭虫。 </li><li> 2小时后每边的紫外线处理，生物安全柜，用无菌镊子取出BDM片，再由前至后，避免将手和胳膊已经去污工作转换后的电影。将BDM电影成无菌，干燥，有盖的容器中，如铝箔覆盖烧杯。储存在室温下在黑暗中。 </li></ol> 4。板式生物测定设置在无菌转移罩使用无菌技术执行所有步骤。 <ol><li>奠定了UV-灭虫BDM片琼脂平板的表面上。第一设置下的一个角，轻轻让与琼脂接触的方辊的其余部分。避免皱纹的产生。有些影片在储存期间会形成皱纹，这是不可避免的。特别薄膜可能需要使用两个镊子在为了打下一块平坦的。 </li><li>广场上盖的塑料薄膜，提供水和营养源的初始定植疏水塑料珠半固态FMM CHL 30琼脂。水可渗透的薄膜，有孔玻璃珠是没有必要的。重新在微波炉融化的琼脂，并用移液器转移到覆盖（在每个相应的四个10微升滴琼脂液NER）。请勿触摸吸管尖穿刺，因为它可能直接覆盖。不要超过10微升每一滴水，因为多余的体重可以拉塑关闭琼脂表面覆盖板反转时。 </li><li>让板坐在右侧的盖子上过夜完全凝固，然后将密封袖子琼脂端在4℃黑暗。当准备进行生物测定（ 第七步 ）， 使用i）一盘FMM CHL 30（阴性对照），2）一个生物测定板包含四个10微升滴在塑料薄膜上，半固态FMM CHL 30及iii）一盘GMM（阳性对照）。以这种方式，四个重复的各菌株可以进行测试每盘介质。 </li></ol> 5。 “液体生物活性设置<ol><li>按照步骤3.1 BDM切割。在切割塑料薄膜液体生物测定，选择大小，融入文化管。将取决于所需要的面积，实现了几种塑料的类型之间的质量均匀的膜厚度。我们发现那件相当于0.0175克适合在15×150毫米的文化管。 </li><li>净化膜表面用UV光，上述的步骤3中所描述的。 </li><li>对于每个重复的分离微生物，准备三根管接种：）5毫升FMM的含有一个BDM片段的预定尺寸（实验），ⅱ）5毫升FMM没有碳源（阴性对照），和iii）5毫升GMM（阳性对照）。此外，准备复制FMM要测试的每个BDM管，但不接种。这些控制管会发现任何微生物的生长造成污染。 </li></ol> 6。真菌的分离以下土壤培养和样品取走，真菌从土壤中坚持BDM薄膜隔离。如果需要，细菌可以simultaneou狡黠孤立用同样的方法，使用适当的媒体分离的土壤细菌，如1/10X稀胰蛋白酶大豆酵母琼脂补充有50微克/毫升放线菌酮，以防止真菌生长37。当定义中是必需的细菌隔离在步骤5和7，M9 38（加放线菌酮）是一个不错的选择。 <ol><li>来样加工之前，准备PBS和PDA CHL 30。允许PDA叶绿素30板干燥（unbagged）在室温下放置约24小时，以消除冷凝的盖和琼脂表面上。前标签PDA CHL 30板。 </li><li>挖掘网格袋含有BDM片墓地步骤1中选择。船舶和储存在4℃不长于48小时后，从土壤中提取。 </li><li>使用无菌压舌板和镊子，轻轻取出土壤，直到露出塑料，但不刷抱住BDM薄膜表面的土壤。 BDM片切为1厘米2个0.5克的材料被回收，直到总共四个复制。 0.5克BDM膜和已连接的土壤转移到25毫升培养管中，含有9.5毫升的PBS。如果覆盖是完全退化或正在处理土壤的控制，加0.5克土的PBS，如果可能的话，选择仍然残留地膜变色片。 </li><li>旋涡振荡培养管中含有9.5毫升PBS和0.5克30秒覆盖高速，超声在超声处理水浴中10分钟，并涡旋30秒。搅拌是为了掰开生物膜，物理去除细胞嵌入或秉承覆盖件，创造均匀的悬浮液。 </li><li>准备连续稀释PBS /土壤的解决方案，以获得单个菌落形成单位镀层。对于每个样品，将在层流罩培养管中充满了4.5毫升的无菌PBS中。对于每一个样本，填写三口井的深井96孔板充满了450微升PBS。 </li><li>原来的塑料/土壤悬浮液（0.5克地膜加PBS液9.5毫升）5.0×10 -2稀释秉承塑料的微生物材料。从这种原始的管中加入0.5毫升的悬浮在文化管4.5毫升无菌PBS获得5.0×10 -3稀释。在高速，持续30秒的涡流。在96 - 孔板中，加入50微升至450微升PBS。重复/重复所有样品。使用多通道移液器来稀释样品大批 。每一个新的稀释混合上下吹打十倍。更改稀释之间的技巧。进行稀释为5.0×10 -6。 </li><li> （可选）比本文所述样品中的真菌菌株的细菌分离物更丰富，因此，如果同时隔离细菌，然后执行连续稀释至5×10 -8在上面的步骤6.6中 。 </li><li>均匀地涂抹加入100μl5.0×10 -3至5.0×10 -6稀释整个琼脂表面PDA叶绿素30板的火焰灭菌的弯曲的玻璃棒。商店板直立30分钟让液体浸泡，然后用封口膜封板，以避免逃生不明菌株的孢子。孵育倒板在20℃黑暗5天，以便快速和缓慢增长的真菌菌落形成。 </li></ol> 7。塑料降解真菌的初步选择 重要提示：从这个角度提出应打开所有文化，只有在生物安全柜（层流罩），以避免对环境的污染与身份不明的孢子。某些土壤真菌和细菌是潜在的人类病原体。 <ol><li>检查稀释板隔离的殖民地。选择一个不触及另一个殖民地的殖民地。 </li><li>使用单一的消毒牙签，轻轻触摸一个殖民地，然后触摸琼脂FMM（ 步骤2.3 </str翁），FMM +塑料（在步骤4中），和GMM板（在步骤2.3），以该顺序。接种到琼脂有孔玻璃珠，塑料薄膜上盖，轻轻擦牙签，在整个塑料薄膜的表面上的琼脂点，然后轻扫。重复接种，从相同的源的细胞集落，直到每块板有四个复制条纹。 </li><li>重复步骤7.1至7.2，直到几个选择的每一个视觉独特的集群式重复从每BDM样品。 </li><li>用封口膜密封板，反转，孵育在20°C黑暗环境中，连续5天。 </li><li>确定菌株生长的比他们更好的成长FMM BDMS。如果增长并不明显，查看标本在解剖显微镜下验证定植或缺乏（ 图1）。保持盖子，盖子上有结露，更换一个新的盖子，但这样做在生物安全柜。 </li><li>潜在的BDM降解菌一旦被选中，使用terile牙签刮接种从木耳殖民塑料本身，分离单个菌落形成单位PDA叶绿素30板的连胜。 </li><li>塑料降解菌株接种在20℃与封口膜孵育PDA CHL 30板的密封板在黑暗中，连续5天。 </li><li>现在单一孤立菌落纯化，确保他们能够真正的去殖民化的塑料薄膜被测试。收集用灭菌牙​​签从一个单一的菌落形成单位的接种物（孢子，酵母细胞，或菌丝）。重复步骤7.2至7.5。经过5天的潜伏期在20°C，检查真菌生长的生物测定板。如果在BDM膜优于它生长在FMM在第一和第二（7.5）（7.8）试验菌株生长，然后将其存储在步骤8中，如下所述的隔离。 </li></ol> 8。长期储存的塑料降解菌</p&gt; 总体来说，塑料降解菌株将被测试板生物测定（7.5）中，重新纯化，以单个孤立的菌落形成单位（7.6），测试在板生物测定（7.8），以及最后，在液体中的生物测定测试（ 9.1 - 9.5）。在测试过程中，隔离应转移到新鲜媒体每个月储存在4°C的工作股票板块，只要有足够的增长是可见的。分离物也应在-80℃，和/或干燥的孢子在无菌过滤器上，在4℃下保存于甘油中这两种存储方法描述如下。 <ol><li>每个菌株生长在BDM膜比它更好增长FMM在这两个第一和第二（7.5）（7.8）板的生物测定，准备两个PDA叶绿素30板具有两个或三个灭菌的滤纸片，直径约1厘米的的表面上。接种这两个板块在上面的步骤7.8中选择的相同的单菌落。草坪均匀地扩散接种琼脂最大化增长。密封板用封口膜和孵化，在20°C的5天。这一步可以同时开始执行步骤7.8的效率。 </li><li>菌丝/孢子琼脂表面上应覆盖过滤器磁盘。琼脂表面消毒镊子，到无菌Eppendorf管取下过滤器的磁盘。空气干燥过滤器磁盘Eppendorf管内过夜（与帽子摘下）在生物安全柜或其他干燥，无菌环境中以最小的空气流动，以避免交叉污染。然后密封用封口膜，并储存在4°C。 </li><li>使用无菌棉签或牙签收获的孢子和菌丝体的草坪板。挂起孢子和菌丝在30％甘油中冻存管，闪光冻在液氮中，并储存于-80℃。 </li></ol> 9。严格有限公司通过液体生物测定塑料利用nfirmation <ol><li>生成新鲜孢子和/或细胞接种液体培养：接种PDA叶绿素30板与潜在的塑料降解菌株的孢子。用封口膜密封板，孵育在20°C，直到真菌孢子可见。 </li><li>在生物安全柜，收获孢子洗板0.01％的Triton X-100（5毫升，每15 x 100毫米培养皿效果很好）和擦洗孢子（或酵母细胞）从表面用无菌弯曲的玻璃棒。吸液孢子（或单元）的悬浮液转移到无菌容器中。 </li><li>用血细胞计数器计数孢子或细胞，并确定适当的容体，FMM肉汤加至5毫升1×10 6个孢子/管，总。溶剂量要小于10微升。稀释浓的孢子悬浮液，以0.01％的Triton X-100的可能是必要的。 </li><li>在一个无菌移罩，接种培养管编制<stro纳克&gt;步骤5 1×10 6个孢子（或酵母细胞）。用封口膜密封的上限，以避免任何逃生的孢子，特别是对不明身份的菌株。在20°C孵育在黑暗中观察样本每周增长。 </li><li>在接种后的第一个星期之内，在塑料薄膜上的真菌的菌丝生长可能是可见的，特别是在塑料薄膜的边缘。浮游酵母（证明阴天媒体），生长在600nm处的光密度读数可以监测。由于真菌可能直接连接到所述塑料薄膜，增长可能用肉眼评估，可以确认光镜和/或扫描电子显微镜（SEM）。 </li><li> FMM-唯一的控制，寻找微乎其微的白色斑点是太小，注册光密度的变化，用分光方法。这些斑点可以用巴氏吸管吸出，并观察使用微分干涉显微镜（DIC）。斑点通常是无法识别的精度pitate，但在某些情况下，它们可以是原来的孕育剂的团块，发芽，但基本上没有增长。 </li><li>使用视觉和微观观测实验样品和对照比较定植，并给予评级BDM膜每个样品的增长。因为许多BDMS的颜色深，并允许通过显微镜观察，除了最小的边缘部位，膜厚度禁止油浸镜头下观察，扫描电镜（SEM）估计我是有用的）存在的微生物的生长， 例如，通过评级系统表1中 ，以及ii）BDM降解的程度。 </li></ol> 10。 SEM样品制备<ol><li>使用清洁的，无菌剪刀切小块BDM复制样本。修复BDMS浸渍24-48小时，在2.5％戊二醛的0.1M磷酸钠缓冲液（pH7.2）。 </li><li>虽然固定发生的，100％纯乙醇脱水加入5-10％VOlume明3埃分子筛（激活的干燥炉中，在175-260℃下，并在干燥器中冷却），并允许坐在在室温搅拌过夜，以确保完全脱水。 </li><li>把BDMS浸在0.1M磷酸钠缓冲液（pH7.2）洗涤四次，每次15分钟。 </li><li>在超纯水把BDMS浸，洗三次，每次5分钟。 </li><li>在乙醇系列脱水BDM样品，浸泡20分钟，在各浓度：50％，70％，80％，90％，95％，和100％。 BDMS两次以上，在100％乙醇洗净，每次漂洗20分钟。 </li><li>待样品临界点干燥和溅射镀膜。 </li></ol>

<ol>
	<li>Gregory, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+implications+of+plastic+debris+in+marine+settings+-+entanglement,+ingestion,+smothering,+hangers-on,+hitch-hiking+and+alien+invasions.">Environmental implications of plastic debris in marine settings - entanglement, ingestion, smothering, hangers-on, hitch-hiking and alien invasions.</a> Philosophical Transactions of the Royal Society. 364, 2013-2025 (2009).</li><li>Teuten, E. L., Saquing, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+and+release+of+chemicals+from+plastics+to+the+environment+and+to+wildlife.">Transport and release of chemicals from plastics to the environment and to wildlife.</a> Philosophical Transactions of the Royal Society. 364, 2027-2045 (2009).</li><li>Thompson, R. C., Moore, C. J., vom Saal, F. S., Swan, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastics+the+environment+and+human+health:+current+consensus+and+future+trends.">Plastics the environment and human health: current consensus and future trends.</a> Philosophical Transactions of the Royal Society. 364, 2153-2166 (2009).</li><li>ASTM D 883. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard terminology relating to plastics. , (1991).</li><li>SO 472. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics - vocabulary, amendment 3. General terms and terms relating to degradable plastics. , (1993).</li><li>Krzan, A., Hemjinda, S., Miertus, S., Corti, A., Chiellini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+and+certification+in+the+area+of+environmentally+degradable+plastics.">Standardization and certification in the area of environmentally degradable plastics.</a> Polymer Degradation and Stability. 91, 2819-2833 (2006).</li><li>Shogren, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+mulches+from+renewable+resources.">Biodegradable mulches from renewable resources.</a> Journal of Sustainable Agriculture. 16, 33-47 (2000).</li><li>Takakura, T., Fang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Climate under cover. , 1-10 (2001).</li><li>Miles, C., Hayes, D., Brodhagen, M., Lee, J., Wszelaki, A., Moore-Kucera, J., Wallace, R., Marsh, T., Inglis, D., van Steenbergen, F., Tuinhof, A., Knoop, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+mulches,+biodegradable+alternatives,+China+and+US.">Plastic mulches, biodegradable alternatives, China and US.</a> Transforming Landscapes, Transforming Lives: The Business of Sustainable Water Buffer Management. , (2011).</li><li>Song, J. H., Murphy, R. J., Narayan, R., Davies, G. B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+and+compostable+alternatives+to+conventional+plastics.">Biodegradable and compostable alternatives to conventional plastics.</a> Transactions of the Royal Society B. 364, 2127-2139 (2009).</li><li>Hayes, D. G., Dharmalingam, S., Wadsworth, L. C., Leonas, K. K., Miles, C., Inglis, D. A., Khemani, K. C., Scholz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+agricultural+mulches+derived+from+biopolymers.">Biodegradable agricultural mulches derived from biopolymers.</a> Degradable polymers and materials, principles and practice. ACS Symposium Series. 1114, 201-223 (2012).</li><li>Ojeda, T. F. M., Dalmolin, E., Forte, M. M. C., Jacques, R. J. S., Bento, F. M., Camargo, F. A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abiotic+and+biotic+degradation+of+oxo-biodegradable+polyethylenes.">Abiotic and biotic degradation of oxo-biodegradable polyethylenes.</a> Polymer Degradation and Stability 94. , 965-970 (2009).</li><li>van der Zee, M., Lendlein, A., Sisson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+for+monitoring+biodegradation+processes+of+environmentally+degradable+polymers.">Analytical methods for monitoring biodegradation processes of environmentally degradable polymers.</a> Handbook of Biodegradable Polymers. , 263-281 (2011).</li><li>Narayan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Misleading+claims+and+misuse+of+standards+continues+to+proliferate+in+the+nascent+bioplastics+industry+space.">Misleading claims and misuse of standards continues to proliferate in the nascent bioplastics industry space.</a> BioPlastics. 01/10, (2010).</li><li>ASTM D 5338-98. <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+determining+aerobic+biodegradation+of+plastic+materials+under+controlled+composting+conditions.">Standard test method for determining aerobic biodegradation of plastic materials under controlled composting conditions.</a> Annual Book of ASTM Standards. , 504-509 (1998).</li><li>ASTM D 5988-03. <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+determining+aerobic+biodegradation+in+soil+of+plastic+materials+or+residual+plastic+materials+after+composting.">Standard test method for determining aerobic biodegradation in soil of plastic materials or residual plastic materials after composting.</a> Annual Book of ASTM Standards. , 354-358 (2003).</li><li>ASTM D 6954-04. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+guide+for+exposing+and+testing+plastics+that+degrade+in+the+environment+by+a+combination+of+oxidation+and+biodegradation.">Standard guide for exposing and testing plastics that degrade in the environment by a combination of oxidation and biodegradation.</a> Annual Book of ASTM Standards. , 748-753 (2004).</li><li>ASTM G21-96. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+practice+for+determining+resistance+of+synthetic+polymeric+materials+to+fungi.">Standard practice for determining resistance of synthetic polymeric materials to fungi.</a> Annual Book of ASTM Standards. , 433-437 (2002).</li><li>ISO 846. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics - evaluation of the action of microorganisms. , 1-22 (1997).</li><li>Russell, J. R., Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+of+polyester+polyurethane+by+endophytic+fungi.">Biodegradation of polyester polyurethane by endophytic fungi.</a> Applied and Environmental Microbiology. 77, 6076-6084 (2011).</li><li>Maeda, H., Yamagata, Y., Abe, K., Hasegawa, F., Machida, M., Ishioka, R., Gomi, K., Nakajima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+biodegradable+plastic-degrading+enzyme+from+Aspergillus+oryzae.">Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.</a> Applied Microbiology and Biotechnology. 67, 778-788 (2005).</li><li>Tokiwa, Y., Calabia, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradability+and+biodegradation+of+poly(lactide.">Biodegradability and biodegradation of poly(lactide.</a> Applied Microbiology and Biotechnology. 72, 244-251 (2006).</li><li>Karjomaa, S., Suortti, T., Lempi&#228;inen, R., Selin, J. -. F., It&#228;vaara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+degradation+of+poly-(L-lactic+acid)+oligomers.">Microbial degradation of poly-(L-lactic acid) oligomers.</a> Polymer Degradation and Stability. 59, 333-336 (1998).</li><li>Miles, C., Wallace, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deterioration+of+potentially+biodegradable+alternatives+to+black+plastic+mulch+in+three+tomato+production+regions.">Deterioration of potentially biodegradable alternatives to black plastic mulch in three tomato production regions.</a> HortScience. 47 (9), 1270-1277 (2012).</li><li>Hedh, J., Wallander, H., Erland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectomycorrhizal+mycelial+species+composition+in+apatite+amended+and+non-amended+mesh+bags+buried+in+a+phosphorus-poor+spruce+forest.">Ectomycorrhizal mycelial species composition in apatite amended and non-amended mesh bags buried in a phosphorus-poor spruce forest.</a> Mycological Research. 112, 681-688 (2008).</li><li>Wallander, H., Hagerberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+ectomycorrhizal+fungi+have+a+significant+role+inweathering+of+minerals+in+forest+soil?.">Do ectomycorrhizal fungi have a significant role inweathering of minerals in forest soil?.</a> , (2003).</li><li>Hehemann, J. -. H., Correc, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+structural+characterization+of+the+complex+agarolytic+enzyme+system+from+the+marine+bacterium+Zobellia+galactanivorans.">Biochemical and structural characterization of the complex agarolytic enzyme system from the marine bacterium Zobellia galactanivorans.</a> Journal of Biological Chemistry. 287, 30571-30584 (2012).</li><li>Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+marine+agar-digesting+bacteria.">Studies on marine agar-digesting bacteria.</a> Journal of Bacteriology. 42 (4), 527-559 (1941).</li><li>Hirsch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+life+at+extremely+low+nutrient+levels.">Microbial life at extremely low nutrient levels.</a> Advances in Space Research. 6, 287-298 (1986).</li><li>Wainwright, M., Adam, T., Barakah, 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+review+of+the+role+of+oligotrophic+micro-organisms+in+biodeterioration.">A review of the role of oligotrophic micro-organisms in biodeterioration.</a> International Biodeterioration and Biodegradation. 31, 1-13 (1993).</li><li>Wainwright, M., Barakah, R., Al-Turk, I., Ali, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligotrophic+micro-organisms+in+industry,+medicine,+and+the+environment.">Oligotrophic micro-organisms in industry, medicine, and the environment.</a> Science Progress. 75, 313-322 (1991).</li><li>Parkinson, S. M., Wainwright, M., Killham, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+oligotrophic+growth+of+fungi+on+silica+gel.">Observations on oligotrophic growth of fungi on silica gel.</a> Mycological Research. 93 (4), 529-534 (1989).</li><li>Hill, T., Kafer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+protocols+for+Aspergillus+minimal+medium:+trace+element+and+minimal+medium+salt+stock+solutions.">Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions.</a> Fungal Genetics Newsletter. 48, 20-21 (2001).</li><li>Hutner, S. H., Provasoli, L., Schatz, A., Haskins, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+approaches+to+the+study+of+the+role+of+metals+in+the+metabolism+of+microorganisms.">Some approaches to the study of the role of metals in the metabolism of microorganisms.</a> Proceedings of the American Philosophical Society. 94, 152-170 (1950).</li><li>Affeldt, K. J., Brodhagen, M., Keller, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillus+oxylipin+signaling+and+quorum+sensing+pathways+depend+on+G+protein-coupled+receptors.">Aspergillus oxylipin signaling and quorum sensing pathways depend on G protein-coupled receptors.</a> Toxins. 4, 695-6171 (2012).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Marzluf, G. A., Reddy, C. A., Beveridge, T. J., Breznak, J. A., Marzluf, G. A., Schmidt, T. M., Snyder, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology,+metabolism,+and+molecular+aspects+of+filamentous+fungi.">Physiology, metabolism, and molecular aspects of filamentous fungi.</a> Methods for General and Molecular Microbiology. , 952-964 (2007).</li><li>Peters, J. E., Reddy, C. A., Beveridge, T. J., Breznak, J. A., Marzluf, G. A., Schmidt, T. M., Snyder, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+in+Gram-negative+bacteria.">Gene transfer in Gram-negative bacteria.</a> Methods for General and Molecular Microbiology. , 735-755 (2007).</li><li>Yabannavar, A. V., Bartha, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+assessment+of+biodegradability+of+plastic+films+in+soil.">Methods for assessment of biodegradability of plastic films in soil.</a> Applied and Environmental Microbiology. 60 (10), 3608-3614 (1994).</li></ol>

作者宣称，他们有没有竞争经济利益。

这里所描述的步骤，表示第一通潜在BDM从土壤中降解菌的分离技术，并已成功用于分离真菌从埋在土壤中为7个月的BDMS。真菌生长时，再接种到新鲜的的BDM材料的相同类型的，表明分离的真菌确实殖民者，薄膜不抑制真菌生长。塑料降解真菌和细菌的分离可能导致它们的使用，单独或组合使用，修订土壤或堆肥塑料需要被降解。 在他们的原生土壤环境中，微生物物种中不存在...

历来被认为是降解塑料聚合物的不良属性，，因为击穿缩短产品的寿命和耐久性。近日，废塑料在自然环境1,2,3环境问题的意识已经由生物可降解塑料有吸引力的替代传统的塑料材料。退化（定义为结构的变化，碎裂，和分子量降低，完整性，和强度4,5）通过一系列的事件，包括非生物过程（热应力，光氧化，水解，侵蚀和机械应力）发生，和生物降解。虽然非生物过程可以改变片段的大小和特性的塑料，微生物所需的最终矿化水和二氧化碳（在有氧条件下）和/或甲烷（在厌氧条件下）。 有相当利基生物降解性塑料存在于农业，塑料覆盖物的使用，以防止杂草生长，保持土壤水分，增加土壤温度7,8。仅在美国，数千英亩的数百覆盖有塑料覆盖物，包括可生物降解的塑料覆盖物组成。在作物生长季节，出售的可生物降解的覆盖物（BDMS）的选项包括出售在一个垃圾填埋场，焚烧能量回收10，通过堆肥降解或降解在土壤耕作后11。其中，至少劳动力密集型的命运春耕BDMS到土壤中，但没有有效的降解和矿化在非作物个月期间（一般在冬季），塑料碎片可能保持和干扰农业设备在春季耕作和种植，坚持环境，让他们显着影响的野生动物，植物的生命和微生物1,2,3,10。 12。在冬季，大多数地区的土壤温度将低于这些温度，可能导致甚至更低的微生物的活动，因此，矿化。除了 ​​减缓退化率，误用术语“生物降解”，导致这些产品的不信任，消费者13,14，包括那些在农业产业。生物降解转换二氧化碳（和/或甲烷）和水14由自然发生的微生物4的聚合物。因此，生物降解必须的化学计量;微生物与基材的物理缔合，并不意味着该材料的微生物降解。 审查可持续利用农业BDMS的努力的一部分，这个研究主要集中在发现原生微生物农业定植和降低市售BDMS的的的的土壤。已经出版了化学测量破裂的生物降解塑料，生物和非生物的手段15,16,17的标准试验方法。然而，这些方法都没有解决个别微生物物种的降解塑料，或它们的隔离方法。本文的方法更接近于标准方法旨在真菌孢子接种标本后评估抗微生物击穿塑料18,19。 当塑料的配方是已知的，原料的来源提供的，粉状塑料，可以暂停由可视化结算区13在以琼脂为基础的媒体和退化。该方法已被用于先前识别微生物降解的聚（丁二酸丁二醇酯- 共 -己二酸酯）的聚合物，如聚氨基甲酸酯20，21，和聚（乳酸）22。类似的方法，包括纯塑料粉末悬浮在液体培养基中，塑料是唯一碳源20,23。虽然这些方法有一个定义的系统的优点是，他们不良模仿BDMS 原位降解。首先，BDMS，因为没有分散在整个土壤基质中的小颗粒，而是，出售，用作薄膜的表面积不同的分布。其次，不同于纯聚合物的化学组成BDMS的。 BDMS通常含有添加剂，如填料，增塑剂，着色剂，这些添加剂可能会影响微生物的生长，从而，矿化率。出于这个原因，因为在这项研究中的某些商业片的组成是专有的，在现场准备的塑料薄膜，是用来隔离真菌和细菌。为简单起见，在下面的方法，仅描述为真菌，经过修改的注意到在适当的情况下对细菌隔离。 在最近的一项研究24，三市售BDMS的一个实验电影在美国的三个不同区域的农业网站，一个生长季节，随后放置在目（250微米）袋和埋在土壤中的一个冬天在同一站点。 250微米筛孔允许真菌菌丝穿透，同时不包括根和大多数土壤动物，并尽量减少土壤侵蚀25,26。尼龙材料，防止塑料袋在​​土壤中的降解。经过挖掘，Fungal菌株中回收由BDM件和没有碳的来源除了琼脂和5厘米×5厘米表面的灭菌新的未使用的的BDM膜，预灭虫平方评估在基本培养基上生长。大多数塑料用作电影不能高压灭菌不丧失诚信，所以UV光被用来杀死任何塑料上的微生物细胞。 ISO 846 19建议在70％乙醇和随后的干燥表面灭虫，但如果使用此方法，必须确保没有的薄膜的组分或添加剂带来不利影响，乙醇。由于BDMS大概制造承受阳光，紫外线被选为去污方法。 BDM片优于仅在基本培养基上生长的菌株作进一步研究。琼脂，海藻产生的多糖，是用来凝固微生物的介质，因为它通常是利用代谢农业和医学上不能力的微生物，但是，琼脂水解酶已被分离自海洋细菌27和28从土壤中也已被隔离的水解琼脂细菌。有BDM聚合物与琼脂均是罕见的土壤真菌，包含这些聚合物作为潜在的营养源的环境中，还没有发展所分泌的酶的底物，但本文所述的（ 步骤7）中的板生物测定，存在两个基板。使用的BDMS，但不琼脂作为碳源的真菌可以区别于真菌只使用琼脂，通过比较生长在琼脂固体培养基含有琼脂培养基（阴性对照）， 二除外）没有添加碳源）BDM片（实验）以及iii）葡萄糖（阳性对照）。预期生长的影响所有菌株在基本培养基上附加葡萄糖真菌所产生的葡萄糖的板可能有其他的实验中所用的特定的基本培养基中生长的能力。电位人BDM降解菌生长在琼脂固化的基本培养基+ BDM电影更好，比他们单独琼脂凝固的基本培养基上生长。培养基平板上生长的真菌是琼脂降解菌或oligotrophs的，预计也将生长在琼脂与BDM电影在生物测定板，而不是在自己的电影（除非他们偶然也会降低BDM聚合物）。 为了消除可能看到到利用琼脂和不BDMS的由于微生物的生长，我们遵循我们的初步检测BDM定植琼脂平板上定义的肉汤培养基（ 第9步 ）生物测定。 BDM枚较的生物测定管中已知的唯一的碳源。 经过初步筛选，并呼吁恢复甘油股票的菌株，有些形成很少，但可见菌丝体液体介质中含有任何已知的碳源。这些结果表明，一些所获取的菌株oligotrophs -生物体生长的清除非常少量的碳，氮，和其他营养物质溶解在含水环境中的或现有的在空气中的挥发物29,30,31。物种鉴定通过18S核糖体DNA分析支持这一观点，许多相匹配的菌株真菌属先前报道表现出贫营养32。 ，这是常见的腐生oligotrophs，范围广泛的环境中30的范围内的底物利用的代谢能力的要求。因此，这并不奇怪，我们孤立从BDMS（大概需要不寻常的酶的能力）相同的真菌表现出贫营养的能力，并能够成长的痕量污染物如皮肤沾染指纹，灰尘，或在空气中的微量挥发油。由于隔离oligotrophs，我们得出的结论是不能单独用来推断BDM击穿增长一个BDM表面上。本文所述的方法反映我们的努力，SCREEN本地的BDM殖民者从农业土壤善意 BDM击穿。

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potato Dextrose Agar</td>
 <td>Becton Dickinson</td>
 <td>8X05491</td>
 <td></td>
 </tr>
 <tr>
 <td >Agar</td>
 <td>Fisher</td>
 <td>BP 1423-2</td>
 <td></td>
 </tr>
 <tr>
 <td >Chloramphenicol</td>
 <td>Acros Organics</td>
 <td>200-287-4</td>
 <td></td>
 </tr>
 <tr>
 <td >Glutaraldehyde</td>
 <td>Electon Microscopy Sciences</td>
 <td>16216-10</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td >Molecular sieve</td>
 <td>Fisher </td>
 <td>M-8892</td>
 <td></td>
 </tr>
 <tr>
 <td >Ethanol</td>
 <td>Pharmco-Aaper</td>
 <td>E200</td>
 <td></td>
 </tr>
 <tr>
 <td >Contrex</td>
 <td>Decon Labs, Inc.</td>
 <td>5204</td>
 <td></td>
 </tr>
 <tr>
 <td >Parafilm M</td>
 <td>Pechiney Plastic Packaging</td>
 <td>S37440</td>
 <td></td>
 </tr>
 <tr>
 <td >Mineral salts for buffers and media</td>
 <td>Fisher</td>
 <td>Various</td>
 <td>Various vendors sell these reagents</td>
 </tr>
 </tbody>
</table>

isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture

原产于的农业土壤殖民市售的生物降解地膜（BDM）薄膜的真菌的分离和降解塑料的潜力进行评估。通常，当塑料的配方是已知的，原料的来源提供的，粉末状的塑料可以悬浮在以琼脂为基础的媒体和退化，由可视化的信息交换区。然而，这种方法不佳模仿BDMS 原位降解。首先，BDMS不分散作为整个土壤基质的小颗粒。其次，BDMS商业销售作为纯的聚合物，而是作为含有添加剂（ 如填料，增塑剂，染料），可能会影响微生物的生长的薄膜。本文所述方法，用为收购土埋地膜的菌株。真菌菌株从挖掘BDMS获得被单独测试的增长奠定了新的，灭虫BDMS的定义中不含碳源电子顶上件琼脂XCEPT。菌株进一步测试增长BDMS的液体培养基中，BDMS唯一碳源。约10周后，真菌和BDM退化评估通过扫描电子显微镜。通过核糖体RNA基因序列的分析，分离和鉴定。本报告介绍了真菌隔离的方法，但细菌也适合细菌代媒体使用这些方法进行分离。我们的方法应该是有用的研究完整的塑料薄膜或产品，塑料原料是未知或不可用故障。然而我们的方法并不提供定量的方法比较BDM降解率。

在最近的一项研究24个重复每三个市售BDMS的标有“可降解”，再加上一部实验电影和传统塑料控制，放在土壤覆盖番茄生产在2010年的春天在华盛顿州Mount Vernon，诺克斯维尔，田纳西州，德克萨斯州拉伯克。在2010年的秋天，BDM电影广场被切断每个风化覆盖4个重复的情节，乡土辗转直接从下方的区域覆盖物样品已切除。每个风化BDM方形夹在土壤内的尼龙网袋，如步骤1所述。只?...

Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

塑料薄膜标有“可降解”市售作为覆盖物用于农业用途。耕作是一个有吸引力的处置方法，但在野外条件下的降解是知之甚少。这项研究的目的是开发用于隔离本地土壤真菌和细菌后场埋葬殖民塑料地膜的方法。

隔离原住民土壤微生物潜力打破生物降解塑料地膜应用于农业

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

isolation-native-soil-microorganisms-with-potential-for-breaking-down

Research

JoVE Journal

Biology

30.4K 次观看.  Western Washington University. 塑料薄膜标有“可降解”市售作为覆盖物用于农业用途。耕作是一个有吸引力的处置方法，但在野外条件下的降解是知之甚少。这项研究的目的是开发用于隔离本地土壤真菌和细菌后场埋葬殖民塑料地膜的方法。

视频: Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

塑料嵌入和非洲爪蟾胚胎切片

科研

生物学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

复合植物代苜蓿truncatula</em用于检测结瘤

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously applied herbicides has presented the need for additional management methods. Allelopathy is a physiological process that some plant species possess that provide the plant with an advantage over its neighbors. Allelopathic crop varieties would be equipped with the ability to suppress the growth of surrounding competitors, thus reducing potential yield loss due to weed interference. This paper focuses on the construction and operation of a stair-step assay used for the screening of the allelopathic potential of a donor species (Oryza sativa) against a receiver weed species (Echinochloa crus-galli) in a greenhouse setting. The structure described in this paper serves as a stand for the plant samples and incorporates a timed watering system for the accumulation and distribution of allelochemicals. Allelochemicals produced by the plant roots are allowed to flow downward through a series of four pots separately into a collection tank and recycled back to the top plant through electric pumps. This method of screening provides an avenue for the allelochemicals from the donor plant to reach receiver plants without any resource competition, thus allowing quantitative measurement of the allelopathic potential of the selected donor plant. The allelopathic potential is measurable through the height reduction of the receiver plants. Preliminary screening data for the effectiveness of this method demonstrated height reduction in the receiver species, barnyardgrass (E. crus-galli), and thus the presence of allelopathic residues from the donor plant, weedy rice (Oryza sativa).

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

Allelopathy has shown promise as a useful supplemental weed control strategy in cropping systems. To determine the allelopathic potential of a desired plant specimen, a stair-step screening method is provided.

可重复的阶梯步骤测定，以访问杂草水稻的过敏性潜力（Oryza sativa ssp.）

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...