This work was funded through the U.S. Army Engineer Research and Development Center, Basic Research Program Office. Permission for publishing this information has been granted by the Chief of Engineers.

1.设备的准备和冻土核心种质<ol><li>设备的准备和现场样品采集和保存减速<ol><li>通过将驱动器适配器插入筒的顶端和转动杠杆锁定到位组装为样品采集螺旋钻。针适配器管到驱动适配器和电机引脚到适配器管。插入桶上的刀具。 </li><li>穿轻型服，丁腈手套，口罩和任何污染，减少对样品。耳罩和在进入隧道冻土（ 图1）安全性的安全帽。 </li><li>进入隧道，并选择收集样本（ 图1B）的位置。 注意:对于垂直或水平钻孔位置，选择已知有证据表明，该材料被冻结（ 例如 ，冰或多年冻土）的区域，也没有已知的大根系统，并且没有已知的砾石。移除所有从生活区的植物材料收集样品之前。如果垂直或水平钻孔中，选择相对平坦，并与螺旋推运器可访问的区域。 </li><li>通过与已用70％的异丙醇，DNA的去污溶液，和RNase去污溶液的塑料材料成层地制备工作站。 </li><li>放置纵向在工作站上半个直径10厘米的聚氯乙烯（PVC）管切割，以提供一个槽，因为它们是从螺旋推运器除去以保持芯。除污PVC管，用70％的异丙醇，DNA净化解决方案，和RNase净化解决方案。 </li><li>附近的工作站，用70％的异丙醇，DNA净化解决方案，并核糖核酸酶净化解决方案喷洒消毒螺旋。从与擦拭螺旋钻移除的解决方案。 </li></ol></li><li>冰和冻土核心的采集和存储<ol><li>选择墙样品的面积（参见1.2.1注意）。 </li><li>用70％的异丙醇，DNA净化解决方案，并核糖核酸酶净化解决方案擦拭消毒冻结壁面积约直径10厘米。 </li><li>提升净化螺旋推运到感兴趣的区域，使得其垂直于样品区，并开始钻入壁（ 图1C，D） ​​的清洁面。 </li><li>小心地从样品采集区域的螺旋。断开电机螺旋并将无菌的PVC管上面的螺旋在洁净工作台。小心倾斜螺旋推运器，使得冷冻芯滑出到无菌的PVC管（ 图1E）。 </li><li>净化用70％的异丙醇，DNA净化解决方案，并核糖核酸酶净化解决方案手套。 </li><li>拿起冰和冻土内核和无菌手套，将它们放入无菌袋。 </li><li>放置在核心凉爽或寒冷的房间里，直到它们被运或处理。 </li><li>嘘的ip使用干冰以保持它们在低于0℃的温度下冷冻芯。 </li><li>紧随核储存在-80℃。 </li></ol></li></ol> 2，多年冻土和冰芯处理<ol><li>备料<ol><li>通过在450℃下进行4小时的烘箱中烘烤制备无菌的，核酸游离重负荷铝箔，金属架，玻璃器皿，金属镊子，和玻璃棉。直至使用抛开这些材料。 </li><li>通过0.22微米过滤器传递溶液消毒95％乙醇和超纯水。 </li><li>在-20℃和水商店乙醇溶液在4℃下。 </li><li>通过用70％乙醇，DNA的去污溶液，和RNase去污溶液喷洒和各溶液后，立即擦拭消毒一个塑料尺。 </li></ol></li><li>细菌培养制备<ol><li>准备肉汤和板成长粘质沙雷氏菌 。 <ol><li>对于肉汤，结合5克试ptone，1克葡萄糖，5克酵母提取物和1g磷酸氢二钾，然后加蒸馏水至达到1L。为了使板，琼脂15克的加入到上述混合物。对于肉汤和板材，调节pH至7和高压釜在121℃下15分钟。 </li></ol></li><li>通过组合8克氯化钠，0.2克氯化钾，1.44加入磷酸氢二钠的，及0.24g的磷酸二氢钾制备1X磷酸盐磨光盐溶液（PBS）中，并加蒸馏水至达到800毫升调节pH至7.4，并添加蒸馏水于121℃以达到1升高压灭菌15分钟。 </li><li>通过浸入S.接种用无菌环肉汤沙雷氏菌培养以前存储在-80℃下冷冻。在无菌条件下，串行稀加入1 ml培养至9毫升的PBS并手动培养反转溶液。 1ml该稀释液加入到9毫升的PBS并手动反转溶液。重复八点多，直到10 -9 DILU达到化。 </li><li>通过1毫升肉汤散发到板，并用细胞吊具扩频传播10 -6，10 -7，10 -8和10 -9解到琼脂平板上。每稀释三次做到这一点。在4℃保存原有的文化。 </li><li>在30℃孵育板24小时。计数菌落数来计算在原始培养细胞的数目。注意:菌落在原始培养的数量将依赖于细菌的生长速度。 </li><li>吸取250微升原生态文化的进入无菌1.5 ml离心管。通过添加1×PBS中的足够量如通过菌落计数在先前步骤中确定的，以获得约10 9个细胞/ ml稀释原始培养。通过以2,500 xg离心离心10分钟沉淀在离心管的细胞。 注:1×PBS中的量将依赖于细菌的生长速度各不相同。 </li><li>在无菌biohood，吸取了肉汤和悬浮细胞在1毫升1×PBS缓冲液。储存在4℃直至使用或约2个月。 </li><li>上处理的当天，淡化S.通过加入1ml的原始S的从步骤2.2.7 沙雷氏菌培养沙雷氏菌培养至39毫升1×PBS缓冲液在50毫升离心管中。 </li></ol></li><li>准备冷室空间，核心处理和保存齿轮<ol><li>前冷却的冷室，洁净的墙壁，地板和金属表用1％漂白溶液中。 </li><li>冷室的设定温度至约-11℃。 </li><li>一旦冷室已达到所需温度时，穿轻型服，腈手套和口罩，以任何污染减少到冷室和样品。 注:两个身着正常个人需要这种方法。 </li><li>把无菌材料 （ 如铝箔，金属架，玻璃器皿，盘， 粘质沙雷氏菌文化，MIcrotome刀片）和样品入冷室，地点在无菌表。 </li></ol></li><li>多年冻土和冰核处理在寒冷的房间设施<ol><li>放置一个永久冻土芯上的无菌的，核酸自由铝箔的片材。 </li><li>轻轻接种芯的外侧与S的稀培养沙雷氏菌使用无菌泡沫塞（ 图2B）。 </li><li>放在一个盘上方坐在无菌金属机架的核心。 </li><li>消毒用70％乙醇，DNA的去污溶液，和RNase去污溶液钢切片机刀片。 </li><li>有70％的乙醇，DNA净化解决方案，并核糖核酸酶净化解决方案个体干净丁腈手套，并在45°角的盘上方持有的核心。 </li><li>有个人乙轻轻刮去约5mm的整个芯的外侧的，包括端部，使用无菌刀片而个人A熄灭各刮后的核心（ <str翁&gt;图2C和图3A，B）。根据需要擦拭用70％的乙醇，DNA的去污溶液，和RNase去污溶液刀片。 </li><li>有个人乙小心，并迅速倒入过滤除菌的95％的乙醇对核心，而个人A熄灭的溶液倾倒芯（ 图2D，3C）。 </li><li>有个别乙立即冲洗用过滤灭菌的水的核心。 </li><li>用新的无菌金属架更换托盘使用的金属架。 </li><li>有个别A以45°角的托盘上方清理他或她的丁腈手套用70％乙醇，DNA净化解决方案，并核糖核酸酶净化解决方案和保持核心。 </li><li>有个别乙喷洒DNA净化解决方案的整个核心。 </li><li>有个别乙立即冲洗用过滤灭菌的水的核心。 </li><li>有个别乙喷洒核糖核酸酶净化解决方案的整个核心。 </li><li>有Individu人乙立即冲洗用过滤灭菌的水的核心。 </li><li>放置在铝箔的无菌片芯和轻轻包。 </li></ol></li><li>解冻外核<ol><li>将在无菌玻璃皿无菌金属架在层流无菌biohood。 </li><li>将具体到S. 2琼脂平板沙雷氏菌在无菌机架下面的玻璃皿从核心（ 图2E）收集液体。 </li><li>放置在无菌的金属架的核心。 </li><li>允许约2-5毫米芯的外表面，以在23℃解冻（平均，这将约10分钟内发生）。转芯大约每2分钟90°。 </li><li>使用无菌镊子和无菌玻璃棉拭芯的整个表面上，并接种2琼脂平板特定至S.由这些材料擦拭到板的表面沙雷氏菌 。接种与原来的文化两个新的板块用于掺杂的外芯，将其暴露于冷室的低温条件下。 </li><li>通过将无菌标尺附近，但不触及核心测量解冻圆柱形芯的外形尺寸。 </li><li>将核心成大无菌袋，并将其存储在-80℃。 </li></ol></li><li>检查增长<ol><li>在23℃孵育琼脂平板一周。 </li><li>检查S的生长琼脂平板沙雷菌菌落。 <ol><li>如果有在平板上没有可见的菌落，继续执行步骤2.5.3。 </li><li>如果菌落出现在平板，从步骤2.1.1第2条"冻土和冰芯处理"重复。 </li></ol></li><li>获得从冷冻芯，并无菌转移到一个无菌袋。 </li><li>存储在无菌袋中的芯在4℃下约24-48小时，解冻整个核心。 </li></ol></li></ol> 3.获取子样本从冰芯和冻土核酸提取<ol><li>从冰芯核酸提取<ol><li>轻轻摇动袋（ 图2G）混合从冰芯解冻材料。 </li><li>倒入解冻材料放入无菌容器用0.22微米的过滤器在真空下收集的微生物。 </li><li>无菌取出用无菌镊子过滤，并将其放置在无菌2毫升陶瓷珠管（1.4毫米）。 </li><li>储存在-80℃的过滤器。 </li></ol></li><li>冻土核核酸提取<ol><li>通过轻轻揉捏袋子从混合冻土核心解冻材料。 </li><li>多年冻土已充分混合后，得到大约为0.5 g整体土壤的子样本用无菌scoopula并将其放置在该直立坐在一个平衡配衡无菌2毫升陶瓷珠螺旋盖管（1.4毫米）。 </li><li>店子样本在-80℃。 </li><li>获取样本的重量含水量。 <ol><li>措施10克永久冻土带与不孕在天平上去皮重的锡Ëscoopula和地点。测量永久冻土和锡的湿重。 </li><li>在24小时设定在105℃的烘箱孵育永久冻土。测量永久冻土和锡干质量。 </li><li>由永久冻土层的干质量减去冻土的湿重和多年冻土的干燥质量除以计算重量含水量。 </li></ol></li><li>在-80°C储存永久冻土带。 </li></ol></li></ol> 4.永久冻土和冰芯中提取核酸<ol><li>材料准备<ol><li>制备琥珀小瓶通过用0.1％焦碳酸二乙酯（DEPC）处理，在37℃，高压灭菌孵育O / N保存的解决方案。 </li><li>制备240 mM磷酸钾缓冲液。添加2.5克氢磷酸二氢和38.7克磷酸氢二钾的。通过加入无菌水调节终体积为500毫升。 </li><li>准备溴化十六（CTAB）提取BUFFER 4.1 5g氯化钠溶解在80毫升水中，慢慢加入10克CTAB同时加热和搅拌。通过加入无菌水调节最终体积至100ml。 </li><li>添加的磷酸缓冲液等体积​​的CTAB缓冲液和过滤用0.22微米的过滤器消毒。盖瓶用铝箔和储存在室温。 </li><li>通过组合9.35克氯化钠和100ml无菌水制备1.6M的氯化钠（NaCl）溶液。加入10克聚乙二醇8000至1.6M的NaCl溶液和过滤消毒。保存在4℃。 </li></ol></li><li>根据Griffiths 等人的修改的版本的核酸提取22和TOWE 等人 23 <ol><li>穿轻型服，丁腈手套，和口罩。干净的实验室空间并用70％的乙醇，DNA的去污溶液，和RNase去污溶液移液管。 </li><li>在2毫升删除子样本（来自冰芯或永久冻结带的土壤样品过滤器），陶瓷珠螺-CA从-80℃冷冻室和解冻在RT p管。 </li><li>将0.5ml溴化十六（CTAB）提取液，涡旋简要的。 </li><li>将0.5ml酚 - 氯仿 - 异戊醇（25:24:1）（pH 8）中，并使用上的涡旋平板适配器水平摇晃试管10分钟。 </li><li>在4℃以下珠跳动，离心管在16100 XG 5分钟。 </li><li>到一个新的无菌1.5毫升管除去水层，并用等体积的氯仿 - 异戊醇混合（24:1）。离心16100 XG在4℃下5分钟。 </li><li>到一个新的无菌1.5毫升管除去水层，加入30％的聚乙二醇8000和1.6 M氯化钠的两卷。孵育在4℃下2小时。 </li><li>离心机在16100 xg离心在4℃下10分钟。 </li><li>于4℃在16100 xg离心用约500微升冰冷的70％乙醇和离心机的洗涤核酸沉淀10分钟。 </li><li>在2小时的无菌biohood空气干燥颗粒。重悬在50μlDNA酶/无RNase的TE缓冲液（pH8.0）中。 DNA可用于下游应用如PCR和qPCR。 </li><li>确定的DNA的浓度用分光光度计。 </li><li> DNA稀释用无菌，不含DNA的水达到每20反应纳克。 </li></ol></li></ol>

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The authors have nothing to disclose.

冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。虽然恢复类群可能不是完整的历史社会，来自冰川和冻土样品的分析中回收可产生约选择时间段15-16宝贵的历史资料。举例来说，有意义的生物信息已经从冰研究在格陵兰冰盖考察20厌氧活性和冻土试验研究了碳循环过程的解冻33的结果绘制的，并已在白令陆桥16提供了深入了解真菌的多样性冻土。之前进...

冰雪圈（ 例如 ，永冻层土壤，冰功能，冰川积雪，积雪和冰）提供了一个窥探到过去的环境条件下坚持什么类型的生物。由于这些基材可以是几十到几十万岁，他们的微生物群落，因为沉积在冷冻保存，反映古环境条件。要正确分析这些生态系统和提取冷冻样品的冻土和冰块，正确的收集和处理有意义的生物信息是必要的。这是最重要的，因为气候预测为21 世纪指示北极和亚北极地区1有明显的变暖的潜力。具体来说，内政部阿拉斯加和格陵兰预期升温5℃左右，并分别在2100年2,3 7℃。预计这将影响显著土壤和水生微生物群落，因此，相关的生物地球化学过程。温暖的温度和降水改变政权有望开始在许多领域2-5冻土退化可能导致较厚，季节性解冻（活动）6,7层，冻土的解冻，和大量的冰体，如熔化地下冰，冰楔，和隔离冰8。这将极大地改变除了这些生态系统中的植物和动物的生物多样性生物地球化学属性。 冰川和永冻层同生沉积物和冰的功能已经被困化学和代表的环境中生活是什么在那里形成的功能时的生物学证据。例如，在内部阿拉斯加，既Illinoisan和威斯康星州的永久冻土岁都存在这个永久冻土尤其在本（YBP）包含IMPA的生物地球化学的证据之前，提供了从现代到15万年约会不同的位置对生物多样性过去气候变化克拉。其结果是，这些沉积物提供生物地球化学和生物多样性的记录了数千年。由于该区域具有低的沉积速率，并从来没有被冰川覆盖的，不受干扰的样本是收集和分析，无论是钻访问垂直插入隧道土壤剖面或钻孔水平。更重要的是，大量的记录存在的突出特别是在该地区永久冻土9-14的独特生物地球化学特征。具体来说，DNA分析的应用，同时估计现存的古冰和永冻土样本中的生物多样性的存在和程度使古环境条件特定生物联动和栖息地的职业探索。 以前的研究已经确定在哺乳动物，植物和可追溯至50K YBP 11，15-19样品微生物气候影响，虽然每个研究使用各色一nt的方法来收集和净化冻土和冰芯。在某些情况下，钻芯灭菌16，20-21，虽然具体的方法没有澄清是否外来核酸还从样品中消除。在其他研究中，细菌分离物15（ 例如 ， 粘质沙雷氏菌 ），以及22已被用于测量的净化程序的功效荧光微球。 这个实验是一个大型研究调查冻土样本可以追溯到大约40K YBP微生物群落的组成部分。研究这部分的具体目标是成功地净化冰和冻土内核。就我们所知，没有任何方法集成了使用而设计的，以消除从冷冻芯的外侧部分外来核酸和相关的核酸溶液。尽管这是一个事实，这些解决方案是commonlŸ用于净化实验室设备分子实验。 一旦芯进行了去污，使用由Griffiths等 23和TOWE 等人 24开发的协议提取基因组DNA，使用分光光度计进行定量，并用无菌，无DNA的水稀释至达到每反应20纳克。细菌的16S rRNA基因用引物331F及797R扩增和探针BacTaq 25和古16S rRNA基因用引物拱349F和拱806R和在下列条件下探测TM拱516F 26扩增:95℃600秒，随后45个循环95℃，30秒，57℃60秒，72℃，在40℃，30秒下进行25秒，最后延伸。所有的qPCR反应中重复进行。在20微升的反应体积包含20 ng的DNA，引物10μM的，探针的5微米，和10微升的qPCR反应混合物。 FO标准- [R采用基因组DNA分别假单胞菌荧光和嗜盐salinarum，细菌和古细菌定量PCR制备。两者中生长至对数期。平板计数进行了与DNA从培养物中分离。与一个的假设和每个基因组为H的16S rRNA基因的六份分光光度计基因组DNA进行定量salinarum和P.荧光 ，分别为27-28。细菌和古细菌的基因的拷贝数是基于标准曲线上计算出的，对数转换，以占治疗之间不等方差，并通过ANOVA评估。 群落的组合物通过使用流动池和桥扩增技术测序16S rRNA基因，并与"定量分析上市微生物生态学&#39;（QIIME）29分析该社区确定。正向和反向读取被连接在一起，然后序列进行过滤，索引，并选择高品质的代表从头操作分类单元（OTU）通过序列比对分配有一个参考数据库。比对的序列进行了比较，对分类分配一个单独的参考数据库。一个门级OTU表的建立是为了确定一般群落组成。

<table border="0" cellpadding="0" cellspacing="0" width="1084">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Auger</td>
			<td style="width:300px;">Snow, Ice, and Permafrost Research Establishment (SIPRE), Fairbanks, AK</td>
			<td style="width:161px;">N/A</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropanol</td>
			<td style="width:300px;">Walmart</td>
			<td>551116880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">95% Ethyl Alcohol (denatured)&#160;</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">A407-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA decontamination solution, DNA Away</td>
			<td>Molecular Bio-Products, Inc., San Diego, CA</td>
			<td style="width:161px;">7010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase decontamination solution, RNase Away</td>
			<td>Molecular Bio-Products, Inc., San Diego, CA&#160;</td>
			<td style="width:161px;">7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light Duty Suits</td>
			<td style="width:300px;">Kimberly-Clark Professional, Roswell, GA</td>
			<td style="width:161px;">10606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrile Gloves</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">FFS-700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antiviral Masks</td>
			<td style="width:300px;">Curad, Walgreens</td>
			<td style="width:161px;">CUR3845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Sample Bags&#160;</td>
			<td>Nasco, Fort Atkinson, WI</td>
			<td style="width:161px;">B01445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Steel Microtome Blade&#160;</td>
			<td style="width:300px;">B-Sharp Microknife, Wake Forest, NC</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal Rack</td>
			<td style="width:300px;">Fabricated at CRREL, Hanover, NH</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Tray</td>
			<td style="width:300px;">Handy Paint Products, Chanhassen, MN</td>
			<td style="width:161px;">7500-CC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Aluminum Foil</td>
			<td style="width:300px;">Western Plastics, Temecula, CA</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">500 ml Bottle with 0.22 &#956;m Filter</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">430513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serratia marcescens&#160;</td>
			<td style="width:300px;">ATCC, Manassas, VA</td>
			<td style="width:161px;">17991</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Biosafety Hood</td>
			<td style="width:300px;">NuAire, Plymouth, MN</td>
			<td style="width:161px;">NU-425-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri Dish</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATCC Agar 181- Tryptone</td>
			<td style="width:300px;">Acros Organics, NJ</td>
			<td style="width:161px;">61184-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATCC Agar 181- Glucose</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP381-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATCC Agar 181- Yeast Extract</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP1422-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATCC Agar 181- Dipotassium Phosphate</td>
			<td style="width:300px;">JT Baker, Phillipsburg, NJ</td>
			<td style="width:161px;">3252-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATCC Agar 181- Agar</td>
			<td style="width:300px;">Difco, Sparks, MD</td>
			<td style="width:161px;">214530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">NanoDrop 2000 UV Vis Spectrophotometer</td>
			<td style="width:300px;">Thermo Fisher Scientific, Wilmington, DE</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Lightcycler 480 System</td>
			<td style="width:300px;">Roche Molecular Systems, Inc., Indianapolis, IN</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Halobacterium salinarum</td>
			<td style="width:300px;">American Type Culture Collection (ATCC), Manassas, VA</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Pseudomonas fluorescens&#160;</td>
			<td style="width:300px;">American Type Culture Collection (ATCC), Manassas, VA</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Microbial DNA Isolation Kit</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">12224-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Ear Protection</td>
			<td style="width:300px;">Elvex</td>
			<td style="width:161px;">EP-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hard Hat</td>
			<td style="width:300px;">N/A</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Kimwipes</td>
			<td style="width:300px;">Kimberly-Clark Professional, Roswell, GA</td>
			<td style="width:161px;">34705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Glass Wool</td>
			<td style="width:300px;">Pyrex</td>
			<td style="width:161px;">430330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Ruler</td>
			<td style="width:300px;">N/A</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Weighing Tin&#160;</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">08-732-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sodium chloride</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">S-9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Potassium chloride</td>
			<td style="width:300px;">JT Baker, Phillipsburg, NJ</td>
			<td style="width:161px;">3040-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Potassium phosphate, monobasic</td>
			<td style="width:300px;">JT Baker, Phillipsburg, NJ</td>
			<td style="width:161px;">&#160;3246-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Potassium phosphate, dibasic</td>
			<td style="width:300px;">JT Baker, Phillipsburg, NJ</td>
			<td>3252-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sodium phosphate dibasic, anhydrous</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP332-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">50 ml Centrifuge Tubes</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">4558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">2 ml Microcentrifuge Tubes</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">1200-250-T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">2 ml Ceramic Bead Tubes (1.4 mm)</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">13113-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Scoopula</td>
			<td style="width:300px;">Thermo Fisher Scientific, Wilmington, DE</td>
			<td style="width:161px;">1437520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Balance</td>
			<td style="width:300px;">Ohaus, Parsippany, NJ</td>
			<td style="width:161px;">E12130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diethylpyrocarbonate (DEPC)</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">D5758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexadecyltrimethylammoniabromide (CTAB)&#160;</td>
			<td style="width:300px;">Acros Organics, NJ</td>
			<td style="width:161px;">22716-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol 8000&#160;</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">P5413-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8)&#160;</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP1752-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:300px;">Eppendorf, Hauppauge, NY</td>
			<td style="width:161px;">5417R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform-isoamyl alcohol (24:1)</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">C0549-1PT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">TE Buffer</td>
			<td style="width:300px;">Ambion (Thermo Fisher), Wilmington, DE</td>
			<td style="width:161px;">AM9860</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Pipets</td>
			<td style="width:300px;">Rainin, Woburn, MA</td>
			<td style="width:161px;">Pipet Lite XLS, 2, 10, 200, 1nd 1000ul pipets</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:439px;">Pipet tips</td>
			<td style="width:300px;">Rainin, Woburn, MA</td>
			<td style="width:161px;">Rainin LTS presterilized, low retention, filtered tips, 10, 20, 200, 1000ul</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Vortexor</td>
			<td style="width:300px;">Scientific Industries, Bohemia, NY</td>
			<td style="width:161px;">G-560</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Vortex Adaptor</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">13000-V1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Clear Bottle</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">C1395500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Amber Bottle</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">C5135250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Bottle Top Filters, 0.22um</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">430513</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">60 mL Syringe</td>
			<td style="width:300px;">Becton, Dickenson and Company, Franklin Lakes, NJ</td>
			<td style="width:161px;">BD 309653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Millex Syringe filters, 0.22 &#956;m</td>
			<td style="width:300px;">EMD Millipore, Billerica, MA</td>
			<td style="width:161px;">SLGV033RB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">70% Ethanol</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP2818-500</td>
			<td>diluted &#38; filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Isotemp 100 L Oven</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">151030511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Cell Spreader</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">08-100-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Disposable Inoculating Loops</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">22-363-602</td>
			<td></td>
		</tr>
	</tbody>
</table>

removal of exogenous materials from the outer portion of frozen cores to investigate the ancient biological communities harbored inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

该方法可用于净化各种冰冻圈环境中收集的冷冻样品，从冰川冻土到。在这里，我们提出从工程技术研究开发中心收集冰和永冻土样本专门收集的数据-寒冷地区研究和工程实验室（ERDC-CRREL）冻土隧道位于福克斯，AK（ 图1A和1B）。多年冻土隧道约110米延伸到黄金溪山谷的一侧，并提供访问丰富的冰和淤泥冲积层30-31。从冰楔和冰冻土壤样?...

Watch this Scientific Journal Video about Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside at JoVE.com

从冷冻核心外部去除外源物质以调查内部的古代生物群落生物学 |视频

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。的协议，提出收集和净化土壤和冰的永冻层的核心。缺乏外源菌落和DNA的建议检测微生物所代表的材料，而不是从钻孔或加工污染。

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

removal-exogenous-materials-from-outer-portion-frozen-cores-to

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7.9K 次观看.  US Army Engineer Research & Development Center, Hanover, NH. 冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。的协议，提出收集和净化土壤和冰的永冻层的核心。缺乏外源菌落和DNA的建议检测微生物所代表的材料，而不是从钻孔或加工污染。 

视频: Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

Microbial Communities in Nature and Laboratory - Interview

微生物群落，在自然和实验室 - 面试

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生物学

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

从美国的马蹄蟹，鲎波吕斐摩斯采血车

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

隔离方法弗朗西斯tularensis外膜

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

过程评价絮凝剂的效率的分散粒子的从植物提取物的去除

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

用化学发光法测定硝酸盐和亚硝酸盐，代谢产物中的一氧化氮途径，在生物材料

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...