我们要感谢他的技术支持，严格审查本手稿和Doug汉密尔顿安格林和迈克尔·齐默尔曼。

1。中空纤维切向流超滤程序<ol><li>缓冲区和解决方案的筹备工作： 洗脱液（1升）： 至1 L试剂级水增加0.1 G钠聚，0.1毫升吐温80和0.01毫升Y型30消泡剂。 </li><li>准备在生物安全柜滤清器总成（ 图1）： <ol><li>通过1的Masterflex易于负载泵连接的I / P 1的Masterflex精密无刷驱动器的I / P头插入的Masterflex的I / P 73实验室管。 </li><li> 1 0-60磅，填充甘油压力表1“不扩散核武器条约”的分支T-连接上游的泵用螺丝钳头安装固定油管，1 HDPE T-连接泵头下游。 </li><li>组装装修NALGENE 53-B的灌装/排气的Masterflex的L / S 15实验室管帽和T-连接，并确保其1升NALGENE重税聚丙烯瓶截留瓶。 </li><li>适合的Masterflex的L / S 24油管瓦特第i个一掐钳（掐钳2）和连接从截留瓶HDPE T-连接。 </li><li>用螺丝钳的Masterflex的L / S 24实验室管连接压力表旭化成Rexeed 25S高通量透析器，透析截留瓶。使用定制DIN适配器设计，以适应¼“身份证油管连接管的中空纤维超滤。 </li><li>掐钳（掐钳1）适合的Masterflex的L / S 24实验室油管和连接10毫升的塑料吸管尖端拆下来作为样本的吸收线和棉塞，然后附加油管的高密度聚乙烯ţ连接器。 </li><li>适合的Masterflex L/S-36实验室管的一端与坡道钳和油管连接污水过滤器的出口端附近的港口。放置在废物容器的另一端。 </li></ol></li><li>添加0.01％（W / V）聚磷酸钠10 L的水样和混合3分钟。 </li><li>关闭掐钳2删除排气帽从截留瓶的。所有其他的夹具应是开放的。 </li><li>设置泵的方向移动从T型连接器的流体压力表（从右到左下面的图1）。调节泵的速度，最高车速的25％，并打开泵。 </li><li>当截留瓶是2/3满，开放掐钳2，快速更换排气帽截留瓶。检查所有的线路和配件，以确保没有泄漏。 </li><li>慢慢增加泵的转速所需的滤过率（约1.5升/分钟），检查是否有泄漏。使用量筒和一个计时器或流量计，检查滤液水从污水线（蓝色的L / S 36油管图1）退出率。通常可以使其难以实现稳定的压力和滤过率的过滤器的出口端形成气泡。这是纠正用手按捏了一会儿出水线。这个动作将一般哄气泡，滤液端口和退出通过出水线。根据需要重复，牢记豌豆大小的气泡往往是不可避免的。 </li><li>监测过滤过程。测量和记录需要的压力和滤过率。压力不应超过20磅。据建议，滤过率应不超过2.0升/分钟。重要的是，这水截留瓶的量进行监测，以确保它永远不会清空。它的体积增加或略有下降是正常的。如果截留瓶的水量降到低于1/3满，然后取出通风帽和密切掐钳2上截留瓶线。带来的体积约2/3满，打开夹子和快速更换排气帽，确保密封。如果在截留瓶量迅速下降，并连续，然后确保截留瓶盖很紧，排气帽安全到位，并在tubi吴仍是在接触水样。如果这些问题都没有发生，那么它很可能截留瓶盖密封不好，应及时更换。 </li><li>当样品容器是空的，立即关闭掐钳1，降低泵的转速，其最大的20％，除去从截留瓶通风口帽和关闭坡道钳。 </li><li>斜坡钳拧紧或松动，调整样品截留瓶的体积约200毫升。体积约200毫升后，拧紧洗脱步骤（1.11-1.12）的斜坡钳。 </li><li>洗脱溶液中加入500毫升的样品容器和冲洗容器内。将10毫升吸管连接成洗脱溶液的容器，它包含了样品的吸收线。确保斜坡钳，被关闭。打开掐钳1，密切掐钳随时吸取洗脱液。 </li><li>洗脱溶液500毫升后制定，接近掐钳1和开放掐钳2。让洗脱液流通的20％，其最大的泵的转速为5分钟。 </li><li>出水行坡道钳拧紧或松动，调整样品截留瓶的体积约100毫升。拧紧的坡道钳和允许的样品分发1分钟。避免拉进油管空气，确保在截留瓶的样本量是不够高，覆盖的L / S 15油管进入回流瓶。 </li><li>扭转这迫使的截留瓶的样品泵的方向。为20秒，共截留瓶〜225毫升，使泵反向运行。关闭泵。 </li><li>从泵头的Masterflex的I / P 73油管和断开压力表。断开的L / S“的Masterflex 24油管退出中空纤维超滤。按住管截留瓶以上，强行进入任何剩余的样品截留瓶。 </li><li>断开所有油管从瓶子和更换非排气帽的通风帽。 </li><li>继续〜225毫升截留的IMS / IFA过程。 </li></ol> 2。免疫分离程序<ol><li>缓冲区和解决方案的筹备工作： <ol><li>允许在磁珠包括： 隐/贾第组合套件的缓冲区，以达到室温。 </li><li> 1X SL - 缓冲液A：加入1毫升的10倍SL缓冲到9毫升试剂级水。 </li></ol></li><li>从截留的瓶子转移打成250毫升锥形离心管中的液体〜225毫升。 10毫升试剂水冲洗截留瓶两次，并添加冲洗锥形离心管。在1500 XG离心15分钟，在4°没有刹车ç暂停。 </li><li>小心吸取上清液从空气 - 水界面5毫升以上的包装颗粒，每个颗粒体积为0.5毫升（即吸15毫升以上颗粒体积为1.3毫升，5毫升0.5毫升或更少）颗粒吸。 </li><li>彻底悬浮颗粒涡旋和/或吸管搅拌成上清。每个5毫升体积的液体转移到平板双面DYNAL L10管含有1毫升每10X缓冲的SL-A和10X SL - 缓冲乙冲洗锥形离心管，2.5毫升试剂水的两倍，并添加冲洗的L10管，带来的L10管12毫升的总量，包括缓冲区。 </li><li>加入100μL以及混合再悬浮反隐和反贾第磁珠的L10管。上肩混频器在室温下为1小时18转旋转的L10管。 </li><li>将反对的MPC-6磁铁，轻轻地用手岩管端至年底，180°2分钟的L10管侧扁。 </li><li>保持在MPC的6磁铁与磁铁侧的L10管起来，慢慢倒出上清离珠/（OO）囊肿络合物BOU第二到磁铁。从磁铁的L10管，并加入0.5毫升的管1X SL缓冲。转让暂停使用两个额外漂洗0.5毫升的1X SL - 缓冲举行的磁铁在垂直位置在MPC-S的1.5 ml离心管一个。 </li><li>轻轻摇动1分钟，在MPC-S的磁铁180°管。与地方磁铁，吸出上清液，用巴斯德吸管向离心管的底部。 </li><li>加入1毫升的1X PBS的离心管的前端，取出磁铁和管轻轻晃动，只是直到珠悬浮。更换磁铁在垂直位置，并轻轻摇动1分钟，180°管。吸的PBS漂洗，不扰民的珠球，用巴斯德吸管去除尽可能多的碎片。 </li><li>取下磁铁，并添加50μL试剂水的离心管的背面。旋涡管在50秒的全速，然后在80℃孵育10分钟30秒涡管。更换成MPC-S的磁铁，在倾斜的位置，结合珠磁铁和留在液体（OO）囊肿。 （OO）囊肿暂停申请到SingleSpot以及幻灯片。 </li><li>重复步骤2.10，采用液体含首次分离同井。在37°C间1小时回暖幻灯片干燥暂停幻灯片以及地方幻灯片。 </li></ol> 3。染色和考试<ol><li>缓冲区和解决方案的筹备工作： 工作的DAPI溶液加入25μL的DAPI储备液（甲醇2毫克/毫升）25毫升的1X PBS。商店库存和1°C和10°C在黑暗之间的工作方案。 </li><li>适用于50μL甲醇以及幻灯片，并允许它在室温下晾干。 </li><li>加入50μL工作的DAPI解决方案以及幻灯片和2分钟，在室温下孵育。 </li><li>使用1 KimwiPE灯芯从井的DAPI。应用50μLEasyStain。在35°C孵育30分钟。 </li><li>威克与Kimwipe的染色好，然后慢慢加入微升300的冷EasyStain固定缓冲区，允许它流过了井沿。在室温下孵育2分钟。 </li><li>使用Kimwipe灯芯从井的缓冲区和应用10μL的EasyStain安装介质。 </li><li>仔细申请盖玻片，消除发生任何气泡。封盖玻片透明指甲油。 </li><li> 200X的总放大倍率为卵形或球形类似于卵囊或囊肿的苹果绿色荧光的对象，用FITC标记的过滤器，扫描整个幻灯片。 1000X总放大倍率的DAPI过滤，然后与DIC的所有检查对象，也总放大倍数1000X。使用校准眼微米和形态特征记录的大小。 </li><li>文档结果。 </li></ol>注：附加Infor公司mation有关的原程序，可以发现在2005年12月版本EPA方法1623 12。中空纤维切向流超滤过程描述用于EPA方法1623 12.0节的地方。热分解修改EPA方法1623 13.3.3节。这个过程还介绍了在IMS的过程可分为2005年12月法1623版本13.3.2.16节之后插入一个额外的PBS冲洗。 EPA方法1623包括这些修改所使用的耗材，试剂和设备的完整列表中列出的设备清单。 4。代表结果通过过滤和免疫分离的过程中回收的隐孢子虫和贾第虫囊肿是由微观分析检测。 200X的总放大倍率，每个生物体表现出一个典型的染色图案，大小和形状如图2所示</stronG&gt;应进一步观察使用1000X总放大倍率的油浸。这将使无论是典型的定义特征或非典型的特点，积极识别，排除测量和鉴定。 隐是一个球形物体的卵形直径在4至6微米，具有突出明亮的边缘（ 图3A辉煌的苹果绿色的FITC荧光）。用DAPI紫外线，卵囊将呈现以下典型特征类别之一：淡蓝色与绿色的边缘，并没有明显的原子核（负的DAPI），强烈的蓝色内部染色，或多达四个不同的，天蓝色的细胞核（DAPI染色内部正面- 图3B）。非典型特征，包括颜色，结构，或DAPI荧光（例如，太多的染色细胞核，红色荧光的内部结构）中的偏差。如果荧光对象已符合标准，为典型的FITC和DAPI染色，它是利用微分干涉CON对比度（DIC）。非典型内部或外部形态特征，如细胞壁的纹饰，或一个或两个填充细胞的细胞核大检查的对象。如果不遵守非典型结构，对象被记录在总IFA计数和一个空的无定形结构，或用一到四个孢子出席（ 图3C）分类。同样， 贾第虫状物体研究方面FITC和DAPI染色以及DIC的特征，如axonemes，中位数机构和原子核， 贾第虫囊肿是圆卵形辉煌的苹果绿色的物体，8 - 18微米长5 -宽15微米，突出明亮的边缘（ 图3D）。用DAPI紫外线， 贾第虫囊肿将展出的DAPI染色阴性，或的DAPI阳性的特点（ 图3E）。荧光检查对象为DIC的典型和非典型的功能以同样的方式为隐 。如果不遵守非典型特征，对象被记录在总IFA计数及分类为空含无定形结构，或与一个或多个类型的内部结构（ 图3F）。 任何被观察到的非典型特征的生物体不应该算作一个囊肿（OO）。环境样品的微观分析，可以挑战有生物体，可能会自动发出荧光或交叉反应与FITC标记的反隐和/或抗贾第虫抗体1。据分析师建议熟悉水生微生物和幻灯片的审查数十获得经验识别隐孢子虫和贾第 （OO）至少有三个阳性控制幻灯片上的囊肿，应会议在显微镜前每一个特点。 （OO）的囊肿可能会飙升与质量控制样品，确定％REC安理为每个使用的计算原生动物： （OO）囊肿回收率=（（QC样品计数-无稀释剂法样品的计数）/穗）×100。 <img alt="图1" src="/files/ftp_upload/4177/4177fig1.jpg" /> 图1。切向流中空纤维超滤系统的图形表示。油管的颜色编码，以帮助系统组装。 <img alt="图2" src="/files/ftp_upload/4177/4177fig2.jpg" /> 图2。代表荧光图像的隐孢子虫和贾第 （OO）的囊肿。 隐孢子虫和贾第虫包囊染色与FITC标记的反隐 / 贾第虫抗体。箭， 贾第虫包囊;箭头， 隐孢子虫 。共有四个隐孢子虫和六鞭毛虫孢囊被发现在焦平面。样品observ1146 200X倍率。 <img alt="图3" src="/files/ftp_upload/4177/4177fig3.jpg" /> 图3代表显微图像隐孢子虫隐孢子虫和表征Giaridia囊肿（A类- C）。辉煌的苹果绿色的FITC荧光突出明亮的边缘（一）含有高达四季分明，天蓝色的DAPI核（B）和一至四个孢子（）（三）每卵囊直径在4至6微米球状物体。 贾第虫包囊（四-六）。辉煌的苹果绿色的FITC荧光圆卵形对象8 - 18微米长5 - 15微米，宽强调明亮的边缘（四）含有多达四个天蓝色的DAPI核（五）与一个或多个可辨别的内部结构等作为细胞核（N）中位数的身体（M）和或axonemes（一）（女）。白色箭头，辉煌的苹果绿色荧光染色隐孢子虫和贾第虫包囊瓦特承滴盘，白色箭头的DAPI阳性细胞核。样本观察下1000X倍率。

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+1MDS+electropositive+microfilters+for+simultaneous+recovery+of+multiple+microbe+classes+from+tap+water.">Evaluation of 1MDS electropositive microfilters for simultaneous recovery of multiple microbe classes from tap water.</a> J. Microbiol. Methods. 68, 260-266 (2007).</li><li>Rhodes, E. R., Hamilton, D. W., See, M. J., Wymer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hollow-fiber+ultrafiltration+primary+concentration+of+pathogens+and+secondary+concentration+of+viruses+from.">Evaluation of hollow-fiber ultrafiltration primary concentration of pathogens and secondary concentration of viruses from.</a> 176, 38-45 (2011).</li><li>Shaw, N. J., Villegas, L. F., Eldred, B. J., Gaynor, D. H., Warden, P. S., Pepich, B. 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D., Glass, 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=Influence+of+water+quality+on+enteric+virus+concentration+by+microporous+filter+methods.">Influence of water quality on enteric virus concentration by microporous filter methods.</a> Appl. Environ. Microbiol. 47, 956-9560 (1984).</li><li>USEPA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+1623:+Cryptosporidium+and+Giardia+in+Water+by+Filtration/IMS/FA.">Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA.</a> Office of Water. 815-R-05-002, (2005).</li><li>Ware, M. W., Wymer, L., Lindquist, H. D., Schaefer, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+alternative+IMS+dissociation+procedure+for+use+with+Method+1622:+detection+of+Cryptosporidium+in+water.">Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water.</a> J. Microbiol. Methods. 55, 575-583 (2003).</li><li>Zuckerman, U., Tzipori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portable+continuous+flow+centrifugation+and+method+1623+for+monitoring+of+waterborne+protozoa+from+large+volumes+of+various+water+matrices.">Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices.</a> J. Appl. Microbiol. 100, 1220-1227 (2006).</li></ol>

美国环境保护署资助这里描述的研究，通过其与地表水和饮用水的技术支持中心办公室合作研究和开发办公室。所有工作的支持，美国俄亥俄州辛辛那提，在美国环保局的网站上。虽然在这篇文章中所描述的信息已根据合同，美国环境保护局（EP-06-031合同）肖环境和基础设施，公司的全部或部分资助，它并不一定反映的意见应推断机构并没有正式认可。它一直受到机构的审查和批准出版。

切向流中空纤维超滤是替代水隐孢子虫和贾第虫包囊的初始浓度和有效的技术。比传统的过滤器，中空纤维超滤是昂贵。因为它有能力从各种不同的水矩阵集中的隐孢子虫和贾第虫囊肿，它是一个有用的替代目前的过滤技术用于EPA方法1623。与大多数其他过滤方法，中空纤维超滤是容易产生污垢极其浑浊样品。高水压会导致过滤器的污垢;因此建议监控过滤运行过程中的压力。此外隐孢子虫和贾第虫囊肿，中空纤维超滤已被证明是能够集中细菌和病毒1-3,5,8。中空纤维超滤Ø这种方法utlined的可用于集中在单个样品的多种生物。值得注意的是，获得200至250毫升的最后一卷，是在集中过程中的关键的最后一步，使额外的离心步骤，可能导致（OO）囊肿损失，避免（步骤2.2）。然而，允许在瓶子的体积降得过低，可回收率有不利影响，因为不会有足够的液体体积，截留瓶迫使所有的卵囊或囊肿。因此，建议保持200至250毫升的最终体积。 热分解是酸解方法1623步的替代方法。已被证明这种替代的步骤，以提高隐孢子虫的卵囊恢复和减少变化时，无论从河流或试剂水9隔离的方法。端侧的酸和热分解方法比较表明，用热，以解离TE从免疫磁珠生物体产生较高的平均回收率为隐孢子虫和贾第 。此外， 隐孢子虫和贾第回收率精度优于热分解处理的样品酸解9。 浓缩步骤的HFUF纳入允许集中多种生物的能力提供更多的灵活性。此外，它是一个较为便宜的替代目前的方法1623过滤选项。

<table border="1"><tbody><tr><td> 设备/试剂 </td><td> 供应商 </td><td> 产品编号 </td></tr><tr><td>旭化成的S / R 25 Rexeed湿中空纤维超滤</td><td>拨号医学</td><td> REXEED25S / </td></tr><tr><td>的I / P 73（的Masterflex的R-3603），或同等学历</td><td>科尔帕默</td><td> EW-06408-73 </td></tr><tr><td> 24 L / s（的Masterflex铂固化），或同等学历</td><td>科尔帕默</td><td> EW-96410-24 </td></tr><tr><td> 15 L / s（的Masterflex铂固化），或同等学历</td><td>科尔帕默</td><td> EW-96410-15 </td></tr><tr><td> 36 L / s（的Masterflex铂固化），或同等学历</td><td>科尔帕默</td><td> EW-96410-36 </td></tr><tr><td>精密的I / P无刷</td><td>科尔帕默</td><td> EW-77410-10 </td></tr><tr><td>易负载的I / P泵头</td><td>科尔帕默</td><td> EW-77601-10 </td></tr><tr><td>高密度聚乙烯黑色T恤，1/4“×3/8”×3/8“ </td><td>美国塑料</td><td> 62064 </td></tr><tr><td>的Masterflexţ连接器的L / S 15-25 </td><td>科尔帕默</td><td> EG-30613-12 </td></tr><tr><td> NALGENE重型PP 1升瓶</td><td>科尔帕默</td><td> EW-06257-10 </td></tr><tr><td> 10毫升移液器</td><td> Fisher Scientific则</td><td> 13-678-11C </td></tr><tr><td>的NALGENE灌装/通风上限为1/4“管，53B </td><td>科尔帕默</td><td> EW-06258-10 </td></tr><tr><td>压力计</td><td>科尔帕默</td><td> -680-46-10 </td></tr><tr><td>直耦合，“不扩散核武器条约”（女），1/4“ </td><td>科尔帕默</td><td> EW-06469-18 </td></tr><tr><td> “不扩散核武器条约”的分支T恤，自然PP </td><td>科尔帕默</td><td> -30610-75 </td></tr><tr><td>捏夹，1/2“ </td><td>科尔帕默</td><td> EW-06833-00 </td></tr><tr><td>铜STOM适合符合DIN适配器</td><td>公司模压产品</td><td> MPC-855NS.250 </td></tr><tr><td>环立场</td><td> Fisher Scientific则</td><td> 14-670B </td></tr><tr><td>环立场夹</td><td> Fisher Scientific则</td><td> 05-769-6Q </td></tr><tr><td>凯克坡道钳，14mm的</td><td>科尔帕默</td><td> EW-06835-10 </td></tr><tr><td>多聚磷酸钠</td><td> Sigma Aldrich公司</td><td> 305553 </td></tr><tr><td>硫代硫酸钠</td><td> Sigma Aldrich公司</td><td> 72050 </td></tr><tr><td>消泡剂Y - 30乳液</td><td> Sigma Aldrich公司</td><td> A5758 </td></tr><tr><td>吐温80 </td><td> Sigma Aldrich公司</td><td> P1754 </td></tr><tr><td> 10升可折叠的高密度聚乙烯cubitainer </td><td>厂商VWR </td><td> IR314-0025 </td></tr><tr><td>离心机瓶架</td><td> Fisher Scientific则</td><td> 05-663-103 </td></tr><tr><td> 250毫升锥形离心管</td><td>康宁</td><td> 430776 </td></tr><tr><td>一次性漏斗</td><td>科尔帕默</td><td>的U-6122-10 </td></tr><tr><td>洗瓶</td><td>科尔帕默</td><td>的U-06252-40 </td></tr><tr><td>离心分离</td><td>贝克曼</td><td> Allegra的X-15R </td></tr><tr><td>摆动转头</td><td>贝克曼</td><td>白羊SX4750 </td></tr><tr><td>为250毫升锥形管离心桶适配器</td><td>贝克曼</td><td> 349849 </td></tr><tr><td> 200μL大口径枪头</td><td> Fisher Scientific则</td><td> 02-707-134 </td></tr><tr><td> VacuShield过滤器</td><td>格尔曼</td><td> 629-4402 </td></tr><tr><td> 5毫升移液器</td><td> Fisher Scientific则</td><td> 13-678-11D </td></tr><tr><td>磁珠： 隐/贾第组合套件</td><td>我DEXX </td><td> 73002 </td></tr><tr><td> 50毫升锥形离心管</td><td>鹘</td><td> 352098 </td></tr><tr><td> DYNAL L10平板双面管</td><td>爱德士</td><td> 74003 </td></tr><tr><td>定时器</td><td>厂商VWR </td><td> 23609-202 </td></tr><tr><td> DYNAL的MPC-6磁铁</td><td>爱德士</td><td> 12002D </td></tr><tr><td> 1毫升移液器</td><td>厂商VWR </td><td> 53283-700 </td></tr><tr><td> 1.5毫升低附着力离心管</td><td> Fisher Scientific则</td><td> 02-681-320 </td></tr><tr><td> 1000微升吸管及相应的屏障提示</td><td>吉尔森</td><td> P1000/DF1000ST </td></tr><tr><td> 100μL移液器及相应的屏障提示</td><td>吉尔森</td><td> P100/DF100ST </td></tr><tr><td> 9英寸的巴斯德移液器</td><td>厂商VWR </td><td> 14672-412 </td></tr><tr><td> DYNAL货币政策委员会 - S磁体</td><td>爱德士</td><td> 12020D </td></tr><tr><td>涡旋</td><td>厂商VWR </td><td> 14216-188 </td></tr><tr><td>磁珠旋转混合器</td><td>爱德士</td><td> 94701 </td></tr><tr><td>热块</td><td> Fisher Scientific则</td><td> 11-718-2 </td></tr><tr><td>实验室护甲珠</td><td>实验室护甲</td><td> 42370-750 </td></tr><tr><td>数字温度计</td><td> Fisher Scientific则</td><td> 15-077-60 </td></tr><tr><td>磷酸盐缓冲液1X pH值7.4（1×PBS） </td><td>西格玛</td><td> P4417 </td></tr><tr><td>单点幻灯片</td><td>爱德士</td><td> 30201 </td></tr><tr><td>盖玻片</td><td>康宁</td><td> 287018 </td></tr><tr><td> EasyStain直接套件</td><td>的BTF </td><td> - </td></tr><tr><td> 10微升吸管及相应的屏障提示</td><td>吉尔森</td><td> P10与DF10ST </td></tr><tr><td> 4&#39;，6&#39;-DiamidINO-2-苯基吲哚盐酸（DAPI） </td><td>西格玛</td><td> D9542 </td></tr><tr><td>透明指甲油</td><td> Fisher Scientific则</td><td> S30697 </td></tr><tr><td>甲醇</td><td> Fisher Scientific则</td><td> L6815 </td></tr><tr><td> kimwipes </td><td>金佰利</td><td> 34155 </td></tr><tr><td>孵化器</td><td>布克尔科学</td><td> 133000 </td></tr><tr><td>滑动回暖</td><td> Fisher Scientific则</td><td> 11-474-521 </td></tr><tr><td>浸油，A型ND = 1.515 </td><td>尼康</td><td> MXA20234 </td></tr><tr><td>尼康90i显微镜与DIC的能力</td><td>尼康</td><td>工商管理硕士77000 </td></tr><tr><td>计划载脂蛋白100X油目标</td><td>尼康</td><td> MRD01901 </td></tr><tr><td>计划Achro 20X </td><td>尼康</td><td> MRL00202 </td></tr><tr><td> FITC标记过滤器</td><td>尼康</td><td> 96302 </td></tr><tr><td>的DAPI过滤器</td><td>尼康</td><td> 96301 </td></tr><tr><td>的X-Cite荧光照明</td><td>尼康</td><td> 87540 </td></tr><tr><td>镜头纸</td><td>尼康</td><td> 76997 </td></tr><tr><td>生化危机一次性袋</td><td> Fisher Scientific则</td><td> 01-829D </td></tr><tr><td>生化危机锐器容器</td><td> Fisher Scientific则</td><td> 14-827-117 </td></tr><tr><td> 3％双氧水</td><td>厂商VWR </td><td> BDH3540-2 </td></tr><tr><td>漂白</td><td> Fisher Scientific则</td><td> 1952030 </td></tr><tr><td> wypall </td><td>金佰利</td><td> 34790 </td></tr></tbody></table>

a modified epa method 1623 that uses tangential flow hollow-fiber ultrafiltration and heat dissociation steps to detect waterborne cryptosporidium and giardia spp.

隐孢子虫和贾第鞭毛虫种原虫，造成水源性腹泻病爆发的全球最流行 ​​的两个。为了更好地描述这些病原体的流行，开发和利用到美国饮用水中这些生物监测水平EPA方法1623提供12。该方法有三个主要部分，首先是样品浓度，其中至少10 L是原始地表水过滤。生物体和被困的碎片，然后洗脱从过滤器和离心样品，以进一步集中。该方法的第二部分使用浓缩水样专门绑定到允许特定拆除集中碎片寄生虫的隐孢子虫和贾第虫包囊的免疫磁珠免疫分离过程。这些囊肿（OO），然后脱离磁珠酸解多项式dure。该方法的最后一部分是囊肿（OO）应用于幻灯片，染色，并通过显微镜列举的免疫荧光染色和枚举。 方法1623有四家上市样品浓度系统捕捉水中隐孢子虫和贾第虫囊肿：过滤器（Envirochek Pall公司，安阿伯，MI），Envirochek高压过滤器（Pall公司），Filta最大的过滤器（爱德士，韦斯特布鲁克，马萨诸塞州），或连续流离心（Haemonetics，布伦特里，马萨诸塞州）。然而， 隐孢子虫和贾第 （OO）囊肿回收率差别很大，取决于水源矩阵和过滤器使用1,14。一种新的切向流中空纤维超滤（HFUF）系统最近被证明是更有效和更强大的恢复隐孢子虫 和贾第虫包囊从各种水矩阵;此外，它是小于其他囊式过滤器“选项可以集中精力和多种病原体同时1-3,5-8,10,11。此外，以往的研究表明山和他的同事的HFUF显着提高回收率隐孢子虫卵囊，直接与Envirochek高压过滤器4相比。也有报道补充修改目前的方法，以提高方法的性能。更换酸解过程与热分解被证明是更有效地从一些矩阵9,13磁珠分离隐 。 这个协议描述修改的方法1623使用热分解步骤的新HFUF过滤系统。使用这种方法的改进与HFUF是一个不太昂贵的替代当前的EPA方法1623过滤选项，允许多种生物的浓度，并提供更多的灵活性。

Watch this Scientific Journal Video about A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

本协议描述了作为替代水性的检测步骤使用的中空纤维切向流超滤样品浓缩系统和热分解<em&gt;隐</em&gt;和<em&gt;贾第</em&gt;物种通过采用EPA Method 1623。

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性<em&gt;隐</em&gt;和<em&gt;贾第虫属。</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

a-modified-epa-method-1623-that-uses-tangential-flow-hollow-fiber

Research

JoVE Journal

Immunology and Infection

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

20.3K 次观看.  Office of Research and Development, US Environmental Protection Agency. 本协议描述了作为替代水性的检测步骤使用的中空纤维切向流超滤样品浓缩系统和热分解隐和贾第物种通过采用EPA Method 1623。

视频: A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

根结线虫的高和低通量筛选根结线虫。</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

为集中的的水性病毒和农业人畜共患病病原体的玻璃纤维过滤器

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

参考微量稀释法达巴万星<em&gt;体外</em&gt;细菌敏感性测试生长有氧

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...