我们要感谢大队实验室的好心提供厌氧室的照片。这项工作是由卫生部授予DK087763（SMM）和一个STEP /霍华德休斯医学研究所课程发展奖学金（ANE）国家机构的支持。

注：C。艰难梭菌是一种人类和动物病原体，可引起胃肠道疾病。涉及C.实验难辨必须以适当的生物安全防范措施（BSL-2）进行。 1。厌氧室使用与维护 艰难梭菌是一种严格厌氧菌和是即使是低浓度的氧的大气中极为敏感。因此，受控的，厌氧环境是必要的，它的成功操作。利用厌氧室中（ 图1A）的提供了最稳定的环境，理想的条件下进行有效的栽培C的艰难梭菌等厌氧菌14。这里，含有气体混合物（5％CO 2，10％H 2，85％N 2）的气氛中，可以稳定地维持。 引入任何物品进入室内无显著氧污染，气闸必须使用（ 图1B）。该装置作为气体交换和自动工作，半手动或手动。气闸有两个门：1提供进入气密室的厌氧室的内部的其他提供访问外部和。取决于模型中，气闸可具有一个附加的门，它可提供访问到一个相邻的厌氧室中，从而节省了单独的气锁的成本。除积极朝着腔或缩小项目，这两个门应保持关闭在任何时间，以避免氧气污染。气闸具有在前面的通/断开关和含有四个按钮面板。气体管道和真空泵软管连接在气闸，面板，其中为单位的全手动操作的开关被靠近的后部。 建议2吹扫周期，使用氮气（N 2），用于与气体混合物（5％CO 2的填充交换之前，10％H 2，85％N 2），以减少氧气的引入室的数量。同样重要的是要永远离开要么门打开，而不是移动项目进出交换的和所需要的量较长时间仔细计划的实验，以减少移动项目在室进出的频率。乙烯基厌氧室的不同操作步骤概述如下。以下这些协议之前，确保这些是与提供与腔室的制造商的说明兼容。 <ol><li>自动模式 这是一个可编程和可定制的模式和完全由气锁进行。编程的气闸，请参阅制造商的说明。对于典型的进入腔推荐的方案涉及用氮气吹洗2次后，再次填充的气闸用混合气体，该湛中保持了密切的氛围相匹配BER。在每个真空循环，标准厂房的程序建议抽真空至20英寸汞柱和清洗，以1英寸汞柱。 <ol><li>确保内气密室门完全关闭。 </li><li>打开外气密室门。 </li><li>将物品放入密封舱，关闭气闸外门。如果引入液体，拧开瓶盖一半，以确保有效的气体交换。密封包装和容器前，应开始循环打开;不会这样做的可弯曲和损坏塑料制品及容器。保持无菌，打开足够的，只允许有效的气体交换包。 </li><li>按下开始按钮。 </li><li>等到气闸已通过程序循环，将显示“厌氧”。 </li><li>打开内气密室门。 </li><li>移动物品进入室内。 </li><li>关闭内门。 </li></ol></li><li>半手动模式 移动在腔室中或放物品时，这种模式下，可以使用与需要当c在腔室中ycling气体是必需的。 <ol><li>确保内气密室门完全关闭。 </li><li>打开外气密室门。 </li><li>将物品放入密封舱，关闭气闸外门。如果引入液体，拧开瓶盖一半，以确保有效的气体交换。密封的包装，如接种环和96孔板，必须在开始之前的周期打开。 </li><li>按功能键。 </li><li>按开始键。气闸将显示每个按钮的功能，不会响应，直到完成这一操作： <ul><li>向上箭头：启动真空泵。 </li><li>向下箭头键：启动氮气（吹扫）气流。 </li><li>启动：启动混合气体流量。 </li><li>菜单：返回到默认的显示。 </li></ul></li><li>按“向上”，从气闸去除气体，直到屏幕显示20英寸汞柱。 </li><li>按“向下”，用氮气填充的气密室，直到显示小于1英寸汞柱。不要OVershoot，因为这将创建正压气密室之内，这可能会影响它的完整性。 </li><li>重复步骤6和7，执行额外的清除周期。 </li><li>按“Up”直到屏幕显示20英寸汞柱。 </li><li>按下“开始”，用混合气体填充交汇处，直到显示小于1英寸汞柱。 </li><li>打开内气密室门。 </li><li>移动物品进入室内。 </li><li>关闭内门。 </li></ol></li><li>手动模式 当自动或半手动模式不工作或没有电源的气闸此模式也可使用。这种模式下不工作时的气闸是上，因此，它是难以确定的气密室内部的压力。由于真空拉和气体吹扫时间取决于真空和气体流速，分别交换中使用压力表需要监控的压力，减少人身伤害和损坏的气闸的风险。 <ol><li>确保内气密室门完全关闭。 </li><li>打开外气密室门。 </li><li>放置物品，包括压力表，进气闸舱，关闭气闸外门。如果引入液体，拧开瓶盖一半，以确保有效的气体交换。密封的包装，如接种环和96孔板，必须在开始之前的周期打开。 </li><li>找到三个开关上标有气闸后面的“手动控制开关。”这些单独标记为： <ul><li>真空</li><li>氮</li><li>混合气</li></ul></li><li>翻转真空切换开关，直到压力表读数约为20英寸汞柱。 </li><li>翻转氮拨动开关，直到压力表读数约为1英寸汞柱。 </li><li>翻转真空切换开关，直到压力表读数约为20英寸汞柱。 </li><li>翻转氮拨动开关，直到压力表读数约为1英寸汞柱。</li><li>翻转真空切换开关，直到压力表读数约为20英寸汞柱。 </li><li>翻转气体混合拨动开关，直到压力表读数约为1英寸汞柱。 </li><li>打开内气密室门。 </li><li>移动物品进入室内。 </li><li>关闭内门。 </li></ol></li></ol> 2。培养，枚举和存储C。从难辨粪便样本这个程序的目的是恢复C。从难辨粪便样品含有孢子，随后保持分离菌落在长期储存的营养细胞或孢子甘油股票。可替换地，该过程可被用于枚举C的数目目前难辨粪便样本（ 例如从动物研究）中。要选择和鉴别富集C。艰难梭菌 ，牛磺胆酸盐，头孢西丁-环丝氨酸果糖琼脂（TCCFA）用于抑制正常粪便菌群生长15-16。环丝氨酸是抑菌对于革兰氏阴性菌，而头孢西丁更广泛地抑制革兰氏阴性和阳性细菌的生长，除C的艰难和最输入球菌菌株。 pH指示剂，中性红，可以被包括在介质中，由于果糖的发酵将导致pH值下降，并从红色/橙色随后的颜色变化到黄色。为了有效地恢复C.孢子作为难辨营养细菌，胆盐牛磺胆酸钠是用于诱导发芽17-18。因为C。难辨形成孢子，醇或样品的热处理可以用来减少或消除营养细胞，限制污染的植物的生长，这可能会增加C的效率艰难复苏19。如上面所提到的，关键是要预先减少使用，以确保除去残余氧事先在厌氧室中的所有板为至少1-2小时。空气dryi纳克在室在使用前板可以减少冷凝。液体培养基中，可能需要高达24小时，以减少视在体积和所用的容器的表面 - 空气比率上。 在开始之前，下列物品应放入厌氧室： <ul><li>粪便样本</li><li>无菌棉签</li><li>无菌接种环</li><li> 1X PBS（见材料） </li><li> TCCFA板（见材料） </li><li> BHIS（脑心浸液培养基酵母抽提物）板和肉汤（见材料） </li><li> 50％甘油（灭菌） </li><li> 10％的L-半胱氨酸（灭菌） </li><li>低温储存瓶</li></ul> *可替换地，分离的菌落可蔓延到BHIS琼脂平板上，随后刮下并重新悬浮于BHIS液体介质，用15％的甘油用于长期贮存于-80℃。 *要注意的是革兰氏染色是不是一种有效的策略，以确定下这是很重要直接从粪便标本23 难辨 </SUP&gt;; C。难辨必须首先从粪便中的其他植物中分离。 <ol><li>重悬在1×PBS中（这可能在有氧条件下进行）的粪便样品，并确保该粪便样品被完全重新悬浮于1×PBS中通过涡旋。如果枚举C.从难辨大便，权衡粪便之前，除了1X PBS中。 </li><li>使在1×PBS中再悬浮的粪便样品进行的集落形成单位（CFU）每克粪便的适当隔离或列举的连续稀释。 </li><li>使用无菌技术，应用100微升每个连续稀释至TCCFA板。 </li><li>采用一系列无菌接种环，条痕为孤立的殖民地或均匀分布的应用文化TCCFA板表面上的菌落计数的应用文化。 </li><li>厌氧孵育板48小时，在37°C。它可以检测并识别C。在24小时内难辨殖民地的基础上扁平，不规则，毛玻璃殖民地15的外观，虽然更准确的识别和枚举48小时后达到。 </li><li>用无菌接种环，传代，似乎是下任殖民地难辨到预还原BHIS琼脂平板上，补充有0.03％的L-半胱氨酸20。挑一个单独的殖民地，和条纹上板象限，采用了新的无菌接种环的每一个象限，以获得单菌落。另外，算上C。每个稀释度的菌落难辨 （这可以在有氧条件下进行），并计算菌落形成每克粪便样品的单位数。 </li><li> C.确认经革兰氏染色鉴定难辨 。经过革兰氏染色，C。难辨将显示为紫色杆和一些细胞可能包含终端孢子。聚合酶链反应（PCR）鉴定编码毒素B的基因可以提供进一步的确认C的产毒菌株21 艰难而多位点序列分型（MLST）是成功的识别和未知和产毒的菌株C的准确分型难辨 22。 </li><li>为了保持C的股票艰难梭菌在-80℃下，选择一个个体，孤立菌落从用无菌接种环的BHIS琼脂平板上，重悬菌落在10ml预还原BHIS液体培养基中添加0.03％的L-半胱氨酸。* </li><li>孵育过夜厌氧，在37℃，或直到文化变得混浊。 </li><li>添加333微升50％的甘油和666微升的C的难辨文化的1.8毫升低温管来创建分离株的15％甘油。 </li><li>盖紧瓶盖低温管，拌匀并立即从厌氧室和地点除去股票在-80℃冷冻长期保存。 </li></ol> 3。 C.培养难辨 </eM&gt;从冷冻甘油股票这个程序提供了C的恢复难辨从保存于-80℃。甘油原液因为反复冻融可能会杀死营养细胞，保持冻结在任何时候都甘油股票是很重要的。我们不建议使用干冰在干冰蒸发可以在室内改变环境室的进出转移株。相反，我们建议使用冷冻冷却架上，以保持运输过程中冷冻甘油股票。对于不同的选择性和鉴别的目的，通常用于培养C.三种媒体难辨 。 TCCFA，如上面所讨论的，是选择性为C。难辨并含有牛磺胆酸钠，一个开端。脑，心脏浸剂补充有酵母提取物（BHIS）是一种常用的，富集的，非选择性培养基中，允许对多种生物体的（ 图2A）24的生长。通常情况下，L-半胱氨酸e的加入BHIS作为还原剂20。最后，将另外的血液向培养基中（ 图2B）可以更有效地形成孢子比TCCFA，并提供检测的独特绿色或黄绿色荧光由C.表现出根据难辨长波紫外线（UV）光15（ 图2C）。当C.培养从孢子种群难辨 ，重要的是要记住，牛磺胆酸钠，必须向培养基中加入以保证发芽。 在开始之前，下列物品应放入厌氧室： <ul><li>冷冻甘油股票（冷却机架） </li><li>无菌接种环</li><li> TCCFA或BHIS板（见材料） </li><li> BHIS肉汤（见材料） </li><li> 50％甘油（灭菌） </li><li>低温储存瓶</li></ul><ol><li>将C的冷冻甘油股票艰难梭菌在已被存储在-80℃PR，一个散热架IOR用来防止解冻。 </li><li>把干冰冷冻甘油股票进入厌氧室。 </li><li>使用无菌技术和无菌接种环，放置少量库存到整个板的一个象限中的板和条纹。 </li><li>旋转板90°，并使用一个新的无菌接种环，继续穿过第二象限条纹。 </li><li>重复第三和第四象限内，以确保单个菌落的分离。 </li><li>立即从厌氧室中取出冷冻甘油贮存，并返回到-80℃。 </li><li>孵育板厌氧过夜，在37℃下单个孤立菌落过夜生长后进行观察。 </li></ol> 4。从C净化孢子难辨 由于产孢需要生存在富氧环境和疾病8的高效传输，孢子股票的准备是ofteÑ​​必要的下游应用，不限于显微镜和动物试验。要注意的是列举孢子需要重复，以确保计数的再现性是非常重要的。稀释度之间上下吹打几次也减少了损失，因为孢子附着的塑料好。 C.孢子化难辨是不是快速或同质其他产孢子的物种。为了优化产孢和恢复，无论是产孢培养基（SMC）17,25或70:30媒体26推荐。其它常用的介质是BHIS，这需要4-5天增长前高效率的孢子被认为是27，和Clospore，产生孢子的高滴度的液体介质- 72小时增长后（10 7每毫升10 8孢子）。其它协议使用冰冷的水，而不是1×PBS 20，但是，使用的等渗溶液可从相互粘连和塑料表面减少孢子。另外，一些研究者进一步用蔗糖梯度，以完全消除营养细胞和碎片29净化他们的孢子。 在开始之前，下列物品应放入厌氧室： <ul><li>无菌接种环</li><li> BHIS，SMC和/或70:30板（见材料） </li><li> BHIS肉汤（见材料） </li><li> 1X PBS，过滤灭菌（见材料） </li></ul><ol><li>从冷冻甘油储用培养菌株到预还原BHIS板孵育厌氧过夜，在37℃下</li><li>重新划到几个预先降低SMC或70:30板和厌氧培养，在37℃下24-48小时。孢子的形成可以通过相衬显微镜遵循。孢子会出现明亮的阶段，而营养和母细胞会出现黑暗期。 *作为替代方案，孢子可从70:30液体培养基进行纯化后24-48小时，产生每毫升10 5 -10 6个孢子，这取决于TG1的用于n。 </li><li>用无菌接种环，刮片和重悬细胞于10ml无菌的1×PBS。 </li><li>丢弃板和从厌氧室中取出孢子悬浮液。沉淀细胞在3000×g离心15分钟。在1×PBS中洗涤细胞两次，每次完全重悬细胞沉淀。 </li><li>孵育过夜，在4℃在营养和母细胞的溶解，以帮助。 </li><li>孵育在70℃下20分钟以杀死任何残余营养细胞。 </li><li>以确定形成单位每毫升（CFU）菌落，连续稀释各孢子制剂在1x PBS及盘上BHIS +0.1％牛磺胆酸钠。列举菌落之前孵育板为至少24小时。 </li><li>孢子可以存储在1×PBS中于室温或4℃下长期贮存。如果储存在4℃下，它可能是有用的再热的孢子制剂在55℃下15分钟，以恢复有效的发芽。 </li></ol>

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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=The+Clostridium+difficile+spo0A+gene+is+a+persistence+and+transmission+factor.">The Clostridium difficile spo0A gene is a persistence and transmission factor.</a> Infect. Immun. 80, 2704-2711 (2012).</li><li>Drasar, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+anaerobic+intestinal+bacteria.">Cultivation of anaerobic intestinal bacteria.</a> J. Pathol. Bacteriol. 94, 417-427 (1967).</li><li>Leach, P. A., Bullen, J. J., Grant, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+CO+2+cabinet+for+the+cultivation+of+strict+anerobes.">Anaerobic CO 2 cabinet for the cultivation of strict anerobes.</a> Appl. Microbiol. 22, 824-827 (1971).</li><li>Killgore, G. E., Starr, S. E., Del Bene, ., Whaley, V. E., N, D., Dowell, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+anaerobic+systems+for+the+isolation+of+anaerobic+bacteria+from+clinical+specimens.">Comparison of three anaerobic systems for the isolation of anaerobic bacteria from clinical specimens.</a> Am. J. Clin. Pathol. 59, 552-559 (1973).</li><li>Bacic, M. K., Smith, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+and+cultivation+of+bacteroides+species.">Laboratory maintenance and cultivation of bacteroides species.</a> Curr. Protoc. Microbiol. Chapter 13, Unit 13C 11 (2008).</li><li>Doan, N., Contreras, A., Flynn, J., Morrison, J., Slots, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proficiencies+of+three+anaerobic+culture+systems+for+recovering+periodontal+pathogenic+bacteria.">Proficiencies of three anaerobic culture systems for recovering periodontal pathogenic bacteria.</a> J. Clin. Microbiol. 37, 171-174 (1999).</li><li>Socransky, S., Macdonald, J. B., Sawyer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+Treponema+microdentium+as+surface+colonies.">The cultivation of Treponema microdentium as surface colonies.</a> Arch. Oral. Biol. 1, 171-172 (1959).</li><li>George, W. L., Sutter, V. L., Citron, D., Finegold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+and+differential+medium+for+isolation+of+Clostridium+difficile.">Selective and differential medium for isolation of Clostridium difficile.</a> J. Clin. Microbiol. 9, 214-219 (1979).</li><li>Wilson, K. H., Silva, J., Fekety, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+Clostridium+difficile+by+normal+hamster+cecal+flora+and+prevention+of+antibiotic-associated+cecitis.">Suppression of Clostridium difficile by normal hamster cecal flora and prevention of antibiotic-associated cecitis.</a> Infect. Immun. 34, 626-628 (1981).</li><li>Wilson, K. H., Kennedy, M. J., Fekety, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sodium+taurocholate+to+enhance+spore+recovery+on+a+medium+selective+for+Clostridium+difficile.">Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile.</a> J. Clin. Microbiol. 15, 443-446 (1982).</li><li>Bliss, D. Z., Johnson, S., Clabots, C. R., Savik, K., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cycloserine-cefoxitin-fructose+agar+(CCFA)+and+taurocholate-CCFA+for+recovery+of+Clostridium+difficile+during+surveillance+of+hospitalized+patients.">Comparison of cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA for recovery of Clostridium difficile during surveillance of hospitalized patients.</a> Diagn. Microbiol. Infect. Dis. 29, 1-4 (1997).</li><li>Marler, L. 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=Comparison+of+five+cultural+procedures+for+isolation+of+Clostridium+difficile+from+stools.">Comparison of five cultural procedures for isolation of Clostridium difficile from stools.</a> J. Clin. Microbiol. 30, 514-516 (1992).</li><li>Sorg, J. A., Dineen, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Clostridium+difficile.">Laboratory maintenance of Clostridium difficile.</a> Curr. Protoc. Microbiol. Chapter 9, Unit 9A 1 (2009).</li><li>Bouillaut, L., McBride, S. M., Sorg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+Clostridium+difficile.">Genetic manipulation of Clostridium difficile.</a> Curr. Protoc. Microbiol. Chapter 9, Unit 9A 2 (2011).</li><li>Lemee, L., Pons, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilocus+sequence+typing+for+Clostridium+difficile.">Multilocus sequence typing for Clostridium difficile.</a> Methods. Mol. Biol. 646, 77-90 (2010).</li><li>Shanholtzer, C. J., Peterson, L. R., Olson, M. N., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+study+of+gram-stained+stool+smears+in+diagnosis+of+Clostridium+difficile+colitis.">Prospective study of gram-stained stool smears in diagnosis of Clostridium difficile colitis.</a> J. Clin. Microbiol. 17, 906-908 (1983).</li><li>Smith, C. J., Markowitz, S. M., Macrina, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferable+tetracycline+resistance+in+Clostridium+difficile.">Transferable tetracycline resistance in Clostridium difficile.</a> Antimicrob. Agents Chemother. 19, 997-1003 (1981).</li><li>Permpoonpattana, P., 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=Surface+layers+of+Clostridium+difficile+endospores.">Surface layers of Clostridium difficile endospores.</a> J. Bacteriol. 193, 6461-6470 (2011).</li><li>Putnam, E. E., Nock, A. M., Lawley, T. D., Shen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpoIVA+and+SipL+are+Clostridium+difficile+spore+morphogenetic+proteins.">SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins.</a> J. Bacteriol. , (2013).</li><li>Burns, D. A., Minton, 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=Sporulation+studies+in+Clostridium+difficile.">Sporulation studies in Clostridium difficile.</a> J. Microbiol. Methods. 87, 133-138 (2011).</li><li>Perez, J., Springthorpe, V. S., Sattar, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clospore:+a+liquid+medium+for+producing+high+titers+of+semi-purified+spores+of+Clostridium+difficile.">Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile.</a> J. AOAC Int. 94, 618-626 (2011).</li><li>Sorg, J. A., Sonenshein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+the+initiation+of+Clostridium+difficile+spore+germination+using+analogs+of+chenodeoxycholic+acid,+a+bile+acid.">Inhibiting the initiation of Clostridium difficile spore germination using analogs of chenodeoxycholic acid, a bile acid.</a> J. Bacteriol. 192, 4983-4990 (2010).</li><li>O'Connor, J. 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=Construction+and+analysis+of+chromosomal+Clostridium+difficile+mutants.">Construction and analysis of chromosomal Clostridium difficile mutants.</a> Mol. Microbiol. 61, 1335-1351 (2006).</li><li>Buggy, B. P., Wilson, K. H., Fekety, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methods+for+recovery+of+Clostridium+difficile+from+an+environmental+surface.">Comparison of methods for recovery of Clostridium difficile from an environmental surface.</a> J. Clin. Microbiol. 18, 348-352 (1983).</li><li>Koch, C. J., Kruuv, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+release+of+oxygen+from+polystyrene+Petri+dishes.">The release of oxygen from polystyrene Petri dishes.</a> Br. J. Radiol. 45, 787-788 (1972).</li><li>Ethapa, T., 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=Multiple+factors+modulate+biofilm+formation+by+the+anaerobic+pathogen+Clostridium+difficile.">Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile.</a> J. Bacteriol. , (2012).</li><li>Speers, A. M., Cologgi, D. L., Reguera, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+cell+culture.">Anaerobic cell culture.</a> Curr. Protoc. Microbiol. Appendix 4, Appendix 4F (2009).</li><li>Lyras, D., 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=Toxin+B+is+essential+for+virulence+of+Clostridium+difficile.">Toxin B is essential for virulence of Clostridium difficile.</a> Nature. 458, 1176-1179 (2009).</li></ol>

没有利益冲突的声明。

这里描述的方法允许简单和快速C的恢复从各种粪便样品，包括人类，小鼠和仓鼠，以及在长期贮存下的难辨难辨甘油或孢子的股票。C.难辨可以是一个困难的有机体培养，但细心保养的厌氧环境和无菌技术的应用可以提供强劲的增长和污染的减少。 厌氧室：注意事项和维护有两种类型的厌氧室：刚性室或乙烯基室。刚性腔通常由?...

艰难梭菌是一种革兰氏阳性，形成孢子的细菌是专性厌氧菌以及人类和动物的一种潜在的致命胃肠道病原体。在1935年最初描述为粪便样本中发现从新生儿1，C的共生有机体难辨后来被证明是伪膜性肠炎的病原体使用抗生素治疗2有关。C.艰难梭菌感染（CDI），通常先通过抗生素治疗而导致的正常结肠菌群的破坏，形成一个利基C。难辨茁壮成长2。C.难辨传输为通过粪-口途径休眠孢子，并随后在胃肠道内发芽，生产营养细胞能够产生多种毒素，引起严重的疾病，结肠炎3。 CDI往往是难治常规治疗而这些fections经常经常性的4。因此，课程发展处负责可达至48十亿在医疗保健费用在美国5-7。 艰难梭菌是即使低氧气的环境中的电平非常敏感。对于C。难辨坚持环境和有效地从主机到主机的传输，一个代谢不活跃孢子的形成是至关重要的8。因为C的实验室维护和操作难辨需要控制，无氧的环境中，这些技术需要使用在厌氧室。利用厌氧室已导致增加的回收和专性厌氧菌9-11的隔离，并允许一些要在厌氧气氛中进行的分子技术。 除了​​C。难辨 ，厌氧室使用和维护这里描述适用其它专性厌氧菌如其他梭菌物种（ 如产气荚膜梭菌 ），其它胃肠物种（ 如拟杆菌属种12）和牙周病原体（ 如消化链球菌属 13种）。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Proteose Peptone no. 2</td>
			<td>BD</td>
			<td>212120</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher</td>
			<td>S373</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>BP362</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S27</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>&#7429;-Fructose</td>
			<td>Fisher</td>
			<td>L96</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma</td>
			<td>T4009</td>
			<td></td>
		</tr>
		<tr>
			<td>&#7429;-cycloserine</td>
			<td>Sigma</td>
			<td>C6880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefoxitin</td>
			<td>Fluka</td>
			<td>C4786</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion medium</td>
			<td>BD</td>
			<td>237300</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose Peptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sigma</td>
			<td>A5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7755</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoPeptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbian sheep blood agar</td>
			<td>Fisher</td>
			<td>L21928</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S27</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP2291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile inoculating loops</td>
			<td>Fisher</td>
			<td>22363596</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile swabs</td>
			<td>Fisher</td>
			<td>1495990</td>
			<td></td>
		</tr>
		<tr>
			<td>Coy Vinyl Anaerobic Chamber and Accessories</td>
			<td>Coy Laboratory Products, Inc</td>
			<td>Customer Specified</td>
			<td>These items are custom ordered per laboratory needs</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TCCFA agar 
			 
			Proteose peptone no. 2 (Difco) 40 g 
			Na2HPO4 5 g 
			KH2PO4 1 g 
			NaCl 2 g 
			MgSO4 (anhydrous) 0.1 g 
			Fructose 6 g 
			Agar 20 g 
			 
			Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			After autoclaving, add: 
			10 ml of 10% (w/v) sodium taurocholate, filter-sterilized (dissolve in water; final concentration, 0.1%) 
			25 ml of 10 mg/ml &#7429;-cycloserine, filter-sterilized (dissolve in water; final concentration, 250 &#956;g/ml) 
			1.6 ml of 10 mg/ml cefoxitin, filter-sterilized (dissolve in water; final concentration, 16 &#956;g/ml) 
			 
			BHIS Medium 
			 
			Brain heart infusion 37 g 
			Yeast extract 5 g 
			 
			For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			Optional (add after autoclaving): 
			 
			3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 
			10 ml of 10% (w/v) sodium taurocholate (dissolve in water; final concentration, 0.1%) 
			 
			SMC Sporulation Medium 
			 
			BactoPeptone 90 g 
			Protease peptone 5 g 
			(NH4)2SO4 1 g 
			Tris base 1.5 g 
			Agar 15 g 
			 
			Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			Optional (add after autoclaving): 
			3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 
			 
			70:30 Medium 
			 
			BactoPeptone 63 g 
			Protease peptone 3.5 g 
			Brain heart infusion 11.1 g 
			Yeast extract 1.5 g 
			(NH4)2SO4 0.7 g 
			Tris base 1.06 g 
			 
			For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. After autoclaving, add 3 ml of 10% (w/v) L-cysteine (final concentration, 0.03%). 
			 
			Blood agar 
			 
			The use of premade Columbia anaerobic sheep blood agar plates (Fisher Scientific, L21928)35 is recommended. 
			 
			1X Phosphate buffered saline (PBS) 
			 
			NaCl 8.01 g 
			KCl 0.2 g 
			Na2HPO4 1.44 g 
			KH2PO4 0.27 g 
			 
			Bring to 1 L with deionized water and adjust pH to 7.4 with HCl. Filter sterilize before use.</td>
		</tr>
	</tbody>
</table>

culturing and maintaining clostridium difficile in an anaerobic environment

艰难梭菌是一种革兰氏阳性，厌氧，产孢子的细菌，主要负责抗生素相关性腹泻（AAD）和一个显著院内病原体。C.艰难是出了名的难以分离和培养，并是即使是低氧气的环境水平极为敏感。这里，对于隔离C.方法从难辨粪便样品，随后C.培养难辨用于制备甘油股票长期贮存介绍。编制和列举孢子股票在实验室用于各种下游应用，包括显微镜和动物实验技术也有所说明。这些技术的必要厌氧室，它保持一致的厌氧环境，以确保最佳的C。适当的条件艰难成长。我们提供的协议在室的进出转印材料未经CA使用显著氧污染以及用于维持适当的厌氧环境，高效的和一致的要求下定期保养建议难辨栽培。

C.示例艰难梭菌生长在BHIS和哥伦比亚厌氧绵羊血琼脂培养基上可以看出，在图2中 。C.难辨形成不规则的菌落是平的，拥有一个毛玻璃的外观，这是显而易见的两种培养基上。这里，C的红霉素敏感型临床分离难辨 ，630E 30，生长在BHIS琼脂，一个富集，非选择性培养基中，放置24小时，在37℃（ 图2A）。哥伦比亚厌氧羊血琼脂上菌落...

Watch this Scientific Journal Video about Culturing and Maintaining Clostridium difficile in an Anaerobic Environment at JoVE.com

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

艰难梭菌是一种致病细菌是严格厌氧并导致抗生素相关性腹泻（AAD）。这里，用于分离，培养和维持C.方法艰难梭菌营养细胞和孢子的描述。这些技术的必要厌氧室，这就需要定期保养，以确保最佳的C。适当的条件难辨栽培。

培养和维护<em&gt;难辨梭状芽孢杆菌</em&gt;在厌氧环境

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

culturing-maintaining-clostridium-difficile-an-anaerobic

Research

JoVE Journal

Immunology and Infection

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

38.3K 次观看.  Emory University School of Medicine.  艰难梭菌是一种致病细菌是严格厌氧并导致抗生素相关性腹泻（AAD）。这里，用于分离，培养和维持C.方法艰难梭菌营养细胞和孢子的描述。这些技术的必要厌氧室，这就需要定期保养，以确保最佳的C。适当的条件难辨栽培。 

视频: Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

科研

免疫与感染

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

头孢哌酮治疗小鼠模型的临床相关的<em&gt;艰难梭菌</em&gt;应变R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

表征形态变化的议定书<em&gt;艰难梭菌</em&gt;应对抗生素治疗

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...