We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1.启动生物膜<ol><li>成长生物膜的产生菌的培养在营养丰富的培养基。接种金黄色葡萄球菌菌株Newman中从一个甘油10毫升的Mueller-Hinton培养基的。执行涉及处理S.各项工作金黄色葡萄球菌手套和生物安全柜中。 </li><li>在37℃下孵育16小时，在旋转摇动器（100-200转）。 </li><li>决定使用分光光度计和标准比色皿（路径长度:1厘米）的文化的OD 600。 </li><li>计算使用下面的公式中chelexed罗斯韦尔园区纪念研究所介质（CRPMI）9来制备20种菌的溶液具有0.1的OD 600（接种物）所需的培养的体积（V）:[V = 20毫升* 0.1 / OD 600（培养）] 注:参考9 CRPMI准备提供详细信息。 <ol><li>通过在10,000×g下旋转1分钟加入计算培养物体积（从上方）到离心管中，并沉淀细胞。为了准备工作重新生物膜接种物，吸在20ml CRPMI所述废生长培养基和悬浮细胞沉淀。 </li></ol></li><li>倾接种到25ml的贮存器。 </li><li>小心地向外径直从无菌96孔挂钩板取下盖子。 注意:必须小心不要污染栓挂从盖向下。盖子可以倒放在干净的表面上的生物安全柜中。 </li><li>用200μl多通道移液器，加入125微升接种物到A行底板的G各孔中。 </li><li>填写排H的每个以及无菌控制125微升无菌CRPMI网上平台。 </li><li>取栓盖并保持其上的板底部朝下钉。小心地将各自的水井个人挂钩。避免在板和钉之间的物理接触。一旦对准，降低盖子仔细了。避免错位的钉后进行调整已经触及底板可续行H.氨化无菌控制</li><li>密封盖子平板用石蜡膜，防止蒸发发生在一个密封的塑料袋，封口严密。保持在袋尽可能低以减少蒸发的空气量。 </li><li>孵育没有在一个5％CO 2培养箱中于37℃振荡48小时。 注:48小时增长已经最佳为S.金黄色葡萄球菌 ，但会发生变化根据所使用的生物体。 注意:虽然静态孵化是一个很好的起点，以优化测定法，以150rpm在微孔板摇床轻轻摇晃是一个可能的变化，可能会提高一些S.生物膜的形成金黄色葡萄球菌菌株。 </li></ol> 2.生物膜挑战板的制作<ol><li>上生物膜挑战的天采取一个新的96孔板中并加入100微升CRPMI的，平衡至室温，以行的每个孔B至H.为了避免不一致的结果，利用96孔的板，其可容纳200μl的媒体当钉在里面，并设有尺寸完全相同生物膜启动板的孔中。 </li><li>稀释最多4试验化合物分别于1.0ml CRPMI的2倍的所需最终浓度，以占步骤2.7的最终稀释度。 注意:一个好的起点是8折浮游细胞的抑菌浓度以上。 </li><li>井A1加入200μl化合物1至A3，化合物2井A4至A6，化合物3井A7至A9和化合物4井A10至A12。 </li><li>通过使用多道移液器连续稀释测试化合物，并从A行转移100微升排B和混合。 </li><li>以这种方式，继续传输100微升很好很好。每次传输后混匀，停止行F. </li><li>混合后，从排出F行100微升到废物容器中。不要任何液体转移到行G和H </li><li>添加100微升CRPMI向板的每个孔用多道移液器向使体积为200微升。 图1所示为最终的板布局。 </li></ol> 3.生物膜挑战<ol><li>加入200μl的磷酸缓冲盐水（PBS）的到设有尺寸完全相同生物膜起始板的孔中新鲜无菌96孔板中。 </li><li>取下培养生物膜启动盘的塑料袋和石蜡膜。 </li><li>掀开锅盖直线上升。避免这些桩和板底部，因为这之间的接触可能会损坏生物膜。保持底部为后续OD 600分析（见3.8）。 </li><li>通过将钉盖到含PBS（步骤3.1）的平板冲洗掉浮游细胞。淹没钉5秒，吊出PBS，并淹没一次，小心不要撞到以及两侧的挂钩。 </li><li>放置冲洗钉盖成挑战板。避免钉和底板，因为这之间的不必要的接触会损伤生物膜导致不一致的结果。 </li><li>密封板用石蜡膜并放置到密封的塑料袋中步骤1.10中描述。 </li><li>孵育所述板没有在5％ 的 CO 2培养箱中于37℃振荡24小时。 </li><li>以生物膜启动盘底3.3。重悬在每个细菌用多道移液器井并测量OD 600与适宜酶标仪确认生长在所有孔中，但在排H的无菌控制发生</li></ol> 4.确定生物膜<ol><li>使用配有孵育室和能够采取动力学荧光读数酶标仪检测刃天青转换。使用530nm的激发和590 nm发射设立了24小时动能荧光测量。 </li><li>设置腔室温度至37℃，并有板阅读每20分钟。设置板器根据制造商的instruc到从板的底部测量系统蒸发散。 </li><li>通过加入200微升的PBS用多道移液器到无菌的96孔板中制备洗涤板。 </li><li>通过加入400微升的0.8毫克制备生物膜回收介质/ ml的刃天青原液至20ml CRPMI培养基至16微克/毫升刃天青的终浓度。 注:刃天青是一种已知的皮肤刺激。在处理使用白大褂，防护眼镜和手套。 CRPMI作为生物膜回收培养基使用，因为它提供了最低的背景信号，但它可以通过的Mueller Hinton培养基如果适当被取代。 </li><li>添加150微升生物膜回收培养基用多道移液器到一个新的96孔板的每个孔中。 </li><li>转移的挑战，从板块到孵化一个生物安全柜，小心地取出塑料袋，封口膜。 </li><li>从挑战板拆下钉盖和3.4中描述的冲洗掉浮游细胞在PBS。保持挑战板的底部后续OD 600 ANA裂解（见4.10）。 </li><li>冲洗后，将钉盖到含有生物膜回收介质板底部。 </li><li>包装用石蜡膜板的一侧，小心不要妨碍周边井的底部。立即包裹后盘，将其放置在酶标仪，并通过启动从步骤4.1以前刃天青动能协议作出重新开始进行24小时的动力读。 </li><li>量化浮游细胞的生长可能会或可能不会挑战过程中发生的是，读取用微型板读数器在整个挑战板底部的OD 600。按照步骤3.8的指示。 注意:仔细悬浮每个的内容以及细胞脱落掉的生物膜倾向于在井的底部中团块生长。阱内的任何气泡会强烈的OD 600读数干扰，必须避免或之前读除去。 </li></ol>

<ol>
	<li>Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M., Hoiby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+insights+from+biofilm+biology+to+drug+development+-+can+a+new+approach+be+developed.">Applying insights from biofilm biology to drug development - can a new approach be developed.</a> Nat Rev Drug Discov. 12, 791-808 (2013).</li><li>Song, Z., 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=Prosthesis+infections+after+orthopedic+joint+replacement:+the+possible+role+of+bacterial+biofilms.">Prosthesis infections after orthopedic joint replacement: the possible role of bacterial biofilms.</a> Orthop Rev (Pavia). 5, 65-71 (2013).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases).">Bacterial biofilms: from the natural environment to infectious diseases).</a> Nat Rev Microbiol. 2, 95-108 (2004).</li><li>Bordi, C., de Bentzmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hacking+into+bacterial+biofilms:+a+new+therapeutic+challenge.">Hacking into bacterial biofilms: a new therapeutic challenge.</a> Ann Intensive Care. 1, 19 (2011).</li><li>Fux, C. A., Shirtliff, M., Stoodley, P., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+laboratory+reference+strains+mirror+'real-world'+pathogenesis?.">Can laboratory reference strains mirror 'real-world' pathogenesis?.</a> Trends Microbiol. 13, 58-63 (2005).</li><li>Kwasny, S. M., Opperman, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+biofilm+cultures+of+Gram-positive+pathogens+grown+in+a+microtiter+format+used+for+anti-biofilm+drug+discovery.">Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery.</a> Curr Protoc Pharmacol. Chapter 13, Unit 13A 18 (2010).</li><li>Ceri, H., 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+MBEC+Assay+System:+multiple+equivalent+biofilms+for+antibiotic+and+biocide+susceptibility+testing.">The MBEC Assay System: multiple equivalent biofilms for antibiotic and biocide susceptibility testing.</a> Methods Enzymol. 337, 377-385 (2001).</li><li>Ceri, H., 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+Calgary+Biofilm+Device:+new+technology+for+rapid+determination+of+antibiotic+susceptibilities+of+bacterial+biofilms.">The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.</a> J Clinical Microbiol. 37, 1771-1776 (1999).</li><li>Baker, 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=Copper+stress+induces+a+global+stress+response+in+Staphylococcus+aureus+and+represses+sae+and+agr+expression+and+biofilm+formation.">Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation.</a> Appl Environ Microbiol. 76, 150-160 (2010).</li><li>Haeili, 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=Copper+complexation+screen+reveals+compounds+with+potent+antibiotic+properties+against+methicillin-resistant+Staphylococcus+aureus.">Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.</a> Antimicrob Agents Chemother. 58, 3727-3736 (2014).</li><li>O'Brien, J., Wilson, I., Orton, T., Pognan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Alamar+Blue+(resazurin)+fluorescent+dye+for+the+assessment+of+mammalian+cell+cytotoxicity.">Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.</a> Eur J Biochem. 267, 5421-5426 (2000).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+the+minimal+bactericidal+concentration+of+an+antimicrobial+agent+for+planktonic+cells+(MBC-P)+and+biofilm+cells+(MBC-B).">Establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (MBC-P) and biofilm cells (MBC-B).</a> J Vis Exp. (83), e50854 (2014).</li><li>Junker, L. M., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+for+small-molecule+inhibitors+of+Pseudomonas+aeruginosa+biofilm+development.">High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.</a> Antimicrob Agents Chemother. 51, 3582-3590 (2007).</li><li>Shah, 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=Persisters:+a+distinct+physiological+state+of+E.+coli.">Persisters: a distinct physiological state of E. coli.</a> BMC Microbiol. 6, 53 (2006).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riddle+of+biofilm+resistance.">Riddle of biofilm resistance.</a> Antimicrob Agents Chemother. 45, 999-1007 (2001).</li></ol>

The authors have nothing to disclose.

这里，我们已经描述了修饰的生物膜测定为S上确定测试抑制剂活性葡萄球菌生物膜注重生物膜有关的细胞的代谢状态。尽管所述生物膜起始和挑战的程序大多模仿制造商的建议，使用刃天青染料来检测和量化存活24小时抑制剂挑战生物膜相关的细胞显着地简化细胞回收（通过水浴超声处理），并随后列举的建议的过程通过的CFU计数的活细胞。该制造商推荐的程序涉及至少5个额外的操作步骤，他们每个人容易出错，劳动强度大，而且不切实际的自动化。简化了栓板测定的读出过程的优点增加了其对于高吞吐量操作和应用的吸引力。 虽然通过的步数冗长协议，该再修改的e是相当简单的概念，很少关键步骤。最关键的是，反复种植同样质量的生物膜的能力是获得可重复的结果至关重要。此外，必须拆卸或更换钉盖时小心。在一个良好的墙上任意点的联系会破坏生物膜，倾斜的结果。更何况，充足的洗濯是至关重要的:失败在任何时候以去除浮游细胞移除任何测量生物膜相关细菌的能力。相反，尽管，洗涤不能太严格;额外的洗涤步骤可以开始删除生物膜，产生不一致的结果。 栓柱板方法具有多种用途，这取决于所期望的结果数据。该法在这里描述为代谢刃天青转化的一个信息丰富的动能测量到的荧光染料11。这种转换是用从蓝色到粉红色的色移（ 图2C），其中，在additi相关到定量荧光读，可以容易初始质读出简单地通过观察转换。除去塞销盖后，这种转换也可以通过读取在600nm的吸光度应该荧光能力不可用定量。虽然荧光最终在每个时最大染料转化高原以及（幸存者），染料转化的发作可以使用相对于未处理的对照（ 图3）的代谢活性幸存者人口来估计。在我们手中，我们已发现，每个〜100分钟的延迟在刃天青转换的开始对应于开始相对于未处理的对照的接种（作为测定浮游细胞培养串联在代谢活性细胞的数量约10倍的降低在使用生物膜恢复介质10倍递增稀释（数据未显示））。 超越动力学读数，虽然，改性栓板方法更适合于高通量药物筛选项目比原始协议。通过测量刃天青的转换作为一个端点代替动力学，用户可以产生样品的二元分析来跟踪化合物简单的活动（ 即，染料转换/不转换），这消除了荧光板读数器的需要24小时，允许传统的孵化。这种方法允许确定的最小生物膜根除浓度无需声处理和电镀。动力学读还跟踪根据处理条件和抑制剂浓度生物膜的代谢状态。数目减少的操纵步骤，增加的再现性，定量读出方法（相对于电镀各孔单独地12），用户可以潜在地筛选数以千计每天化合物在单一浓度屏幕。事实上，栓式板先前已在高通量博士使用微克屏幕生物膜分散剂，虽然刃天青（如这里使用的）是一个更经济的选择比利用13专有的荧光素酶试剂。 而在现有的制造商协议的普遍改善，这里的特色化验确实有一定的局限性必须牢记。本质上，我们只衡量生物膜相关细胞的代谢能力。此法不测量生物膜质量或以其他方式削弱生物膜结构和完整性。这些影响不一定被检测到。代谢不活跃，但仍存活，持留细胞，这已被证明在生物膜14,15存在也将被检测到。进一步，诱导静止而不实际灭菌生物膜可能错误地显示为误报，取决于诱导状态下的长度的化合物。可行性指标是最终替代物更铁一般的测量方法，如流式细胞仪或CFU枚举，并且最终用户必须确定哪些方法是合适的。最后，由于刃天青转换依赖于内源NADH，细胞内贮存的耗竭可能是有害的细菌。虽然我们还没有看到对S.有害影响金黄色葡萄球菌 ，其他细菌物种可能会作出不同的反应。用户必须经验测试和验证此协议的可靠性和重复性给予不同的生物体和实验室环境。

致病微生物可在体内形成生物膜导致非急性慢性感染1。生物膜相关的感染是涉及异物（ 例如，人造骨替换，乳房植入物）或气管导管或导尿管2的安装植入医疗过程的严重危险因素。在这些情况下，抗感染治疗几乎总是必要的，因为基于生物膜的感染很少自行清除，甚至在免疫的个体。 金黄色葡萄球菌是在生物膜相关并发症牵连的最常观察到的病原体的使用期间发生的1微创医疗器械3。 不幸的是，生物膜的性质作为保护屏障，使他们对治疗比浮游细胞1,4-更耐磨，并预测临床疗效的评价是两个初始的关键部分药物开发以及耐药性监测。有越来越多的承认，实验室条件下，专注于浮游文化，可能无法忠实地代表现实世界的疾病5。进一步使问题复杂化，生物膜表型的复制是困难的，现有的生物膜模型繁琐和高之间和内部检测变异6受苦。因此，许多研究人员，通过必要性，默认为药物敏感性的浮游细胞的测定，从而潜在地忽略细菌毒力和疾病的一个重要方面。 在这里，我们描述了协议用 ​​于测定细菌，特别是S.金黄色葡萄球菌 ，在利用96孔基于生物膜系统-7,8-预生长的生物膜。而对生物膜形成和挑战的协议基本上遵循制造商的建议，我们提出一种替代信息丰富的方法用于量化biof的可行性攻击后ILM。简要地说，细菌是在栓板，其中生物膜上附着在板的盖的突出栓形成中培养。经过生物膜形成，钉在一个新的盘子装满PBS去除浮游细胞的井轻轻蘸。栓柱盖，具有附着生物膜，被转移到一个新的挑战板，含有不同浓度的抗生素进行检测。第二次温育后，盖子被再次除去，洗净，并转移至含有刃天青染料，这里它们经受一个最终温育恢复板。刃天青转化动力学上能够记录或作为一个定义的恢复期后端点读数。此染料型定量生物膜的可行性的方法从繁琐CFU（菌落形成单位）有很大不同的原始协议7所述计数基于方法学。药物的挑战板和刃天青转化动力学OD 600测量结果作为可行性读浮游和生物膜细胞，分别出局，提供快速，可靠，且技术上简单的信息丰富的生物膜存活试验。

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">&#160;Mueller-Hinton Medium</td>
			<td style="width:89px;">Oxoid</td>
			<td style="width:103px;">CM0405</td>
			<td style="width:195px;">Follow recommendations of manufacturer</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">RPMI-medium</td>
			<td style="width:89px;">Corning&#160;</td>
			<td style="width:103px;">17-105-CV</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:227px;">CRPMI</td>
			<td style="width:89px;">Ref 9</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">RPMI-1640 medium chelexed for 1h with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Chelex 100 Resin</td>
			<td style="width:89px;">Bio Rad</td>
			<td style="width:103px;">142-2822</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">MBEC-plates</td>
			<td style="width:89px;">Innovotech</td>
			<td style="width:103px;">19111</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Resazurin Sodium Salt</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">R7017</td>
			<td style="width:195px;">800&#181;g/ml in DI water&#160;&#160;&#160;&#160; Filter sterile</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Micro Plate Shaking Platform&#160;</td>
			<td style="width:89px;">Heidolph Titramax 1000</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Cytation 3 Plate Reader</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:58px;width:227px;">Gen5 software</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Recording and analysis of resazurin conversion</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Neocuproine</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">N1501&#160;</td>
			<td style="width:195px;">prepare 10 mM stock in 100% Ethanol, store at -80&#186;C</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Copper sulfate</td>
			<td style="width:89px;">Acros Organics</td>
			<td style="width:103px;">7758-99-8</td>
			<td style="width:195px;">prepare a 100 mM stock solution in water, store at 4&#186;C</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:227px;">Cu-Neocuproine</td>
			<td style="width:89px;">Self-Made</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Gentamicin</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">G3632-1G</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

刃天青试验是非常灵敏可靠地检测能力攻击后复制的药物培养基很少活细胞。在这种方法中，我们定义的最小生物膜根除浓度在哪些没有刃天青转化24小时内看到的最低浓度。刃天青检测依赖于它转换染料的颜色由蓝色变为粉红色代谢活跃的细胞中发现的氧化分子。染料转换是这样的细胞生长的一个指标，并且可以使用荧光微板读数器来量化。在本文中，亚铜灵和Cu-亚铜灵（铜亚铜灵络合）为他们的活动进行了测试对生物膜有关S.金黄色葡萄球菌施加是基于图1的布局。该协议的执行过程中获得的数据的OD 600读数生物膜起始板的，它作为一种质量控制（数据未示出）中，板底部的挑战后的OD 600读数已经完成（ 图2A），和刃天青变换的动能记录到回收板（ 图2B）的其荧光代谢物试卤灵。进一步的挑战板的终点图像可以用于刃天青转换（ 图2C）的目视检查，如果有/无刃天青转换评价足够用于实验的目的。另外，如果动能录制功能不可用或多个板被并行运行，荧光可以之后通过单个端点读取24小时来确定。如在图2A中看到的那样，从挑战板的OD读数表明，单独亚铜灵不抑制浮游细胞的生长;然而，铜亚铜灵以1.3μM抑制生长，如预期10。一个类似的模式被从生物膜板的刃天青动力学观察到的，指示只有铜亚铜灵能够消除代谢活性生物膜相关的细胞（ 图2B）。 除了提供生物膜根除浓度，动力学测定法提供了在其上指示的活菌数的生物膜的代谢状态的信息。当比较用庆大霉素的浓度增加对未处理的对照处理的生物膜，刃天青变换的滞后可以看出（ 图3）。为2.5微克/毫升庆大霉素，有一个13小时的延迟，直到井达到刃天青的相同的转换作为未处理的对照。这种滞后可能来自存在于处理生物膜与未处理的活细胞的数目减少。 <img alt="图1" src="/files/ftp_upload/53925/53925fig1.jpg" /> 图1.挑战板布局。为例挑战板电子布局在六个不同浓度的容纳一式三份四种化合物并行测试。未处理的和不育对照也包括在内。布局设计支持方便板准备使用多管移液器连续稀释液。浓度是μM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53925/53925fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53925/53925fig2.jpg" /> 图2.确定最小生物膜根除浓度。新铜试剂和Cu-亚铜灵被消灭生物膜电位测试。 （A）中的OD 600从挑战板采取除去钉盖的后量化生物膜脱落的细胞的生长。 （B）刃天青转化率在INDIVI动力学监测双口井在24小时的时间内。个人minigraphs显示一段时间（24小时）荧光（RFU）。缺乏刃天青转化表示没有幸存者。刃天青转换表示存活细胞脱落掉的生物膜和在回收培养基中生长的存在。 （3）如果动能读出是不可能或不需要，那么一个简单的是/否的答案活细胞的存在，可以直观地从蓝色刃天青到粉色，简化形式的颜色转换决定。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53925/53925fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53925/53925fig3.jpg" /> 图3.动力学测定刃天青揭示生物膜对细菌有抑制作用。化合物可能无法完全根除生物膜嵌入细胞，但仍可以减少IR可行性。这可以比较刃天青用不同浓度的庆大霉素处理的孔的转化延迟时可以看到。具有在刃天青转换延迟井表示存活细胞的数量减少。黑色（仅媒体），橙（0微克/毫升），绿（0.3微克/毫升），蓝（0.6微克/毫升），紫（2.5微克/毫升），和灰色（5微克/毫升）。 ΔT是指在大致一半最大荧光值未处理和庆大霉素处理的样品呈指数刃天青转换的时间差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53925/53925fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

12.9K 次观看.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

视频: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

科研

免疫与感染

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

皮下感染耐甲氧西林金黄色葡萄球菌（MRSA）的

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

耐甲氧西林实验性心内膜炎模型<em&gt;金黄色葡萄球菌</em&gt;（MRSA）的大鼠

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

金黄色葡萄球菌生长使用人血红蛋白作为铁源

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

木炭琼脂刃天青分析的可视化分枝杆菌的半定量，中等吞吐量枚举

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

方法基因水平转移的研究<em&gt;金黄色葡萄球菌</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...