Name: visualization of the charcoal agar resazurin assay for semi-quantitative, medium-throughput enumeration of mycobacteria
Uploaded: 2016-12-14T13:00:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria at JoVE.com

我们感谢克里斯汀伯恩斯黄的手稿和J.戴维·沃伦（威尔康乃尔医学院）的化学援助，同时开发CARA的专家评审。这项工作是由比尔和梅林达·盖茨基金会，艾比和霍华德P.米尔斯坦计划在转化医学的结核病药物加速器计划，以及美国国立卫生研究院TB研究组（U19 AI111143）支持。微生物学和免疫学系是由威廉·伦道夫·赫斯特基金会的支持。 SSK是由美国国立卫生研究院资助K08AI108799支持。

1. CARA微孔板的制备<ol><li>高压釜900毫升含有0.2％的甘油和0.4％活性炭在2升Erlenmeyer烧瓶的Middlebrook 7H11琼脂的，或可替代地450毫升的1升玻璃烧杯中。包括烧瓶或烧杯大型高压灭菌搅拌棒。覆盖烧瓶或烧杯用铝箔的开口并连接到玻璃高压釜胶带。 </li><li>冷却到触摸（约55-65℃）在磁力搅拌板上设置以低速维持在悬浮液中的木炭。 </li><li>无菌地执行在具有磁搅拌板上的生物安全罩的所有后续步骤。 </li><li>删除箔。加入100毫升OADC补充（OADC补充，当在10％的人使用，0.2％葡萄糖，0.5％白蛋白，0.085％的NaCl，0.0005％的油酸，和0.4毫克/毫升过氧化氢酶的产率最终媒体浓度）的2升Erlenmeyer烧瓶，或者50μLOADC到1升烧杯中，继续混合。 </li><li>如果使用锥形瓶中，倒入约25-40毫升7H11-OADC木炭成无菌试剂水库。如果使用的是烧杯中，这是没有必要使用一个试剂容器。 </li><li>填充在96孔微量培养板用200μl/孔的从试剂贮存器或烧杯7H11-OADC炭。迅速开展工作，以避免琼脂凝固和引入气泡。避免溅孔的固体介质以外，作为可以被真菌污染的来源。使用多道移液器，使用一组12个过滤嘴对7H11-OADC木炭转移到96孔板的8行（AH）。 注:媒体快速凝固。为了避免堵塞枪头，经常改变它们。 </li><li>另外，倒CARA微孔板使用P1000电子多道移液器与过滤嘴协助编写众多板块。 注:由于微孔板的低量，在CARA微孔板琼脂浇分钟内凝固。 </li><li>在可重复密封的塑料BA CARA微孔板地点栈GS以避免干燥。 </li><li>商店CARA微孔板在4℃。 </li></ol> 2.设置复制和非复制MIC 90测定<ol><li>接种结核分枝杆菌 ， 牛分枝杆菌 BCG或耻垢分枝杆菌在一个OD的0.01-0.1 580和扩大到米德尔7H9-ADN数中期（OD 580〜0.5）（含有0.2％甘油的Middlebrook 7H9，0.2％右旋糖，0.5％白蛋白和0.085％NaCl）中或7H9-OADC（含有0.2％甘油和10％OADC补充的Middlebrook 7H9）。 </li><li>生长的结核分枝杆菌和牛分枝杆菌 BCG的约20毫升站在培养物在细胞培养瓶中和M.在聚丙烯圆底（4毫升培养）或50毫升锥形离心管（10-20毫升培养）振摇耻垢 。在37℃下孵育病原分枝杆菌，用20％ 的 O 2和5％的CO 2，和M，在37℃ 耻垢 ，用20％ 的 O 2。 </li><li>建立一个最小抑制浓度（MIC 90）根据复制和非复制的条件（ 图2）式的实验。 注:测试剂通常测定在重复或一式四份，以允许测试4，或2的化合物，分别为每96孔微孔板。例如，在一个96孔板，一个测试药剂可以以行AD和另一行中的EH测定。二甲基亚砜（载体）是在2列1和12，和测试试剂稀释系列从塔3（最低浓度），运行到塔11（最高浓度）。 MIC 90式的测定法通常采用2倍稀释系列。有可用于分枝杆菌14-16,18,24,25众多非复制模型，并出于说明的目的，我们使用非复制6,8,9的多应力模型。 <ol><li>为复制测定法，散布在7H9-ADN200μl的细胞以一个OD的0.01 580到透明底，组织培养物处理的96孔板的所有孔中。 </li> &lt;利&gt;对于非复制测定法，洗涤细胞两次在含有0.02％四丁酚醛的磷酸盐缓冲盐水（PBS），和重悬的细胞在非复制培养基（0.05％KH 2 PO 4，0.05％ 硫酸镁 ，柠檬酸铁铵0.005％ ，0.0001％的ZnCl 2，0.1％ 氯化铵 ，0.5％BSA，0.085％氯化钠，0.02％四丁酚醛，0.05％丁酸;调节pH至5.0，用2N的NaOH）。 <ol><li>稀释细胞的OD 0.1 580中的非复制介质和从新鲜制备的1M库存添加的NaNO 2至0.5mM的最终浓度。 </li><li>分发200微升的细胞以的OD 0.1 580到透明底，组织培养物处理的96孔板的所有孔中。 注:准备化合物稀释液为100倍储备溶液在DMSO中。因此，典型的MIC板在最终浓度0.4-100微克测试分子的冲击/ ml的将需要0.04-10毫克/毫升的DMSO储备溶液。 </li></ol></li><li>加入2微升迪测试剂1的决方案成行AE和2微升测试剂2稀释成排的EH。调匀。 </li><li>加入2微升车辆控制（通常DMSO）为对照孔，列1 2，12（行AH）。调匀。 </li><li>对于每一个实验中，包括至少一个阳性对照，例如从0.004至1微克/毫升（复制法）和/或0.08至20微克/毫升（非复制测定）利福平。 注意:在0.1至25微克/毫升的使用6-溴-1H-吲唑-3-胺9的被推荐为具有选择性的控制化合物，纳米2依赖性活性在非复制的多应力模型。 </li><li>用于复制测定法，在37°C孵育微孔板在20％O 2和5％CO 2的7天（ 结核分枝杆菌和牛分枝杆菌 BCG）或1-48小时（ 耻垢分支杆菌 ）。非复制的多应力模型，孵育微孔板7天，在37℃在1％ 的 O 2和5％的CO 2（&lt;EM&gt;米结核病和牛分枝杆菌 BCG）。 </li></ol></li></ol> 3. CARA微孔板接种<ol><li>在该CARA将被用作一个读出的时间点，小心悬浮孔用P200多道移液器设定在50-75微升的MIC 90式的测定板的内容。移液器上下至少5-10倍，并且轻轻摇动用枪头圆周运动的好内容。 </li><li>将10微升检测以及内容的CARA微孔板。确保对测定板的孔内容的顺序的CARA微孔板的孔内容的顺序相匹配。在转移过程中避免飞溅，并确保10微升被发现到CARA微孔板的中间。确认10微升吸收到CARA微孔板。 注:没有察觉的CARA微孔板细胞之前，必须稀释。 </li><li> CARA的绑定栈微孔板与板带和然后放入一个密封的塑料袋。孵育CARA微孔板在37℃下，用20％ 的 O 2（ 耻垢分枝杆菌 ），或1％ 的 O 2和5％的CO 2（ 结核分枝杆菌和牛分枝杆菌 BCG）。 </li><li> 结核分枝杆菌和牛分枝杆菌 BCG，复制测定法:孵育7天;对结核分枝杆菌和牛分枝杆菌 BCG非复制测定，孵育10天;对耻垢分枝杆菌 ，复制测定，孵育1-2天;对耻垢分枝杆菌 ，非复制实验，孵化2-3天。 注:时间为估计值，可以针对不同的复制，非复制和压力条件下相应的修改。 </li></ol> 4.制定CARA微孔板<ol><li>开发CARA微孔板时细菌的生长，或更大，宏观菌落的膜，是在负（载体）对照孔可见。 注意:长时间培养后，CARA微孔板常出现干燥，我们建议预湿以及内容用无菌PBS。这用于防止木炭从吸收刃天青，从而导致低荧光或油井到井变化。 </li><li>使用一组12个P200提示用多道移液器，取40微升无菌PBS沿着孔的侧面，并允许PBS横跨琼脂/细菌小菌落的顶部分布。 </li><li> CARA准备通过混合5毫克刃天青（0.01％最终），并在PBS50毫升5％吐温80的显色剂。涡和无菌过滤器。 注:1（体积/体积）用在PBS中的10％吐温80:另一种CARA显影试剂可通过混合刃天青在1液体溶液的商业制备来制备。 </li><li>使用12通道移液器加入50μl新鲜制备CARA显影试剂的CARA微板的各孔中。岩石板来回了几次，帮助每孔跨分配琼脂和细菌垫试剂。 </li><li>将平板我呐再密封的塑料袋中，并孵育在37℃下为分枝至少30分钟耻垢和45-60分钟为结核分枝杆菌或牛分枝杆菌 BCG。 注:如果在车辆对照孔未能在第一个小时内变成粉红色，板可以被重新袋装并孵育时间较长。 </li><li>阅读荧光之前，放置CARA微孔板在生物安全橱中在室温下与盖子去掉15分钟。当使用生物安全柜外BSL3分光光度计，坚持光学品质PCR贴在板上，并用柔软的纸巾轻轻按压贴纸表面上的密封严密。 </li><li>确定经由顶端与在530nm激发和发射在590nm读取荧光。这是没有必要为空白盘。 </li></ol> 5.数据分析<ol><li>积抑制剂浓度上对数10的规模和荧光在Y轴的X轴上的线性刻度。可以使用分散采用曲线图符合如"日志抑制剂与响应可变斜率（4参数）"。绘制数据点，平均值±标准差。 </li></ol>

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有没有利益冲突披露。

卡拉最初被开发为缓解进展非复制或双活性分子7的瓶颈。该CARA作为初筛安打和CFU测定（ 图1）的浓度-应答确认之间的中间步骤。由于单个CARA板可以代替用来列举存活的细菌准备连续稀释需要大量的微孔板，以及含有琼脂培养皿中，CARA提供了一个简单的方法来快速评估分子的活动，并测试一次多个变量，包括化合物浓度和暴露于化合物的时间。 在一个CFU试验中，除了系列稀释经常范围高达10 6倍，琼脂板通常稀释由附加〜800倍（10微升到8毫升在一个三-式培养皿）分子。我们的两阶段，对活性化合物的多应力筛选测定非复制分枝涂berculosis具有5倍的结转因素，那就是，一个存在于该测定的非复制阶段化合物以五分之一的原始浓度在生长测定6-9的（复制）相。卡拉减轻了通过隔离的小分子用活性炭结转的效果。多数结核病药物和临床候选结合活性炭迅速而完全，用氨基糖苷类链霉素7的异常。在CFU测定琼脂平板活性炭纳入防止细菌生长抑制，或细菌杀死，被结转TMC207和PA-824 7,21,22。因而，借助于在细菌学琼脂掺入活性炭，该CARA不依赖于细胞和测试试剂的系列稀释液。 卡拉有助于预测复制活性和非活性复制杀菌影响的抑菌或杀菌的影响。 IN GENERAL，卡拉是并行使用标准的MIC 90测定。的CARA的预测能力来自比较的MIC 90和CARA的结果（ 图2和表1）。当用于研究的抗分支杆菌剂，该CARA能准确地预测被复制杀菌（ 图4a）的活性，复制抑菌（ 图4b），非复制杀菌（ 图4c），或双活性（ 图4d）。卡拉还允许在这两个时间和剂量复合的活动简单的评价。 CFU测定单独需要大量的努力和材料，以评估在宽范围的剂量和时间的化合物的活性，但是当CARA结果缩小的条件范围的那些任务变得易于管理，其中所述化合物可以证明是活性（ 图5 ）。 虽然CARA在学习的行动工具在分枝杆菌NTI感染治疗，化验有其局限性。该CARA具有窄的动态范围（2-3日志10）和用于在其中一个预计可能不存在超过2至3个对数10细菌杀灭条件可能不适合。 CARA预测需要使用更严格和准确的方法来枚举细菌数量，诸如CFU试验进一步研究。一些抗感染药，如PAS的抗生素后效应，可以混淆CARA作为预测工具针对复制的结核分枝杆菌 7与杀菌和抑菌活性的化合物之间进行区分。 有两个建议，以提高CARA的质量。首先，必须倾CARA琼脂凝固之前微孔板迅速，同时保持空间精度和避免微孔以外飞溅。无论所需板的数量，我们建议在做介质的0.5至1升批次，以帮助维持对Medium液体形式为倒板所需要的时间的持续时间。更改提示经常却使微孔板用中使用部分堵塞的提示避免。其次，必须最大限度地减少重复的荧光人为的变异。分枝杆菌菌落，特别是用于病原性分枝杆菌，例如结核分枝杆菌 ，经常不稳定生长。例如，小菌落在琼脂表面上生长可以在大小，形状，高度变化，或者可以延伸到微孔板的内壁。一个挑战是与显色剂均匀覆盖全部杆菌。另一个障碍是，在37℃下延长温育后，该CARA微孔板的固体细菌学介质可以变得干燥，易吸收的显影试剂。因为活性炭可以结合刃天青和急冷荧光7，有可能是从油井到井的变化，由于干井吸收刃天青显影溶液和活化charcoa升淬刃天青荧光。预润湿立即加入显影试剂之前所有CARA微孔板用PBS表面减轻这两个问题 - 显影试剂将同样达到所有分枝杆菌菌落，并且刃天青活性炭上述安全地保持。该CARA可以具有识别和表征分枝杆菌突变体的表型，或在介质通量药物筛选测定法的其他细菌种类的应用程序。

结核分枝杆菌 ，结核的病原体，可以在处于潜伏状态的主机是难以用抗生素根除生存。表型（非遗传）感染期间结核分枝杆菌的抗性被认为是由于，在部分地非复制杆菌1,2-种群。 I类持久化的表型显示耐药性之间通过大量人口的药物敏感细胞3,4罕见随机产生的机制。 II类持留由宿主免疫的因素，包括在感染过程中遇到的微环境的外部应力呈现非复制。我们最近开发II类的体外模型的非复制的持久性，以模仿由结核分枝杆菌在活化巨噬细胞和肉芽肿面临的条件。 II类的持久性的多应力模型包括条件生长缓慢，如脂肪酸碳源，并完全哈尔吨增长，如轻度酸性（pH值5.0），低氧（1％O 2）和一氧化氮和其它的反应性氮中间体5-9。使用非复制的这种多应力模型大规模筛查，和其它II类非复制模型，已经产生了多种分子，其活性是针对非复制状态5-7,9-18。相同的非复制的屏幕还显示，具有抗既复制和非复制杆菌活性的分子的一类，称为"双活性"7,10,12,19,20。 抗菌药物发现的命中以铅阶段的工作，候选分子的广泛特性来选择命中扩张，初步药理特性，目标识别和初步的体内药效学研究线索。作为早期步骤，抗感染药由行动其抑菌或杀菌机理和分类，如果杀菌，W论是细菌灭杀是时间和/或浓度依赖性。的菌落形成单位（CFU）测定是为解决这些问题的古典，金标准方法。在CFU试验，细菌暴露于测试试剂，之后，等分试样被去除，连续稀释，并稀释的等分试样散布固体细菌学培养基上，并温育，以允许存活细胞的生长。最后，细菌菌落列举。 CFU试验需要大量酶标板稀释细胞和含有琼脂的Petri板枚举幸存的殖民地。对于缓慢生长的分枝杆菌的CFU试验是通过缓慢的生成时间（18-24小时），这需要大约3周菌落平板上出现阻碍。另外，培养箱空间专门生物安全级别为3的设施通常是有限的。 虽然繁琐，CFU试验是黄金标准来表征非复制型和双活性分子对mycobacter的影响IA。非复制的检测方法受到许多连接到复制测定以评估细胞活力6-8的高误报率。例如，具有抗复制结核分枝杆菌有效的活性化合物（一种"复制活性"）可能无法在非复制测定期间杀死，但仍然可以通过凭借结转从的非复制相杀测定到测定，这是支持复制（"复制条件"）的条件下进行的恢复阶段。化合物结转进一步复杂化的双活性分子的分析，使得难以区分的活性是否被复制，非复制，或双。 为了解决上述问题，我们开发的C harcoal 一个噶尔- [R esazurin对分枝杆菌快速，半定量枚举 SSAY（CARA）如M. tubercul肌病， 牛分枝杆菌 BCG和耻垢分枝杆菌 7（ 图1和2）。在体外缓冲溶液，活性炭迅速隔绝大多数用于治疗肺结核7的标准药物。在卡拉活性炭微孔板结合，可能会从一个测定微量结转化合物和CARA的这一特点消除了串联稀释到枚举7,21,22事先测定孔内容的要求。 CARA微孔板的琼脂表面上分枝杆菌菌落的数目通过加入刃天青的估计，蓝色染料，其还原形式，试卤灵，是粉红分子通过一个分光23测量其荧光。 CARA微孔板温育1-2天的耻垢分枝杆菌和7-10天的缓慢生长的分枝杆菌如结核分枝杆菌和牛分枝杆菌 BCG。卡拉有2-3〜10日志的迪菲一个狭窄的动态范围伦斯在CFU。当使用代替CFU试验中，单个CARA微孔板替换大约用于用于电镀连续稀释和120三 - 风格琼脂平板5个96孔板中。 CARA数据的解释有助于确定哪些孵育时间和化合物浓度来测试比较费力基于CFU-试验指导今后的研究。

<table><tbody><tr><td>Middlebrook 7H9</td><td>Beckton Dickinson</td><td>271310</td><td></td></tr><tr><td>Middlebrook 7H11</td><td>Beckton Dickinson</td><td>298810</td><td></td></tr><tr><td>Middlebrook OADC</td><td>Beckton Dickinson</td><td>212351</td><td></td></tr><tr><td>BSA, heat shock</td><td>Roche</td><td>3118958001</td><td></td></tr><tr><td>activated charcoal</td><td>Sigma</td><td>C5510</td><td></td></tr><tr><td>PBS, Dulbecco&#039;s Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; free</td><td>Life Technologies / Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>tween 80</td><td>Sigma</td><td>P8074</td><td></td></tr><tr><td>tyloxapol</td><td>Sigma</td><td>T8761</td><td></td></tr><tr><td>sodium nitrite</td><td>Sigma</td><td>2252</td><td></td></tr><tr><td>rifampicin</td><td>Sigma</td><td>R3501</td><td></td></tr><tr><td>6-bromo-1H-indazol-3-amine</td><td>Alfa Aesar</td><td>H34095</td><td></td></tr><tr><td>potassium phosphate monobasic&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>P0662</td><td></td></tr><tr><td>magnesium sulfate, heptahydrate&amp;nbsp;</td><td>Sigma</td><td>M1880</td><td></td></tr><tr><td>ferric ammonium citrate&amp;nbsp;</td><td>Sigma</td><td>F5879</td><td></td></tr><tr><td>zinc sulfate, heptahydrate</td><td>Sigma</td><td>Z0251</td><td></td></tr><tr><td>ammonium chloride&amp;nbsp;</td><td>Sigma</td><td>A9434</td><td></td></tr><tr><td>butyric acid, liquid</td><td>Sigma</td><td>B103500</td><td></td></tr><tr><td>resazurin powder</td><td>Sigma</td><td>R7017</td><td></td></tr><tr><td>sodium chloride&amp;nbsp;</td><td>J.T. Baker</td><td>4058-01&amp;nbsp;</td><td></td></tr><tr><td>prepared resazurin solution&amp;nbsp;</td><td>Invitrogen</td><td>DAL1100</td><td></td></tr><tr><td>PCR stickers</td><td>Denville</td><td>B1212-5</td><td></td></tr><tr><td>spectrophotometer</td><td>Molecular Devices</td><td>M5</td><td></td></tr><tr><td>96-well, tissue culture treated microplates</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>reagent reservoirs</td><td>VWR&amp;nbsp;</td><td>89094-678</td><td></td></tr><tr><td>resealable plastic bags</td><td>VWR&amp;nbsp;</td><td>395-94602</td><td></td></tr><tr><td>14 ml Polypropylene round-bottom tubes</td><td>Corning&amp;nbsp;</td><td>352059</td><td></td></tr><tr><td>50 ml conical centrifuge tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Cell culture flask</td><td>Corning</td><td>431464U</td><td></td></tr><tr><td>clear, flat bottom tissue culture treated 96-well microplate</td><td>Costar</td><td>3595</td><td></td></tr><tr><td>Prism 6 for OS X</td><td>GraphPad</td><td>http://www.graphpad.com/scientific-software/prism/</td><td></td></tr></tbody></table>

visualization of the charcoal agar resazurin assay for semi-quantitative, medium-throughput enumeration of mycobacteria

有一个迫切需要发现和进展抗感染药的缩短结核病治疗时间。 结核分枝杆菌，结核的病原体，是难治快速和持久的化疗由于表现出的表型耐药性杆菌的存在。的C harcoal 一个噶尔řesazurin 一个 SSAY（CARA）的开发，以表征由高通量筛选广告活动发现的活性分子针对复制型或非复制型结核分枝杆菌的一种工具。在细菌学琼脂培养基活性炭的夹杂有助于减轻化合物结转的影响，并消除了对预稀释之前斑点上CARA微孔板细胞的要求。后在37℃的7-10天的潜伏期，由分枝杆菌菌落CARA微孔板的表面上生长刃天青的还原允许半定量ASSE通过荧光细菌数量的ssment。卡拉约检测细菌数2-3日志10差并预测最低杀菌浓度导致≥99％的细菌杀死（MBC≥99）。卡拉有助于确定分子是否在被复制杆菌，非复制，或既活跃。使用卡拉导频实验促进其中的测试药剂和化合物曝光的时间的浓度需要由菌落形成单位（CFU）的测定进一步评估鉴定。此外，如果复制的活性成分都属于杀菌或抑菌卡拉无法预测。

预期CARA结果在图2中描述，并在表1中总结。对于复制的细胞时，卡拉用的标准的MIC 90测定并行运行，而对于非复制测定法，该CARA与被耦合到一个生长阶段的适于MIC 90测定并行运行。测试试剂，其导致CARA荧光的失败可以上升到高于背景水平的浓度是CARA-MBC≥99（ 图3a）。在"≥99"下标表示该CARA-MBC提供测试剂的浓度估计，使人们产生≥2日志10细菌灭杀（≥99％杀死）。 液体肉汤MIC 90测定是无法杀菌和抑菌活性区分和这种区别必须由CFU试验来解决。通过惯例，从生长缓慢的分枝杆菌抑菌活性区分杀菌的阈值近似超过7天7.26 2-3日志10杀。由于CARA的动态范围也2-3日志10杀，该CARA可以提供杀菌或抑菌活性的估计。该CARA容易识别一些复制活性抑菌由于这些化合物的故障减少CARA荧光背景水平（ 图2）。然而，一些化合物具有抑菌活性对复制的结核分枝杆菌具有有效的抗生素后效应，这意味着它们继续甚至在不存在化合物结转在恢复阶段，以抑制细菌的生长。这种效果可能是困难的卡拉法来识别。化合物被怀疑施加抗生素后效应，如果它们显示一个"静态窗口"，其定义为一&gt; 4倍移向右between中的MIC和CARA曲线（ 图3b）。静态窗口指示不要照搬结核分枝杆菌的活性分子可能是抑菌而不是杀菌。在一些情况下，静止窗户仅膨胀Y轴为CARA荧光（ 图3c和3d）的检查后是显而易见的。 CARA和MIC 90的数据通常被绘制在一起（ 图4）。通过MIC 90和CARA测试replicating-和非复制活性分子的代表性数据示于图4。有对分子4大活动类:1）复制杀菌（证明用异烟肼， 图4a）; 2）复制抑菌（利奈唑胺， 图4b）证明; 3）非复制杀菌（展示用羟基保泰松， 图4c）;并且，4）代表licating活性（抑菌或杀菌）和非复制杀菌（与PA-824， 图4d所示）。重要的是， 图4a和4b表明，虽然异烟肼和利奈出现由MIC 90测定以具有针对非复制的细菌的活性，所述CARA表明它们是所测试的非复制的条件下无活性。为了测试CARA的效用在预测的分子的时间和浓度依赖性的影响，复制耻垢分枝杆菌暴露于增加利福平浓度（ 图5a - d）中 ，并在1-24小时之间的不同时间，将等分试样点样CARA上板。这些数据表明，利福平施加的冲击，早在1小时（ 图5a），第3和24小时（ 图5b-d）中的显示增加的杀菌活性，并且由2在〜10μg/ ml的杀≥2-3日志104小时（ 图5d）。一个类似的实验测试的车辆的控制和9的药物浓度一式四份，并在4个时间点，将是一个标准的基于CFU-测定望而却步。 因此，该CARA在药物发现作用作为介质通量，快速机制来标识一个分子的活性曲线。 CARA预测值应该使用标准CFU试验进行严格的评估。在加入0.4％活性炭至培养皿为CFU分析可能有助于改善相关性CARA数据，并可能导致更准确的CFU计数，校正的幅度通常正比于化合物的效力21,22。 <img alt="图1" src="/files/ftp_upload/54690/54690fig1.jpg" /> 图1:CARA在药物开发的预测工具。此图总结了CARA的效用为药物筛选（单点筛选，挑肥拣瘦，和剂量应答测定）和费时命中到铅测定（CFU测定法和目标识别）之间的中间阶段。 [与黄金等改编的权限，抗菌制剂和化疗，2015年 7] <a href="http://ecsource.jove.com/files/ftp_upload/54690/54690fig1large.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/54690/54690fig2.jpg" /> 图2: 肉汤MIC 90测定和CARA与预期效果示意图。这两个MIC 90和CARA结果中给出了8种可能的活动。颜色编码为MIC 90微孔板为白色（没有增长）和布朗（增长），以及CARA微孔板为黑色（无荧光）和粉色（试卤灵荧光）。数据是假设。 [改编与黄金等权限，抗菌制剂和化疗，2015年 7] <a href="http://ecsource.jove.com/files/ftp_upload/54690/54690fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54690/54690fig3.jpg" /> 图3:CARA 专业术语和定义。导致CARA荧光背景水平的分子的最小杀菌浓度是CARA-MBC≥99（a）中 。下复制的条件下，CARA-MBC≥99到MIC 90的右侧的一个≥4倍移常常表示抑菌活性和被称为"静态的窗口"（b）中 。对于一种有效的抗生素后效应分子，静态窗口可能难以观察（c）和要求Y轴（CARA荧光）扩展可视化（D）。数据是假设。 [与黄金等改编的权限，抗菌制剂和化疗，2015年 7] <a href="http://ecsource.jove.com/files/ftp_upload/54690/54690fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54690/54690fig4.jpg" /> 图4: 说明性MIC 90和CARA结果选择化合物。对于MIC 90（红色）和CARA（蓝色）的数据被证实为（ 一 ）异烟肼（INH），（B）利奈唑胺，（C）羟基保泰松，和（d）的PA-824。野生型结核分枝杆菌H37Rv暴露于化合物为标准复制条件或非复制7-9的多应力模型下7天。<html> 如图2中进行的MIC 90测定和CARA。 [与黄金等改编的权限，抗菌制剂和化疗，2015年 7] <a href="http://ecsource.jove.com/files/ftp_upload/54690/54690fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54690/54690fig5.jpg" /> 图5: 剂量-和利福平的时间依赖性活性。非致病的，快速生长耻垢分枝杆菌暴露于复制条件和等分下增加利福平的浓度分别固定在取样CARA 1（a）中 ，图3（b），6（c）和24（D）小时。 [改编与黄金等权限，抗菌制剂和化疗，2015年 7].COM /文件/ ftp_upload / 54690 / 54690fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/54690/54690tbl1.jpg" /> 表1:图2的预期结果的总结 。 [与黄金等改编的权限，抗菌制剂和化疗，2015年 7] <a href="http://ecsource.jove.com/files/ftp_upload/54690/54690tbl1large.jpg" target="_blank">请点击这里查看此表的放大版本。</a>

Watch this Scientific Journal Video about Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria at JoVE.com

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

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 ...

visualization-charcoal-agar-resazurin-assay-for-semi-quantitative

Research

JoVE Journal

Immunology and Infection

10.4K 次观看.  Weill Cornell Medical College. 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. 

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

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

科研

免疫与感染

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

用于宿主靶向治疗结核病临床前检测的自动化培养系统

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

<meta charset="utf8"></head><body>使用双报告基因的 脓肿分枝 杆菌高通量筛选测定

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

<meta charset="utf8"></head><body>作者聚焦:用于高效发现 脓肿分枝杆菌 治疗的可扩展药物筛选方案

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

分枝杆菌高通量药物筛选的分析开发

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Source: Schaaf, K., et al. <a href="https://app.jove.com/v/55453">A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

评估对结核分枝杆菌感染模型的药物疗效的测定

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

A Charcoal Agar Resazurin Assay to Evaluate Test Compound Activity Against Mycobacteria

Source: Gold, B. et al., <a href="https://app.jove.com/v/54690">Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria.</a> J. Vis. Exp. (2016)
This video showcases the Charcoal Agar Resazurin Assay (CARA) for evaluating the activity of antimicrobial compounds against Mycobacterium tuberculosis. CARA aids in assessing the efficacy of these compounds by gauging their impact on bacterial growth and distinguishing between bactericidal and bacteriostatic effects.

评估测试化合物对分枝杆菌活性的木炭琼脂刃天青测定

1. Setting Up Replicating and Non-replicating MIC90 Assays

	Inoculate M. tuberculosis, M. bovis BCG or M. smegmatis at an OD580 of 0.01-0.1 and expand to ...

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

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此视频中的章节

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

系统抑制剂靶向细胞内的疗效和毒性筛选

高通量兼容检测，以评估对巨噬细胞传代药物疗效

用于宿主靶向治疗结核病临床前检测的自动化培养系统

分枝杆菌高通量药物筛选的分析开发

分枝杆菌高通量药物筛选的分析开发

分枝杆菌高通量药物筛选的分析开发

评估对结核分枝杆菌感染模型的药物疗效的测定

评估测试化合物对分枝杆菌活性的木炭琼脂刃天青测定