Name: in vitro biofilm formation in an 8-well chamber slide
Uploaded: 2011-01-20T18:30:00
Duration: 6 min 14 s
Description: Watch this Scientific Journal Video about In vitro Biofilm Formation in an 8-well Chamber Slide at JoVE.com

R01DC003915 NIDCD /美国国立卫生研究院的工作是由劳Bakaletz。

在体外生物膜的形成1。 <ol><li>细菌菌落（已在适当的琼脂隔夜增长），暂停在媒体和外径490调整为0.65 </li><li>由此产生的细菌悬浮液再稀释1:6（1 mL的菌悬液+ 5毫升预热中等），孵育37℃，5％CO 2约3小时，以达到中期日志阶段。 </li><li>中期日志增长悬挂1:2500稀释与预热媒体和200μL稀释成每孔8室幻灯片。 </li><li>在37 ° C孵育5％的CO 2。 </li><li>经过约16小时，吸媒介，从每个会议厅的角落，并添加200μL新鲜，预热介质 - 沿腔壁免除，以免内建立的商会，这可能会破坏生物膜的剪切力。 </li><li>如果孵化时间超过24小时，改变培养基，每12小时或需要保持细菌的生存能力。 </li></ol> 2。生物膜的可视化<ol><li>吸如上所述，从每一个分庭的媒介，并用无菌生理盐水洗两次轻轻居民生物膜。 </li><li>新增200 BacLight活/死染色（3μL的组件A + 3μL每毫升生理盐水组件B）μL，每口井，并在室温下孵育15分钟。从这个点开始，从保护文化。 </li><li>吸干污渍，并用无菌生理盐水洗两次轻轻地像以前一样。 </li><li>加入200μL的中性福尔马林每口井和30分钟，在室温下孵育修复标本。 </li><li>固定液中取出，并妥善处置，按照机构准则。 </li><li>用生理盐水洗两次澡洗的液体含有上述福尔马林和处置。 </li><li>从幻灯片中删除的塑料井，补充足够的生理盐水，每孔使盖玻片置于垫片时，无气泡存在。 </li><li>盖玻片和密封的安装介质盖玻片边缘。指甲油可以用密封的幻灯片，但幻灯片不会成为永久。 </li><li>允许密封胶干燥约1小时前，通过显微镜检查。 </li></ol> 3。结果<img src="/files/ftp_upload/2481/2481fig1.jpg" alt="图1" /> 图1 8室幻灯片中形成生物膜复合代表性的三维图像。细菌活/死染色其中活菌荧光标记的绿色和死菌荧光红色。 （一）综合整个呈现出不同的体系结构与塔和水通道的生物膜的形象。 （二）在同一生物膜（一），现在看到的横截面。

<ol>
	<li>Bakaletz, L. O. <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+in+otitis+media:+evidence+and+relevance.">Bacterial biofilms in otitis media: evidence and relevance.</a> Pediatr Infect Dis J. 26, S17-S19 (2007).</li><li>Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146, 2395-2407 (2000).</li></ol>

了解这一机制的生物膜的形成，以及其基质成分特征，是一个浓厚的兴趣领域。该协议可用于帮助识别蛋白质或其他成分，有助于生物膜结构完整性，以及帮助识别和目标蛋白质可能适合自己的治疗应用。 已建立的孵育时间和稀释在上面描述的协议nontypeable流感嗜血杆菌（NTHI）最佳生物膜的形成。其他细菌物种的优化这些步骤可能需要一些前期工作（即初始稀释和孵化所需的时间来实现日志中期阶段增长） 。成立商会的初始播种1:2500稀释，以确保一个强大的生物膜开发这种微生物引起的玻片和孵育时间内表示，没有overgrowing室和/或排气速效养分的生物膜。因此，最初的稀释和孵育时间可能需要调整其他细菌物种。

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<td>Chocolate II agar</td>
<td>&nbsp;</td>
<td>Fisher</td>
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<td>8-well chamber slides</td>
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<td>Fisher</td>
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<td>BacLight Live/Dead bacterial viability kit</td>
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<td>&beta;-NAD</td>
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in vitro biofilm formation in an 8-well chamber slide

许多疾病的长期性，是因为顽抗，以传统的抗生素治疗的细菌生物膜的形成。生物膜是与社会相关的细菌附着在表面上，包裹在一个矩阵。细胞外基质的作用是多方面的，包括促进营养收购，并提供​​对环境压力（如宿主的免疫反应）的显着的保护。在生物膜内细菌如何回应我们已经使用了一个协议，其中，我们有8井室幻灯片形成细菌生物膜的环境压力，努力获得更好的理解。生物膜与BacLight直播/死染色染色和研究使用共聚焦显微镜来表征不同的培养条件下的相对生物膜的大小和结构。 Z - Stack还通过共聚焦显微镜图像采集和分析，由COMSTAT。这个协议可以被用来帮助阐明生物膜形成机制和动力学，以及确定生物膜的结构和稳定的重要组件。

Watch this Scientific Journal Video about In vitro Biofilm Formation in an 8-well Chamber Slide at JoVE.com

In vitro Biofilm Formation in an 8-well Chamber Slide

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