Preparation, imaging, and quantification of bacterial surface motility assays.
Preparation, imaging, and quantification of bacterial surface motility assays.
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DOI：
10.3791/52338
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发表时间：
2015-04-07
期刊：
影响因子：
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通讯作者：
Shrout JD
中科院分区：
文献类型：
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作者：
Morales-Soto N;Anyan ME;Mattingly AE;Madukoma CS;Harvey CW;Alber M;Déziel E;Kearns DB;Shrout JD
细菌表面的运动性，如聚集，通常在实验室使用平板分析来检测，这种方法需要特定浓度的琼脂，有时还需要在生长介质中加入特定的营养物质。这种显性的介质和表面生长条件的制备提供了有利的条件，不仅允许细菌生长，而且允许细菌在这些表面上以薄液膜的形式协调运动。群平板和其他表面动力平板分析的重复性可能是一个主要的挑战。尤其是对于更多只在0.4%-0.8%(重量/体积)琼脂范围内表现出活力的温带群，方案或实验室环境的微小变化可能会对群分析结果产生很大影响。“润湿性”，也就是这些平板分析的液-固-气界面的水分含量，通常是一个需要控制的关键变量。评估蜂群的另一个挑战是如何量化任何两个(或更多)实验之间观察到的差异。在这里，我们详细介绍了一个通用的两阶段协议，以准备和成像蜂群分析。我们提供了一些指导方针，以规避通常与群体检测、试剂盒准备和这些检测的数据量化相关的挑战。我们特别演示了我们的方法，使用表达荧光或生物发光遗传报告的细菌，如绿色荧光蛋白(GFP)、荧光素酶(Lux操纵子)或细胞染色，以实现延时光学成像。在同一实验中，我们进一步展示了我们的方法跟踪竞争种群的能力。
Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.