细菌群的延时成像与集体压力反应
Summary
我们详细介绍了一个简单的方法，以产生高分辨率延时电影的伪单一aeruginosa群，响应噬菌体（噬菌体）和抗生素压力使用平板文档扫描仪。这个过程是一种快速而简单的监测蜂群动力学的方法，可应用于研究其他细菌物种的动能和生长。
Abstract
蜂群是许多细菌物种观察到的表面运动形式，包括伪莫纳斯阿鲁吉诺萨和大肠杆菌。在这里，密集的细菌种群在数小时内在典型的肌腱状社区中远距离移动。蜂群对多种因素敏感，包括中等水分、湿度和营养成分。此外，在由抗生素或噬菌体（噬菌体）强调的P.aeruginosa中观察到的集体应激反应，排斥成群的人接近含有压力的区域。此处描述的方法涉及如何控制影响蜂群的关键因素。我们介绍了一种使用平板文档扫描仪以高时态分辨率监控蜂群动力学和集体应力响应的简单方法，并描述了如何编译和执行群的定量分析。这种简单且经济高效的方法提供精确且控制良好的蜂群定量，可扩展到其他类型的板基生长测定和细菌物种。
Introduction
蜂群是协调细菌活力的一种集体形式，它增加11、2、32,3宿主的抗生素耐药性和毒性因子的产生。这种多细胞行为发生在半固体表面上，类似于覆盖肺部上皮膜的粘膜44，5。5生物活性剂通常由蜂群产生，以克服表面张力，这些物的产生由复杂的细胞-细胞信号系统调节，也称为仲裁感应66，7，8。7,8许多种类的细菌能够蜂拥，包括伪莫纳斯阿鲁吉诺萨，金黄色葡萄球菌，和大肠杆菌99，10，11，12。10,11,12细菌产生的蜂群模式多种多样，受表层的物理和化学性质影响，包括营养成分、孔隙度和水分13、14。13,除了表面特性外，生长温度和环境湿度还影响蜂拥动力学的几个方面，包括蜂拥速率和模式12、13、14、15。12,13,14,15影响蜂群的生长变量会带来挑战，影响实验的可重复性和解释结果的能力。在这里，我们描述了一个简单的标准化方法，通过延时成像来监测细菌群的动态。该方法描述了如何控制显著影响蜂群进展的关键生长条件。与传统的群分析方法相比，这种延时成像方法能够同时跟踪多个群在较长时间内和高分辨率的动感。这些方面提高了从监测蜂群中获得的数据深度，并有助于识别影响蜂群的因素。
通过生产和释放黄脂质和3-（3-羟基甲氧）烷基酸进入周边地区6,6、16，促进在P.aeruginosa的蜂拥。抗生素亚致命浓度或噬菌体病毒感染的压力的引入影响群的组织。特别是，这些应力诱导P.aeruginosa释放仲裁传感分子2-七溴二苯基-4-奎诺酮，也称为伪单一奎诺酮信号（PQS）17，18。17,18在包含两个群群的群检测中，由压力引起的种群产生的PQS阻止未经处理的群群进入含有压力的区域（图1）。这种集体压力反应构成一个危险的通信信号系统，警告P.aeruginosa关于附近的威胁18，19。,19压力对P.aeruginosa的影响，集体应激反应的激活，以及群的排斥，都可以通过本文描述的延时成像方法进行可视化。此处描述的协议解释了如何：（1） 准备加盘以进行蜂拥，（2） 培养P. aeruginosa进行两种检测（传统蜂群测定或集体应力响应测定）（图 1），（3）获取延时图像，（4） 使用 ImageJ 编译和分析图像。
简单地说，在蜂拥的加盘中间发现了来自通宵培养的P.aeruginosa，而在卫星位置发现了感染噬菌体或用抗生素治疗的P.aeruginosa。P. aeruginosa蜂拥的进展在放置在湿度调节的 37 °C 培养箱中的消费者文档平板扫描仪上进行监测。扫描仪由软件控制，该软件可在群生长期（通常为 16-20 小时）定期自动扫描板。此方法可生成多达 6 个 10 厘米的蜂拥板的并发延时视频。这些图像被汇编成电影，压力引起的人群对人群的排斥被使用免费的ImageJ软件量化。特别考虑确保不同蜂拥实验之间的一致性和可重复性。
Protocol
1. 准备加热的加盘P.阿鲁吉诺萨蜂拥延时间推移成像 通过在500 mL双蒸馏水中加入 64 克2Na2HPO4、15 克 KH2PO24和 2.5 g NaCl，在玻璃瓶中制备 1 L 5x M8 最小介质。使用额外的 ddH2O. 高压灭菌器将最终体积调整为 1 L，以在室温下消毒和储存液体介质。 通过在50 mL ddH2O中加入24.6克MgSO 4+7H24O，在玻璃瓶中制?…
Representative Results
生长P.aeruginosa的步骤，应力细胞，和图像的蜂拥的加盘在图1中表示。我们接种了一个野生型P.aeruginosa UCBPP-PA14菌株从LB-agar板在2 mL的LB汤在37°C过夜，并在蜂拥的加板中心发现5μL。这个板块的延时成像显示，在中心以殖民地的形式出现初始生长，然后从殖民地径向传播肌腱（视频1）。对于集体应力反应测定，除了在中心发现P.aeruginosa外，在卫星位置以5…
Discussion
该协议侧重于最小化蜂状板的变异性，并提供一种简单且低成本的方法，以获取P. aeruginosa的延时图像，并响应应力。通过调整介质组成和生长条件，可以扩展到图像其他细菌系统。对于P.aeruginosa，虽然M9或FAB最小介质可用于诱导蜂拥16，21，,21这里介绍的协议使用M8介质与卡氨基酸，葡萄糖和硫酸镁6。P. aeruginosa蜂群…
Disclosures
The authors have nothing to disclose.
Acknowledgements
J.-L.B.、A.S.和N.M.H-K.撰写和修订手稿。所有作者都设计了这些实验。J.-L.B.进行了实验和分析。这项工作得到了NIH授予K22AI112816和R21AI139968赠款给A.S.和加利福尼亚大学的支持。N.M.H-K.得到伦德贝克奖学金R220-2016-860和R251-2017-1070的支持。资助者在将作品提交出版的决定中没有任何作用。我们没有相互竞争的利益可申报。
Materials
| Reagents | |||
| Bacto agar, dehydrated | BD Difco | 214010 | For LB-agar plate and swarming agar plate |
| Casamino acids | BD Difco | 223050 | For swarming media |
| D-Glucose | Fisher Chemical | D16500 | Dextrose. For swarming media |
| Fosfomycin disodium salt | Tokyo Chemical Industry | F0889 | Stock concentration: 200 mg / mL. Dissolved in ddH2O |
| Gentamycin sulfate | Sigma-Aldrich | G1914 | Stock concentration: 3 mg / mL. Dissolved in ddH2O |
| Kanamycin sulfate | Sigma-Aldrich | 60615 | Stock concentration: 100 mg / mL. Dissolved in ddH2O |
| LB-Miller | BD Difco | 244620 | For LB broth and LB-agar plates |
| Magnesium sulfate heptahydrate | Sigma-Aldrich | 230391 | For swarming media |
| Potassium phosphate monobasic | Sigma-Aldrich | P0662 | For 5X M8 media |
| Sodium chloride | Sigma-Aldrich | S9888 | For 5X M8 media |
| Sodium phosphate dibasic heptahydrate | Fisher Chemical | S373 | For 5X M8 media |
| Strains | |||
| Pseudomonas aeruginosa | Siryaporn lab | AFS27E.118 | PA14 strain |
| DMS3vir | O'Toole lab | DMS3vir20 | Bacteriophage |
| Supplies | |||
| Aluminium oxide sandpaper | 3M | 150 Fine | For black lids |
| Black fabric | Joann | PRD7089 | Black fabric |
| Black spray paint | Krylon | 5592 Matte Black | For black lids |
| Erlenmeyer flask | Kimax | 26500 | 250 mL |
| Glass storage bottles | Pyrex | 13951L | 250 mL, 500 mL, 1000 mL |
| 8 inches zip ties | Gardner Bender | E173770 | For attaching black matte fabric |
| Petri dishes (100 mm x 15 mm) | Fisher | FB0875712 | 100 x 15 mm polystyrene plates |
| Wooden sticks | Fisher | 23-400-102 | For streaking and inoculating bacteria |
| Equipment | |||
| Autoclave | Market Forge Industries | STM-E | For sterilizing reagents |
| 25 mL pipette | USA Scientific, Inc. | 1072-5410 | To pipet 20 mL for swarming agar plates |
| Dehumidifier | Frigidaire | FAD704DWD 70-pint | For maintaing room relative humidity at about 45% |
| ImageJ | NIH | v1.52a | Software for image analysis |
| Incubator | VWR | 89032-092 | For growth of bacteria at 37 °C |
| Isotemp waterbath | Fisher | 15-462-21Q | For cooling media to 55 °C |
| Laminar flow hood | The Baker Company | SG603A | For drying plates |
| P-20 pipet | Gilson | F123601 | Spotting on swarming agar plates |
| Pipette Controller | BrandTech | accu-jet | To pipet 20 mL for swarming agar plates |
| Roller Drum | New Brunswick | TC-7 | For growth of bacteria at 100 rpm |
| Scanner | Epson | Epson Perfection V370 Photo | Scanner for imaging plates |
| Scanner automation software | RoboTask Lite | v7.0.1.932 | For 30-min internals imaging |
| Scanner image acquisition software | Epson | v9.9.2.5US | Software for imaging plates |
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Cite This Article
Bru, J., Siryaporn, A., Høyland-Kroghsbo, N. M. Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response. J. Vis. Exp. (159), e60915, doi:10.3791/60915 (2020).