Name: time-lapse imaging of bacterial swarms and the collective stress response
Uploaded: 2020-05-23T13:30:00
Description: Watch this Scientific Journal Video about Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response at JoVE.com

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。

1. 准备加热的加盘P.阿鲁吉诺萨蜂拥延时间推移成像
<ol><li>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。</li>
	<li>通过在50 mL ddH2O中加入24.6克MgSO 4+7H24O，在玻璃瓶中制?...

<ol>
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H., Zinjarde, S. S., Venugopalan, V. P., McLean, R. J. C., Weber, M. M., Rahman, P. K. S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing:+implications+on+rhamnolipid+biosurfactant+production.">Quorum sensing: implications on rhamnolipid biosurfactant production.</a> Biotechnology &amp; Genetic Engineering Reviews. 27, 159-184 (2010).</li><li>K&#246;hler, T., Curty, L. K., Barja, F., van Delden, C., Pech&#232;re, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swarming+of+Pseudomonas+aeruginosa+is+dependent+on+cell-to-cell+signaling+and+requires+flagella+and+pili.">Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili.</a> Journal of Bacteriology. 182 (21), 5990-5996 (2000).</li><li>Pollitt, E. J. G., Crusz, S. A., Diggle, S. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+field+guide+to+bacterial+swarming+motility.">A field guide to bacterial swarming motility.</a> Nature Reviews. Microbiology. 8 (9), 634-644 (2010).</li><li>Tremblay, J., D&#233;ziel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+reproducibility+of+Pseudomonas+aeruginosa+swarming+motility+assays.">Improving the reproducibility of Pseudomonas aeruginosa swarming motility assays.</a> Journal of Basic Microbiology. 48 (6), 509-515 (2008).</li><li>Morales-Soto, N., 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=Preparation,+imaging,+and+quantification+of+bacterial+surface+motility+assays.">Preparation, imaging, and quantification of bacterial surface motility assays.</a> Journal of Visualized Experiments: JoVE. (98), e52338 (2015).</li><li>Ha, D. -. G., Kuchma, S. L., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plate-based+assay+for+swarming+motility+in+Pseudomonas+aeruginosa.">Plate-based assay for swarming motility in Pseudomonas aeruginosa.</a> Methods in Molecular Biology. 1149, 67-72 (2014).</li><li>Tremblay, J., Richardson, A. -. P., L&#233;pine, F., D&#233;ziel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-produced+extracellular+stimuli+modulate+the+Pseudomonas+aeruginosa+swarming+motility+behaviour.">Self-produced extracellular stimuli modulate the Pseudomonas aeruginosa swarming motility behaviour.</a> Environmental Microbiology. 9 (10), 2622-2630 (2007).</li><li>Morales-Soto, N., 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=Spatially+dependent+alkyl+quinolone+signaling+responses+to+antibiotics+in+Pseudomonas+aeruginosa+swarms.">Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms.</a> The Journal of Biological Chemistry. 293 (24), 9544-9552 (2018).</li><li>Bru, J. -. L., 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=PQS+produced+by+the+Pseudomonas+aeruginosa+stress+response+repels+swarms+away+from+bacteriophage+and+antibiotics.">PQS produced by the Pseudomonas aeruginosa stress response repels swarms away from bacteriophage and antibiotics.</a> Journal of Bacteriology. , (2019).</li><li>van Kessel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PQS+signaling+for+more+than+a+quorum:+the+collective+stress+response+protects+healthy+Pseudomonas+aeruginosa+populations.">PQS signaling for more than a quorum: the collective stress response protects healthy Pseudomonas aeruginosa populations.</a> Journal of Bacteriology. , (2019).</li><li>Zegans, M. E., 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=Interaction+between+bacteriophage+DMS3+and+host+CRISPR+region+inhibits+group+behaviors+of+Pseudomonas+aeruginosa.">Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa.</a> Journal of Bacteriology. 191 (1), 210-219 (2009).</li><li>Kamatkar, N. G., Shrout, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+hardness+impairment+of+quorum+sensing+and+swarming+for+Pseudomonas+aeruginosa.">Surface hardness impairment of quorum sensing and swarming for Pseudomonas aeruginosa.</a> PloS One. 6 (6), 20888 (2011).</li><li>Mattingly, A. E., Kamatkar, N. G., Morales-Soto, N., Borlee, B. R., Shrout, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Environmental+Factors+Influence+the+Importance+of+the+Phosphodiesterase+DipA+upon+Pseudomonas+aeruginosa+Swarming.">Multiple Environmental Factors Influence the Importance of the Phosphodiesterase DipA upon Pseudomonas aeruginosa Swarming.</a> Applied and Environmental Microbiology. 84 (7), (2018).</li><li>Boyle, K. E., Monaco, H., van Ditmarsch, D., Deforet, M., Xavier, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+Metabolic+and+Quorum+Sensing+Signals+Governing+the+Decision+to+Cooperate+in+a+Bacterial+Social+Trait.">Integration of Metabolic and Quorum Sensing Signals Governing the Decision to Cooperate in a Bacterial Social Trait.</a> PLoS computational biology. 11 (5), 10004279 (2015).</li><li>Bernier, S. P., Ha, D. -. 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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...

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。

<table border="0" cellpadding="0" cellspacing="0" style="width:844px;" width="844">
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	<tbody>
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			<td height="21" style="height:21px;width:228px;">Reagents</td>
			<td style="width:157px;"></td>
			<td style="width:120px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bacto agar, dehydrated</td>
			<td style="width:157px;">BD Difco</td>
			<td style="width:120px;">214010</td>
			<td>For LB-agar plate and swarming agar plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Casamino acids</td>
			<td style="width:157px;">BD Difco</td>
			<td style="width:120px;">223050</td>
			<td>For swarming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">D-Glucose</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:120px;">D16500</td>
			<td>Dextrose. For swarming media</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:228px;">Fosfomycin disodium salt</td>
			<td style="width:157px;">Tokyo Chemical Industry</td>
			<td style="width:120px;">F0889</td>
			<td>Stock concentration: 200 mg / mL. Dissolved in ddH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:228px;">Gentamycin sulfate</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">G1914</td>
			<td>Stock concentration: 3 mg / mL. Dissolved in ddH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:228px;">Kanamycin sulfate</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">60615</td>
			<td>Stock concentration: 100 mg / mL. Dissolved in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">LB-Miller</td>
			<td style="width:157px;">BD Difco</td>
			<td style="width:120px;">244620</td>
			<td>For LB broth and LB-agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Magnesium sulfate heptahydrate</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">230391</td>
			<td>For swarming media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Potassium phosphate monobasic</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">P0662</td>
			<td>For 5X M8 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Sodium chloride</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:120px;">S9888</td>
			<td>For 5X M8 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:120px;">S373</td>
			<td>For 5X M8 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Strains</td>
			<td style="width:157px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Pseudomonas aeruginosa</td>
			<td style="width:157px;">Siryaporn lab</td>
			<td style="width:120px;">AFS27E.118</td>
			<td>PA14 strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">DMS3vir</td>
			<td style="width:157px;">O&#39;Toole lab</td>
			<td style="width:120px;">DMS3vir20</td>
			<td>Bacteriophage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Supplies</td>
			<td style="width:157px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Aluminium oxide sandpaper</td>
			<td style="width:157px;">3M</td>
			<td style="width:120px;">150 Fine</td>
			<td>For black lids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Black fabric</td>
			<td style="width:157px;">Joann</td>
			<td style="width:120px;">PRD7089</td>
			<td>Black fabric</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Black spray paint</td>
			<td style="width:157px;">Krylon</td>
			<td style="width:120px;">5592 Matte Black</td>
			<td>For black lids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Erlenmeyer flask</td>
			<td style="width:157px;">Kimax</td>
			<td style="width:120px;">26500</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Glass storage bottles</td>
			<td style="width:157px;">Pyrex</td>
			<td style="width:120px;">13951L</td>
			<td>250 mL, 500 mL, 1000 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">8 inches zip ties</td>
			<td style="width:157px;">Gardner Bender</td>
			<td style="width:120px;">E173770</td>
			<td>For attaching black matte fabric</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Petri dishes (100 mm x 15 mm)</td>
			<td style="width:157px;">Fisher</td>
			<td style="width:120px;">FB0875712</td>
			<td>100 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Wooden sticks</td>
			<td style="width:157px;">Fisher</td>
			<td style="width:120px;">23-400-102</td>
			<td>For streaking and inoculating bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Autoclave</td>
			<td style="width:157px;">Market Forge Industries</td>
			<td style="width:120px;">STM-E</td>
			<td>For sterilizing reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">25 mL pipette</td>
			<td style="width:157px;">USA Scientific, Inc.</td>
			<td style="width:120px;">1072-5410</td>
			<td>To pipet 20 mL for swarming agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Dehumidifier</td>
			<td style="width:157px;">Frigidaire</td>
			<td style="width:120px;">FAD704DWD 70-pint</td>
			<td>For maintaing room relative humidity at about 45%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">ImageJ</td>
			<td style="width:157px;">NIH</td>
			<td style="width:120px;">v1.52a</td>
			<td>Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Incubator</td>
			<td style="width:157px;">VWR</td>
			<td style="width:120px;">89032-092</td>
			<td>For growth of bacteria at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Isotemp waterbath</td>
			<td style="width:157px;">Fisher</td>
			<td style="width:120px;">15-462-21Q</td>
			<td>For cooling media to 55 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Laminar flow hood</td>
			<td style="width:157px;">The Baker Company</td>
			<td style="width:120px;">SG603A</td>
			<td>For drying plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">P-20 pipet</td>
			<td style="width:157px;">Gilson</td>
			<td style="width:120px;">F123601</td>
			<td>Spotting on swarming agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Pipette Controller</td>
			<td style="width:157px;">BrandTech</td>
			<td style="width:120px;">accu-jet</td>
			<td>To pipet 20 mL for swarming agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Roller Drum</td>
			<td style="width:157px;">New Brunswick</td>
			<td style="width:120px;">TC-7</td>
			<td>For growth of bacteria at 100 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Scanner</td>
			<td style="width:157px;">Epson</td>
			<td style="width:120px;">Epson Perfection V370 Photo</td>
			<td>Scanner for imaging plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Scanner automation software</td>
			<td style="width:157px;">RoboTask Lite</td>
			<td style="width:120px;">v7.0.1.932</td>
			<td>For 30-min internals imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Scanner image acquisition software</td>
			<td style="width:157px;">Epson</td>
			<td style="width:120px;">v9.9.2.5US</td>
			<td>Software for imaging plates</td>
		</tr>
	</tbody>
</table>

time-lapse imaging of bacterial swarms and the collective stress response

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。

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...

Watch this Scientific Journal Video about Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response at JoVE.com

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

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。

细菌群的延时成像与集体压力反应

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...

该协议可用于制作伪多莫纳斯aeruginosa群的延时电影，并捕获细菌或抗生素对蜂群的排斥。延时电影捕捉了大量对抗生素和噬菌体反应的详细动态，该技术只需要除湿机、计算机、孵化器和文档扫描仪。要为 P.aeruginosa 蜂长延时成像准备蜂拥的阿加片，请使用 25 毫升移液器在每个实验的 10 厘米直径 Petri 盘上添加 20 毫升的蜂拥糖溶液。将板放在一个堆叠中，盖在室温下一小时。打开除湿机，将房间的相对湿度降低至40-50%当加糖凝固时，在层压流罩中再干燥蜂拥的阿加板30分钟，盖上盖子，以每分钟300立方英尺的速度朝上，室温下的相对湿度为40-50%。然后在4摄氏度下储存干蜂的阿加板长达24小时。对于蜂拥的阿加电镀，握一个 P20 移液器尖端，装有五微升从隔夜 P.aeruginosa 培养，在一个蜂拥板中心上方 10-45 度角 2-1/2 厘米处，将柱塞压到仅液体掉落处接触 agar 的第一站。使用模板将点一致地定位在不同的蜂拥盘上。为了对噬菌体感染进行盘，首先将30微升过夜培养的P.aeruginosa与6微升混合，每毫升噬菌体DMS3 vir的12个斑块形成单位，并立即在2.8厘米半径同心圆的6个等距卫星位置添加6微升的P.aeruginosa噬菌体混合物。使用模板将点一致地定位在不同的蜂拥盘上。要进行板式抗生素处理，将30微升过夜培养的P.aeruginosa与感兴趣的抗生素的适当浓度和体积混合，并立即在6个等距卫星位置的6个等距卫星位置，在菜盘中心周围2.8厘米半径的同心圆上添加6微升抗生素处理细菌。使用模板将点一致地定位在不同的蜂拥盘上。当所有培养物都镀上时，用定制的黑色盖子替换清晰的培养皿盖。小心地将蜂拥的阿加板放在扫描仪上，在 37 摄氏度的培养箱中安装 10 升水浴，使湿度保持 75%。若要自动进行图像采集，请使用脚本软件。选择空闲扫描和单次扫描，右键单击空闲扫描，然后左键单击启用。在孵化结束时，将所有扫描的图像导入 ImageJ。在序列选项窗口中，图像数将指示所选图像数。将图像从一个图像开始，从文件夹中的第一张图片开始，将图像缩放为 100%，以保留图像的原始大小。单击"隐藏到 RGB"以保留图像的颜色。保留未选中使用虚拟堆栈，然后单击"确定"以加载映像。单击文件，保存为 和 AVI 将图像合并到 AVI 文件中。将压缩调整为 JPEG，将帧速率调整为每秒五帧。然后将 AVI 延时保存在相应的存储文件夹中。在37摄氏度的LB肉汤中，从LB的一个LB阿加板对一个野生型P.aeruginosa的单一殖民地进行延时成像，并发现进入蜂拥的阿加板的中心，发现在板块中心形成一个殖民地，从菌群径向扩散。沼泽从蜂拥的阿加板中心移动到外围，被感染噬菌体或用根塔霉素治疗的细菌发出的应激信号击退。单独在卫星位置发现的噬菌体或根塔姆霉素不会导致蜂拥而至的人口避开这些区域。使用 25 毫升移液器，确保蜂拥的阿加板包含正好 20 毫升。根据实验室环境调整板材干燥时间也很重要。我们的技术使我们能够可视化伪多莫纳斯亚鲁吉诺萨种群的整体蜂群模式。下一个目标是使用光明场显微镜监测单个细胞的活力。

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Super-resolution Imaging of the Bacterial Division Machinery

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...