Name: investigating the detrimental effects of low pressure plasma sterilization on the survival of bacillus subtilis spores using live cell microscopy
Uploaded: 2017-11-30T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy at JoVE.com

作者感谢安德莉亚在这项工作的部分和 Nikea 在视频拍摄期间对她的帮助的出色的技术援助。我们还要感谢莱尔对她慷慨捐赠的枯草芽孢杆菌菌株: LAS72 和 LAS24。这项工作得到了部分支持, 从德国研究基金会 (DFG) Paketantrag (PlasmaDecon 柏 728) 到 PA (AW 7/3-1) 和 RM (MO 2023/2-1) 和 dlr 赠款 dlr-FuW-Projekt ISS 生活, 射频 FuW, Teilprogramm 475 (f.m F, 大副和 rm)。F.M.F. 由亥姆霍兹空间生命科学研究学校 (SpaceLife) 的博士奖学金在德国的科隆, 德国航空航天中心 (DLR) 支持, 由亥姆霍兹协会 (亥姆霍兹-礼俗) 提供经费在六年期间 (格兰特 No。VH-KO-300) 并获得了 DLR 的额外资金, 包括航空航天执行委员会和航空航天医学研究所。这项研究的结果将被包括在博士论文的费利克斯 m。

1.枯草芽孢杆菌孢子的生产和纯化
<ol><li>为孢子生产, 转移各自的B. 枯草芽孢杆菌菌株的5毫升隔夜文化, 补充适当的抗生素, 到200毫升双强度液体谢弗产培养基 (每公升 16 g 营养素肉汤, 氯化钾 2 g, 0.5 g MgSO4* 7 H2O, 2 mL 1 M Ca (不3)2, 2 毫升 0.1 M MnCl2 * 4 h2O, 2 ml 1 mM FeSO4, 2 ml 50% (w/v) 葡萄糖18), 并培养它与蓬勃发展的曝气在37° c 72 H 或直到 > 95% 的文化有孢子。使用以下菌株的孢子:枯草芽孢杆菌PY79 (野生类型) b. 枯草芽孢杆菌 PY79ΔrecA:: 新 (缺乏 DNA 修复蛋白 recA) B. 枯草杆菌PY79 recA -yfp:: 猫 (recA 与黄融合荧光蛋白 [YFP]19)。</li>
	<li>在50毫升的试管中用离心法收获孢子15分钟 3000 x g, 通过反复洗涤步骤 (最多15次) 净化样品, 使用无菌蒸馏的 H2O, 并通过相衬显微镜检查纯度和发芽状态。确保孢子悬浮物由相明亮的孢子 ( & gt ; 99% ) 组成 , 没有营养细胞 ( 棒 ) , 发芽孢子 ( 黑色 / 灰色外观 ) 和细胞碎片 , 否则进一步的显微实验可以扰。清洗样品, 直到达到预期纯度。</li>
	<li>通过在 LB 琼脂上镀出50µL 的10倍稀释 (即: 使用30µL 样品 + 270 µL 无菌水进行1:10 稀释, 确定孢子滴度。采取30µL 从特别稀释到270µL H2O 为1:100 稀释等等) 计算 CFU (殖民地形成单位) 和孵育板材在37° c 隔夜。CFU 测定后, 通过浓缩或稀释无菌水, 将样品调整到每毫升 109孢子。</li>
</ol>2. 气溶胶沉积的枯草芽孢杆菌孢子的样品制备
注意: 孢子的堆积和重叠可能导致在治疗过程中产生遮蔽效应, 最终导致假失活动力学。为了尽量减少这个问题, 用气溶胶沉积技术准备孢子样品20。简要地, 控制高精确度二物质喷管与电子定时器调控液体吞吐量与压力的载体气体的流动 (这里 N2)。使用氮气流量将注入的液体样品通过喷嘴出口分散。
<ol><li>将样品载体以灭菌的显微幻灯片 (用于生存动力学) 或圆形25毫米片 (用于荧光跟踪 DNA 修复过程/cLSM; 共聚焦激光扫描显微镜) 置于电控气溶胶喷涂单元内与喷嘴对准。所用的孢子浓度需要达到百倍的最终浓度。</li>
	<li>将孢子培养的1毫升转移到喷嘴流体入口, 并在1.3 巴压力下启动 0.1 s 的喷涂过程。喷洒的孢子悬浮 (1 x 107) 形成一层薄薄的薄膜, 在几秒钟内迅速干燥, 形成均匀分布的孢子单层。在室温下将处理过的样品载体储存在无菌容器中。</li>
</ol>3. 低压等离子处理
<ol><li>制备生物样品的等离子体处理系统, 在 5 Pa 用氩等离子体在 500 W 处操作系统, 5 分钟。这样, 系统中的所有曲面都将被清洗和预热。这减少了分子的黏附从环境空气, 即氮气, 氧气和水, 当排泄系统。在对系统进行预处理后, 在玻璃货架的帮助下, 将样品放在反应堆容器的中心, 并将其仔细地放在反应器内。</li>
	<li>使用至少三生物复制。关闭会议厅并在 2 Pa. 后疏散, 将过程气体填入会议厅。调节系统压力至 5 Pa。</li>
	<li>经过规定的工艺时间, 关闭电源和供气, 并仔细排气系统, 以防止从样品持有人吹样品。通风后, 取出样品并将样品放置在系统中的下一个参数中。对于非等离子处理的控制暴露样品到真空仅 (5 Pa) 在过程气体的存在等效于最长的应用的血浆时间。</li>
</ol>4. 孢子存活的恢复和评价
<ol><li>准备一份蒸压10% 聚乙烯醇 (PVA) 的溶液, 用大约500的µL 仔细地盖住样品托架, 并让它们风干 4 h. 用无菌钳将干燥的 PVA 层 (现在含有孢子样本) 剥离, 并将其转移到2毫升反应管。将1毫升无菌水加入试管, 通过涡流溶解 PVA 层。此过程导致孢子的 > 95% 的恢复, 不影响其萌发能力21。</li>
	<li>在96井板 (即270µL 无菌 H2O + 30 µL 样品/前稀释) 中, 连续地稀释1:10 的无菌水中的样品。在性汤营养琼脂 (LB) 上每稀释50µL, 在夜间孵育37° c 的盘子, 并列举生长的菌落数量 (CFU)。</li>
</ol>5. 活体细胞显微镜和 DNA 修复过程在萌发孢子中的追踪
<ol><li>对于发芽实验, 准备一个1毫米厚的1.5% 磅琼脂垫, 煮沸700µL 培养基和吸管它成一个无菌显微镜培养皿。10分钟后, 切出一个8毫米 x 8 毫米 x 1 毫米磅琼脂垫与无菌手术刀和转移琼脂仔细在顶部的孢子单分子是休息在25毫米玻璃片。 注意: 这一步对于个体孢子的可视化是至关重要的, 并允许跟随它们的反应, 对营养琼脂诱导的发芽激活。因此, LB 琼脂服务两个目的, (1) 固定的孢子在表面上, 避免孵育沿表面和光学焦点, 和 (2) 激活孢子发芽。</li>
	<li>用琼脂覆盖样品后, 用 63 x/1.3 平面物镜油浸泡物镜, 将玻璃片迅速转移到成像室, 并通过自动共焦激光扫描显微镜与倒置光学显微镜对样品进行显微观察。</li>
	<li>
执行荧光成像 (YFP) 与激发波长 514 nm 和发射可以检测到520和 560 nm。
	<ol>
<li>使用照片乘数 (透射光路) 之一在扫描模式下记录明亮的图像。</li>
		<li>记录时间序列与激光功率为 2.6%, 并设置共焦孔径为5通风单位和在采样频率1帧每三十年代从 0 h 到 5 h, 根据实验。特别值得注意的是, 高剂量的单色激光照射在 514 nm 完全抑制发芽 (图 1A, B)。</li>
	</ol>
</li>
	<li>在整个成像过程中, 在加热阶段保持样品在37° c (环境空气湿度)。每个条件至少使用三生物复制。在孢子聚集的情况下, 多层孢子分布或尘埃粒子污染, 堵塞等离子体处理 ("阴影") 可能发生, 并使阴影孢子发芽 (图 1C, D)。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56666/56666fig1.jpg"> 图 1: 等离子体处理的孢子共焦活细胞荧光显微镜观察到的潜在问题(A, B)高剂量单色 (514 nm) 激光照射对孢子萌发的抑制作用。(A) 概述 (3 x 3 缝合框架) 的枯草芽孢杆菌(LAS72, RecA-YFP) 孢子萌发后180分钟。中间的框架被暴露在三十年代间隔时间到高剂量的激光 (514 nm, 70% 激光功率), 而周围地区 (= 帧) 没有照亮 (光明场通道合并图像和 RecA YFP 荧光; 有序结构由使用35毫米成像盘子与印有500µm 网格).(B) 演示了一个4X 的放大视图, 它显示了光照和非光照区域之间的边界, 表明孢子暴露在高剂量的单色激光照射下没有发芽和生长, 而孢子非光照区域完全恢复到表达明亮 RecA-YFP 荧光 (绿色信号) 的植物细菌。(C, D)被污染的微粒或多层孢子 (箭头) 覆盖的孢子似乎可以通过等离子体处理来保护底层孢子免遭失活, 并允许它们的萌发和生长 ("遮蔽效应")。(C) 孢子在六十年代被等离子体处理, 在萌发后180分钟或在 (D) 中成像, 120s 和成像后240分钟.<a href="//cloudflare.jove.com/files/ftp_upload/56666/56666fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
6. 扫描电子显微镜 (SEM)
<ol><li>使用扫描电子显微镜, 提供有关等离子体处理孢子的表面形貌的超微结构信息, 与未经治疗的对照相比。用溅射涂布机在片上用金钯 (3 nm) 将干燥的孢子单层膜涂上。使用场发射扫描电子显微镜对样品进行成像, 在5伏加速度电压下操作, 包括镜头内二次电子探测器, 以揭示地形对比。</li>
</ol>7. 数据分析
<ol><li>确定孢子生存从商数 n/n0, 其中 n 是平均 CFU 的处理样本和 n0是平均 CFU 的未经治疗的真空控制。用氩等离子体处理孢子失活的时间函数 (秒)。将所有数据表示为平均值和标准偏差 (n = 3)。</li>
	<li>使用成像软件分析活细胞成像获得的图像。在实验开始时以及在4小时后, 对孢子萌发和超过在等离子体处理后的比例进行量化, 在代表性的框架中计数孢子。为了在孢子存活测定中的意义确定, 使用统计软件对方差分析进行单因素方差检验。P值 < 0.05 被视为具有统计学意义。</li>
</ol>

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Phys. D: Appl. Phys. 40, 2826 (2007).</li><li>Stapelmann, K., 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=Utilization+of+low-pressure+plasma+to+inactivate+bacterial+spores+on+stainless+steel+screws.">Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws.</a> Int. J. Astrobiol. 13, 597-606 (2013).</li><li>Raguse, M., 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=Understanding+of+the+importance+of+the+spore+coat+structure+and+pigmentation+in+the+Bacillus+subtilis+spore+resistance+to+low-pressure+plasma+sterilization.">Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low-pressure plasma sterilization.</a> J. Phys. D: Appl. Phys. 49, 285401 (2016).</li><li>Pandey, R., 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=Live+cell+imaging+of+germination+and+outgrowth+of+individual+Bacillus+subtilis+spores;+the+effect+of+heat+stress+quantitatively+analyzed+with+SporeTracker.">Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker.</a> PloS one. 8, e58972 (2013).</li></ol>

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使用低温、低压等离子体进行表面灭菌是一种很有前途的替代品, 例如用电离辐射、化学药品 (例如,2O2之类的气体进行处理) 和传统的灭菌程序环氧乙烷) 或干燥和潮湿的热量23。一般的杀菌方法主要是提供有效的灭菌, 但已知会影响处理过的材料, 并对操作者构成潜在的风险。低压等离子体提供了一个快速和均匀的生物失活, 使用的成分, 如紫外线光子, 自由...

太空探索的一个主要目标是在太阳系的其他行星体和卫星上寻找生命形式和生物分子的特征。将地球起源的微生物或生物分子转移到重要的勘探领域, 对火星和欧罗巴1等行星机构的生命探测任务的发展和完整性产生了特别的影响。1967年由空间研究委员会设立的《行星保护国际准则》对载人和机器人任务向其他行星、卫星、小行星和其他天体实施了严格的管制, 并对在发射前对航天器和关键硬件部件进行清洗和灭菌, 以消除对陆地微生物的污染, 防止天体的交叉污染2。在过去的十年中, 非热等离子体的应用在生物医学和营养学研究以及航天应用中得到了广泛的关注,3,4,5。等离子体灭菌是一种有前途的替代传统的杀菌方法, 因为它提供了快速和有效的微生物失活6, 同时对敏感和热不稳定材料温和。等离子体放电含有活性剂的混合物, 如自由基、带电粒子、中性/激发原子、紫外线 (UV) 中的光子和真空紫外 (真空) 谱, 导致快速的微生物失活3。本研究采用双电感耦合低压等离子体产生的低压等离子体 (DICP) 源7,8灭活枯草芽孢杆菌孢子在玻璃测试表面上分布。
家庭的革兰氏阳性菌Bacillaceae广泛分布在土壤、沉积物和空气的自然栖息地以及在不寻常的环境中, 如洁净室设施和国际空间站9,10 ,11。最明显的特点,芽孢杆菌是能力形成高抗性休眠孢子 (以下简称孢子) 生存不利的条件, 如养分枯竭12。孢子通常比它们的营养细胞相对于各种处理和环境压力更具抵抗力, 包括热、紫外线、γ辐照、脱水、机械破坏和有毒化学物质, 如强氧化剂或pH 变化的试剂 (在参考文献13,14), 因此是测试微生物失活方法效率的理想对象。由于基因组 DNA 是细菌的等离子体治疗的主要目标15,16, 修复的血浆诱导的 dna 损伤 (如 dna 双链断裂) 孢子复活是至关重要的生存细菌13, 17。
因此, 我们研究了孢子萌发能力和 dna 修复在孢子萌发和生长过程中的作用, 处理后的孢子与低压力氩等离子体的个别孢子和它们的表达荧光标记 dna 修复蛋白质 RecA 与时间分辨共焦荧光显微镜。我们给出了在单分子膜中制备枯草芽孢杆菌孢子的步骤说明, 以实现重现性试验结果, 用低压等离子体处理芽孢膜灭菌, 制备等离子体处理的孢子超微结构评价使用扫描电子显微镜 (SEM) 和现场细胞显微镜分析的个人孢子的水平与监测的活性 DNA 修复过程中发生的细胞内反应等离子体治疗。

<table><tbody><tr><td>Two substance nozzle (model 970-8)</td><td>Schlick</td><td>14,404</td><td>230 V, 50 Hz, D 4.484/8, 0.8 mm bore diameter</td></tr><tr><td>Luria Bertani Medium</td><td>Sigma Aldrich</td><td>70122-100G</td><td></td></tr><tr><td>Tube connectors</td><td>Festo</td><td>n/a</td><td>G 1/8</td></tr><tr><td>Magnetvalve DO35-3/2NC-G018-230AC</td><td>Bosch Rexroth</td><td>820005100</td><td></td></tr><tr><td>PLN Polyamid tube</td><td>Festo</td><td>558206</td><td>d = 6 mm</td></tr><tr><td>Glass slides</td><td>VWR</td><td>48300-026</td><td></td></tr><tr><td>Electric Timer 550-2-C</td><td>Gefran</td><td>F000074</td><td>220 V</td></tr><tr><td>attofluor cell chamber</td><td>Menzel, Fisher Ref.</td><td>3406816</td><td>d=25 mm, round</td></tr><tr><td>MgSO4*7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>13152</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>202967</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt; * 4 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>244589</td><td></td></tr><tr><td>FeSO&lt;sub&gt;4 * 7H2O&lt;/sub&gt;</td><td>AppliChem</td><td>13446-34-9</td><td></td></tr><tr><td>Glucose</td><td>Merck</td><td>215422</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9541-500G</td><td></td></tr><tr><td>Nutrient Broth (NB)</td><td>Merck</td><td>105443</td><td></td></tr><tr><td>Luria-Bertani (LB)</td><td>Merck</td><td>110283</td><td></td></tr><tr><td>96-wellplate</td><td>ThermoFisher</td><td>243656</td><td></td></tr><tr><td>Zeiss LSM 780, Axio Observer Z1</td><td>Carl Zeiss Microscopy GmbH</td><td>n/a</td><td></td></tr><tr><td>Leo 1530 Gemini</td><td>Carl Zeiss Microscopy GmbH</td><td>n/a</td><td></td></tr><tr><td>ZEN 2 and ZEN lite 2012 (Software)</td><td>Carl Zeiss Microscopy GmbH</td><td>n/a</td><td></td></tr><tr><td>SigmaPlot, version 13.0 (Statistic software)</td><td>Systat GmbH, Erkrath, Germany</td><td>n/a</td><td></td></tr><tr><td>Attofluor cell chamber</td><td>Invitrogen</td><td>A7816</td><td></td></tr><tr><td>&amp;micro;-Dish 35 mm, high Grid-500 Glass Bottom</td><td>ibidi</td><td>81168</td><td></td></tr></tbody></table>

investigating the detrimental effects of low pressure plasma sterilization on the survival of bacillus subtilis spores using live cell microscopy

等离子体灭菌是一种有前途的替代传统的杀菌方法的工业, 临床和航天的目的。低压等离子体 (LPP) 放电含有广泛的活性物种, 导致快速的微生物失活。为了研究 LPP 杀菌的效率和机制, 我们使用试验生物体的孢子枯草芽孢杆菌, 因为它们对常规灭菌程序的抵抗力非常高。我们描述了枯草芽孢杆菌的生产孢子单分子膜, 低压等离子体在双电感耦合等离子体反应器中的杀菌过程, 用扫描电子显微镜 (SEM) 表征孢子形态, 以及用活细胞显微镜分析孢子的萌发和生长。血浆物种的一个主要目标是基因组材料 (dna) 和修复的等离子体诱导 DNA 损伤孢子复活是至关重要的生存的有机体。在这里, 我们研究了孢子萌发能力和 dna 修复在孢子萌发和生长后 LPP 的作用, 通过跟踪荧光标记的 dna 修复蛋白 (RecA) 与时间分辨共聚焦荧光显微镜。经过治疗和未经处理的孢子单膜激活发芽和可视化的倒置共聚焦活细胞显微镜随时间的反应, 个别孢子。我们的观察表明, 萌发和超过孢子的分数取决于 LPP 的持续时间, 在120s 后达到最低限度. RecA-YFP (黄色荧光蛋白) 荧光检测仅在少数孢子和发展在所有超过细胞, LPP 处理孢子略有升高。此外, 从 LPP 处理的孢子中获得的一些植物性细菌在细胞质中呈上升趋势, 并趋于溶解。所描述的个体孢子分析方法可以作为研究孢子萌发和生长其他方面的典范。

等离子体处理的枯草芽孢杆菌孢子的存活
等离子体处理的枯草芽孢杆菌在本研究中使用的孢子显示, 随着等离子体处理时间的延长, 存活率下降 (图 2)。表达recA-基因的菌株的孢子 YFP 显示了类似于野生型菌株孢子的存活曲线, 表明基因修饰对细菌活力没有显著影响。与野生型菌株的孢子?...

Watch this Scientific Journal Video about Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy at JoVE.com

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

这项协议说明了评估在用低压等离子体处理后恢复枯草芽孢杆菌孢子后, 监测活力参数和 DNA 修复过程的相关性所需的重要连续步骤。荧光标记的 DNA 修复蛋白质通过时间分辨共焦显微镜和扫描电子显微镜。

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

investigating-detrimental-effects-low-pressure-plasma-sterilization

Research

JoVE Journal

Biology

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

9.4K 次观看.  German Aerospace Center (DLR e.V.). 这项协议说明了评估在用低压等离子体处理后恢复枯草芽孢杆菌孢子后, 监测活力参数和 DNA 修复过程的相关性所需的重要连续步骤。荧光标记的 DNA 修复蛋白质通过时间分辨共焦显微镜和扫描电子显微镜。

视频: Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

在表面定植微生物种群监测空间分异

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

科研

遗传学

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

免疫与感染

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.&#160; In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

建立浮星细胞（MBC-P）和生物膜细胞（MBC-B）抗菌剂的最小杀菌浓度

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

光诱导 原 位透射电子显微镜观察液-软物质相互作用

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

工程学

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

枯草芽孢杆菌孢子计数和标记分析在流式细胞术中的改进

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

生物化学

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

生物工程

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

使用开源液滴微流控进行质粒稳定性分析

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

摘要

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在表面定植微生物种群监测空间分异

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

建立浮星细胞（MBC-P）和生物膜细胞（MBC-B）抗菌剂的最小杀菌浓度

光诱导原位透射电子显微镜观察液-软物质相互作用

方法学研究

枯草芽孢杆菌孢子计数和标记分析在流式细胞术中的改进

枯草芽孢杆菌中细菌体和内膜的可视化

单细胞物种间细菌相互作用的动力学可视化

使用开源液滴微流控进行质粒稳定性分析

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

摘要

探索更多视频

此视频中的章节

在表面定植微生物种群监测空间分异

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

建立浮星细胞（MBC-P）和生物膜细胞（MBC-B）抗菌剂的最小杀菌浓度

光诱导 原 位透射电子显微镜观察液-软物质相互作用

方法学研究

枯草芽孢杆菌孢子计数和标记分析在流式细胞术中的改进

枯草芽孢杆菌中细菌体和内膜的可视化

单细胞物种间细菌相互作用的动力学可视化

使用开源液滴微流控进行质粒稳定性分析

光诱导原位透射电子显微镜观察液-软物质相互作用