在JWV组的工作是支持由欧盟玛丽居里重返社会奖学金，一个Sysmo2格兰特（NWO-ALW/ERASysBio），在豪华授予（ZonMW）由VENI奖学金（NWO - ALW）。 OPK组是由几个污水处理厂补助（NWO），SYSMO1（IGdeJ）SYSMO2批，英基EUROCORES SynBio授予（SynMod）Kluyver工业发酵的食品和营养的顶部研究所的基因组学中心的支持。

1。准备B 的枯草文化<ol><li>接种细胞从-80 ° C的股票在10毫升的时间推移显微镜（TLM），中期（62毫米K 2 HPO 4，44mm的KH 2 PO 4，15毫米（NH 4）2 SO 4，6.5 mM柠檬酸钠，0.8毫米硫酸镁 ，0.02％casamino的氨基酸，27.8毫米的葡萄糖，0.1毫米L -色氨酸，pH值设置为7使用一个氢氧化钾溶液）补充抗生素，如有必要。 </li><li>成长的细胞在摇瓶（30 ° C时，225 RPM）过夜。 </li><li>第二天早晨，1:10稀释细胞在预热化学定义介质（CDM）（62毫米K 2 HPO 4，44mm的KH 2 PO 4，15毫米（NH 4）2 SO 4，柠檬酸钠6.5毫米，0.8毫米硫酸镁 ，2.2MM葡萄糖，2.1毫米L -谷氨酸，L -色氨酸6微米，7.5微米，0.15 x金属混合（准备50X吨股票组合（编号8），其中包含：0.2 M MgCl 2的70毫米MnCl 2 氯化钙 ，5毫米，0.1毫米氯化锌 ，0.2毫米硫胺素盐酸盐，盐酸2毫米，0.5毫米FECL 3（添加最后一个）），不含抗生素 2 MnCI 。 </li><li>成长的B。枯草芽孢杆菌细胞指数中期阶段（30℃，225 RPM）。通常，这大约需要4个小时。重要的是，准备琼脂糖幻灯片细胞到达前一个小时指数中期阶段（见第2节）。 </li><li>测量在600nm处600（一）文化的吸光度和稀释细胞近似 600 0.035使用清洁发展机制。这外径确保时间推移显微镜的显微镜玻片上发现的单细胞，与适当的间距。 </li></ol> 2。显微镜样品制备（见图2） 细胞到达前一个小时中指数增长，准备在显微镜幻灯片如下： <ol><li>两个显微镜玻片上（例如Knittel玻璃，7.6 × 2.6厘米），用70％乙醇和水的清洁。 </li><li>以一个基因框架（ABgene; 1.7 × 2.8厘米），并仔细清除，而不会造成基因框架的另一侧的塑料盖，拆卸从基因框架的塑料薄膜。 </li><li>将在玻片首先促进只是一个侧面的接触，用指甲其余基因框架的指导附件中的基因框架。而附加的基因帧载玻片防止气泡。 </li><li>使用微波溶解150毫克（1.5％）高清晰度低熔点琼脂糖（Sigma公司），在10毫升的清洁发展机制。琼脂糖需要充分溶解，以获得最小的背景的时间推移显微镜实验所需。如果需要，补充诱导剂或其他化合物，此时琼脂糖CDM </li><li>转移的基因框架中500μL的热情琼脂糖CDM。确保完全覆盖整个地区，包括（边界）。 以下步骤（2.6 - 2.10），以进行迅速，以防止过度干燥的琼脂糖CDM。 </li><li>琼脂糖CDM基因框架填充上放置第二个载玻片。尽量避免气泡。夹着幻灯片放置在水平位置为4分45 ° C的冰箱，让琼脂糖CDM充分巩固。 </li><li>小心地滑出上载玻片。用刀片切出琼脂〜5毫米宽基因框架内，细胞将增长带。每张幻灯片可以使用最多3条〜4毫米的空间分隔一方。这些空间将提供B 的 ，这是不可或缺的空气枯草芽孢杆菌的增长。需要时间，如果有四个不同的菌株，可以和两个带被削减了一半，导致四个小方块。除去残留的固体培养基。 </li><li>小心地从基因框架的第二次，也是最后一次的塑料盖，揭露粘边的基因框架</li><li>用吸管尖而不触​​及负载的固体培养基上的单核细胞（步骤1.5）。整个地带，或1μL一个小广场上使用2.5μL。始终琼脂糖垫之上，并开始让液体分散分配给它的增长领域，同样转动向上和向下滑动。幻灯片做好准备，尽快液体的边缘成为瓦楞纸箱和液体的运动不再可见时打开的幻灯片。 </li><li>将帧从一个侧面向其他基因（避免气泡）上一个干净的显微镜滑盖滑（24 × 50毫米）。保证完整基因框架用指甲沿盖玻片上施加压力的附件。如果盖玻片的细胞中，而不让他们足够长的时间干细胞往往在实验过程中，以增加彼此顶部。另外要小心不要等待时间过长，在提出申请前盖玻片，因为琼脂糖然后将过于干燥。 </li><li>预温的幻灯片，在1小时30 ° C。如果幻灯片瓦特乌尔德直接放在显微镜（见步骤3.1），在预热的环境室的温度波动，可能会导致实验的第一个小时的自动对焦问题。 </li></ol> 3。定时荧光显微镜（也看到图3和电影1） <ol><li>预温时间的环境室（在我们的手中至少在实验开始前2小时）在实验开始后，为了防止自动对焦的问题。所需的时间取决于环境室加热系统和显微镜以及用作。 </li><li>根据你的实验设置，选择适当的目标，过滤器和分色镜。对于确保长期实验的紫外线过滤器是放在光源和样品之间。此外，如果可能的话，阻止一些使用中性密度滤光片，以尽量减少暴露激发光。 </li></ol>用于下列设备（英国DeltaVision，提供），德容等人发表的时间推移显微镜实验2010年5月 ：IX71显微镜（Olympus），CoolSNAP HQ2相机（普林斯顿仪器），300W氙灯光源，60X明亮领域的目标（1.2​​5 NA），GFP filterset（色度，四十零分之四百七十零nm的激发，发射五十零分之五百二十五纳米），mCherry filterset（色度，在35分之572纳米激发，发射六十〇分之六百三十二纳米）。自动对焦是用透射光，并使用自动对焦例行目前Deltavision的Softworx软件。应当指出，现在有一些其他的自动对焦系统，也适合如蔡司定重点，尼康完美的对焦系统和徕卡自适应焦点控制。 <ol start="3"><li>计划您的实验，根据实验设置。它是明智的，其他时间推移显微镜或细菌的实际实验之前确定的特定构造所需的光量，以及自动对焦设置。更短的曝光时间和激发光量较少，将最大限度地减少漂白和光毒性。使用自动对焦常规的透射光。 </li></ol>以下设置德容等人发表的时间推移显微镜实验用于2010年5月 ：电影快照每隔8或12分钟，使用10％APLLC白LED亮场画面的光线和0.05秒曝光， 10％的氙气灯和GFP的检测，32％氙气灯和0.8 S的曝光mCherry检测，分别为0.5秒曝光。原始数据存储，使用softWoRx 3.6.0（应用Presicion）。 0.06微米的步骤和一个1.2微米的总范围设定为自动对焦。 <ol start="4"><li>放置在显微镜的环境室的预热准备幻灯片（第2）和监控成microcolony单层生长的单细胞在30 ° C </li></ol>特别提示： <ul><li>选择的单细胞在琼脂板的中间。琼脂的边缘垫干出更容易。存储的X，Y，Z位置使用显微镜的软件。 </li><li>舞台的大动作，在X，Y和Z方向的可能扰乱琼脂糖，因此妨碍识别自动对焦例行细胞。在一般情况下，尽量减少X，Y，Z轴运动，我们不选择10％以上实验职位，即使幻灯片包含多株。 </li><li>后选择第一个单元格，只调整与软件的Z -焦点。从这一点来说，不改变焦点手动显微镜上使用的“Z -旋钮”，除非这是数字编码的机构。确保每个自动对焦例行后，新的X，Y，Z位置是存储软件。 </li><li>检查是否自动对焦设置适当的实验，在开始运行之前。相衬显微镜的使用可能会提高使用明或DIC显微镜相比，自动对焦常规，由于增强对比度。然而，在相衬目标相环，使他们较不敏感，在收集的荧光光（约10％）。因此，对于弱荧光的样品，无相环的目标是更适合。 </li><li>检查选定的单元格是否仍然在集中，每半小时，直到实验运行稳定。当细胞在这一点上是重点，手动调整。由于温度的变化，以及不好干样品，这可能是在第一时间内所必需的。此外，由于提高对比度，自动对焦更好的作品时，更多的细胞，在视野。 </li></ul><ol start="5"><li>实验结束后，独立电影作为单独的文件和安全的不同渠道（即相衬，GFP，mCherry）（如果需要一定的收购包堆放在一个单一的文件将放置所有通道）。出版物，图片可以增强2D去卷积，尤其是我们蛋白定位研究eful。 Deconvolve使用显微镜的软件或图像，如商业惠更斯（ <a href="http://www.svi.nl">包</a> www.svi.nl） 。 </li><li>使用ImageJ（ <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> ）（使用的原料，未经处理的图像文件）和Microsoft Excel或西格玛剧情分析数据。堆叠可以例如保存为“AVI”ImageJ电影文件。下面给出一个可以在时间测量荧光单细胞的详细描述。 </li></ol> 4。使用ImageJ的启动子活性的动态数据分析我们注意到，其他良好的软件包，这是专门在BHV软件9，4，Schnitzcell 10，PSICIC 11，以及微生物跟踪12，如时间推移显微镜图像分析，但在这里我们重点免费提供的ImageJ包。 <ol><li>下载ImageJ（ <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> ）（如有必要）正确的插件打开你的文件（堆叠） 。例如，电影记录使用deltavision显微镜只能开在ImageJ该DeltaVision首战插件。 ImageJ插件文件​​夹中复制DV插件和启动程序。更改编辑/选项/ 1250内存和线程的内存容量。这允许更大的文件，如那些从时间推移电影获得工作。 </li><li>要评估一个单细胞的细胞历史，打开原始相衬电影microcolony和滚动到最后一帧，在电影的兴趣。选择“分割线”菜单中的选择按钮。 </li><li>在后台绘制的行，然后按“Ctrl”+“T”型。这将打开的权益回报率（ROI）经理区域。 （相应的背景荧光值，可用于手动减去背景 - 见下文或者使用内ImageJ背景减法例行目前）还提请在利益的细胞，增加的投资回报率的投资回报率经理。由于我们是在这种情况下，研究调查子- GFP融合GFP是分散在整个细胞质中，整个小区每个单个像素的整个细胞的长度应该有类似的荧光值。滚动一帧的时间，并选择一个新的投资回报率在同一单元格的兴趣。保存这第三次的投资回报率，已保存的程序继续下去，直到相应的投资回报率在动画的第一帧。 要知道，大大细胞分裂后子细胞的荧光可以有所不同。既可以使用膜可应用于荧光蛋白产生的细胞（如红膜染料调频® 5-95（Invitrogen公司）和GFP的结合），可视隔形成的染料。在这种情况下，相应的荧光通道和不相衬的电影应该被用来追踪细胞的时间。另外，人们可以留在安全方面的投资回报率的选择只有一个一个细胞的一半。 </li><li>在投资回报率Manager，点击“保存”。如果文件的结尾是“投资回报率。”，然后在列表中选择一个投资回报率，仅这一项将被保存。如果文件的结尾是“压缩”，然后将保存一整套（必需）。 </li><li>关闭相衬电影和开原荧光的（如GFP）的电影。点击“显示所有”和“措施”中的投资回报率经理。将打开一个新窗口（结果）。复制到一个Excel工作表的结果，并减去每个介质的背景荧光的单细胞的平均荧光。对由此产生的净荧光可以绘制时间，揭示了细胞在时间上的兴趣的启动子活性。 </li><li>另外，microcolony的所有细胞的荧光，可分析在特定的时间点。为了做这个选择并保存在一帧的投资回报率的如上所述的背景和每一个细胞，荧光值复制到Excel和生产使用的“工具”菜单中的“直方图”功能的直方图。 </li><li>为了获得出版的单帧图片，选择感兴趣的框架，选择“图像” - “复制图片”和改变的图像类型为“RGB”或“8位”选择“图像” - “类型” - “RGB颜色”或“8位”。保存复制帧“。TIFF”。 RGB/8-bit图片可以打开传统的绘图程序，如CorelDraw或Adobe Illustrator。如果需要，可适应在ImageJ使用“图像” - “窗口/水平” - “调整” - “亮度/对比度”或“形象” - “调整”图片。 </li></ol> 5。生产与ImageJ出版电影<ol><li>打开原始的相衬ImageJ相应的荧光电影（S）如上所述。选择矩形选择按钮（1左ST），在这样的方式在第一帧绘制一个矩形的投资回报率，发展microcolony是封闭的投资回报率THROUghout整个电影。选择“图像” - “作物”和安全的下一个新的名字电影的更小的版本。选择在荧光电影通过相同的投资回报率的投资回报率经理和前进行。 </li><li>要水平或垂直结合的电影，选择“插件” - “栈合成”。如果需要，可以增加时间压模，通过“插件” - “时间压模”。保存一次。“DV”或“TIFF”，一旦合并堆栈。“AVI”或QuickTime影片。 </li></ol> 肺炎链球菌替代议定书“的适应化修改（图4和电影2 ）： 6。准备的S.肺炎文化<ol><li>成长S。肺炎细胞（封装应变D39 13，或者未封装应变R6站在文化在C + Y培养基15 14在37 ° C时，直到OD约0.4 600nm处达到离心2分钟14000转的细胞重悬细胞沉淀在一个新鲜的C + Y培养基中含有甘油14.5％（V / V），这会导致在一个600nm处正好0.4分装的细胞，并储存于-80 ° C以供将来使用量。 </li><li>时间推移显微镜，以前养殖南等分肺炎细胞。接种4毫升的新鲜C + Y培养基细胞从-80℃等分1:100。成长，直到中旬指数生长期的细胞一个0.1 600nm处的OD - 0.2 。通常情况下，这需要约2小时，使用时从-80 °彗星等分细胞。 </li></ol> 7。显微镜样品的制备<ol><li>上面所述为 B准备载玻片枯草 ，但确保琼脂糖包含复杂的C + Y介质。由于南肺炎是microaerophile，琼脂糖带之间的气泡应小于B 的枯草芽孢杆菌 （〜1毫米的空间，双方）。 </li><li>在600海里（600）一个文化的吸光度测量，淡化指数增长的肺炎链球菌细胞近似600 0.05使用C + Y介质和使用稀释负载琼脂糖幻灯片。 </li></ol> 8。时间推移相衬显微镜调整显微镜设置肺炎：使用自南相衬显微镜肺炎链球菌是难以识别使用明场显微镜。继续为B中所述的协议枯草芽孢杆菌 （按照步骤2.9 - 3.7） 年肺炎细胞可以生长在任30 ° C或37 ° C（他们在37 ° C生长较快）。 9。代表性的成果： 时间推移荧光实验已成功，如果细菌进入一个microcolony单层，这是完全坐落在实验结束的视野之内（见图5A - C）上升。如果细胞彼此顶部增长，不仅无法准确地追溯他们的历史，但也不能正确测量重叠细胞的荧光水平。细胞往往生长在彼此顶部，如果发现细胞没有足够的干（步骤2.9），或者如果需要进行调整，以获得经济增长放缓的培养基成分。如果microcolony增长的角度，然后殖民地内的荧光信号的分布不能确定。 “microcolony运动”的原因，可以发现细胞的不足，干燥（步骤2.9），或者如果没有编程软件在开发过程中跟踪microcolony。此外，重要的是，增加了荧光当地补丁不是在中期检测到隐藏的荧光信号来自细胞（见图5D - F）。背景相关的问题，可以从媒体的化合物，airbubbles或不溶琼脂糖团块出现。为了形象化这个，我们在图中显示。 5F的这个特定的幻灯片图像时使用红色荧光染料的激发/发射滤​​波器的背景信号。正如所看到的，明亮的autofluorescent点都存在可能妨碍成像。为了防止这样的点，确保琼脂糖完全溶解，放在显微镜载玻片盖玻片时，有没有airbubbles。 <img src="/files/ftp_upload/3145/3145fig1.jpg" alt="图1"/> 图1：实验概述<img src="/files/ftp_upload/3145/3145fig2.jpg" alt="图2"/> 图2：在显微镜样品的制备<img src="/files/ftp_upload/3145/3145fig3.jpg" alt="图3"/> 图3：定时B.荧光显微镜枯草芽孢杆菌细胞窝藏一个P kinB - GFP融合。快照取自电影1。热门面板：明亮的领域，底部面板：GFP的通道。 <img src="/files/ftp_upload/3145/3145fig4.jpg" alt="图4"/> 图图4：定时相衬显微镜的S.肺炎野生型菌株R6。快照取自电影2。 <img src="/files/ftp_upload/3145/3145fig5.jpg" alt="图5"/> 图5：（时移）显微镜成果的插图。交流显示获得的数据与透射光设置要考虑的因素，需要。 （ 一 ）明视显微镜sporulating B.一个microcolony单层（积极的成果） 枯草芽孢杆菌细胞（B）明场图像的一个B。枯草 microcolony彼此顶部增长，其中一些细胞（阴性结果）（ 三 ）明视形象的一个sporulating B.枯草 microcolony增长的重点领域（消极结果）。东风显示与episcopic灯光设置得到的数据，需要考虑的因素（四） 第一阶段B的对比图片枯草芽孢杆菌中描述的可视化，E和F的荧光信号来自（五）GFP D.注意，是在每一个像素（积极的成果）类似的背景信号中的细胞信号的指数阶段的细胞。另外请注意，曝光时间可能会太多，因为一个单元格中显示了一个饱和信号（负的结果）（F）的信号通过红色通道背景，包含增加红色荧光水平（地区注意在D所示的细胞获得负结果）。 电影1。延时B 的荧光显微镜枯草芽孢杆菌细胞窝藏一个P kinB - GFP融合。快照在8分钟的时间间隔。左：明场，右：绿色荧光蛋白通道。 <a href="/files/ftp_upload/3145/3145movie1.mpg">点击这里观看电影。</a> 电影2。定时相衬显微镜的S.肺炎野生型菌株R6。快照，在10分钟的间隔<a href="/files/ftp_upload/3145/3145movie2.mpg">。点击这里观看影片</a> 。

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没有利益冲突的声明。

在许多其他的单细胞技术相比，荧光显微镜方法，这里所描述的时间推移，可用于后续的一个关于它的祖先，其行为和分裂活动的具体细胞的历史。与荧光标记的目标发起人或蛋白质相结合，具体的发展途径的激活，可以跟在时间和蛋白定位以及可以在蛋白质动力学监测细菌的发展。 如上所述，专注于不同的细菌种类的研究可以进行适应的生长条件，根据一个特定的细菌的要求。我们遇到的唯一的限制是有关的生长条件和样本大小。由于密封的环境，介质条件不能改变在实验过程中。此外，每个实验的四个菌株最多可有效的监控。 考虑到几个关键步骤，单细胞分析方法这里描述可以很容易地适用于使用任何自动显微镜。在下面的，将给予这些关键步骤的概述。在正文详细信息，可以发现一般准备：检查一个特定的细菌实验前所需的自动对焦设置，这是明智的。同样，荧光可视化近似最优设置，应在事先确定，如果可能的话。此外，下面的一个准备的时间线将有助于所有材料准备使用时间（预热显微镜室，编程的显微镜设置，幻灯片前一小时准备细胞所需的生长阶段，看到图1） B 的生长枯草芽孢杆菌在TLM和清洁发展机制：TLM和清洁发展机制是化学定义饥饿媒体B.在其中枯草仅增长缓慢。根据特定菌株细胞生长在媒体的时间周期可能要延长。增长缓慢堆放对方阻止细胞在显微镜样品的制备 ：基因框架，载玻片和盖玻片之间的气泡要防止，防止琼脂糖介质的广泛干燥。这同样适用于中/盖玻片接口。至关重要的是让足够的干细胞，防止游泳和/或多个层生长时间推移荧光显微镜：幻灯片以及环境室预热，以防止重大的自动对焦问题是至关重要的。应选择在琼脂板的中间细胞，因为这些具有最高的机会，留在现场和重点在实验过程中（样品干燥不够好）。最多10％的实验地点仍然正常工作。后选择感兴趣的第一个单元格只使用该软件来调整焦距（详情请参阅文本）。检查细胞是否在重点仍然在第一时间在30分钟的间隔实验分析 ：重要的是扩展分析程序之前检查介质的背景是否有类似的价值观，在荧光渠道。小的尘埃粒子，中型组件，脏镜头或微小的琼脂糖团块可以有助于当地增加荧光，使电影很难或根本无法分析故障排除 ：如果细胞在彼此之上的增长，这既表明盖玻片附加太快或介质不适合microcolony单层增长。如果感兴趣的细胞不断过早死亡，而在幻灯片上的其他细胞分裂愉快，你可能要检查你是否把紫外线过滤器的位置。这也可能有助于减少曝光时间，或在长期实验的光照强度。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>基因框架</td><td> ABgene </td><td> AB - 0578 </td><td> 1.7 × 2.8厘米</td></tr><tr><td>高分辨率低熔点琼脂糖</td><td>西格玛</td><td> A4718 </td><td></td></tr><tr><td>大盖玻片</td><td>几个</td><td></td><td> 24 × 50 mm的</td></tr><tr><td>如果需要的话，膜染料，例如FM 5-95 </td><td> Invitrogen公司</td><td> T23360 </td><td>其他膜染料也可用： <a href="http://probes.invitrogen.com/media/pis/mp34653.pdf">http://probes.invitrogen.com/media/pis/mp34653.pdf</a> </td></tr><tr><td>时间推移显微镜与环境室</td><td>几个</td><td></td><td>在相应的章节中看到我们的设备的详细信息</td></tr></tbody></table>

live cell imaging of bacillus subtilis and streptococcus pneumoniae using automated time-lapse microscopy

科学家在过去的几年里变得越来越意识到，从微生物的人口为基础的实验中获得的平均数据是不是代表的行为，状态或单细胞的表型。由于这一新的见解的单细胞的研究数量不断上升（ 见 1,2,3最近的评论） 。但是，许多应用技术的单细胞不容许一个特定的时间的单细胞（如流式细胞仪或标准显微镜）的发展和行为监测。 在这里，我们提供了一个在最近的几项研究4，5，6，7，允许以下和录音（荧光） 枯草芽孢杆菌和链球菌肺炎通过许多代人的增长和分工个别细菌细胞使用显微镜方法的详细描述。由此产生的电影可以被用来构造内的人口，从一个共同的祖先起源追溯历史的一个单细胞进化谱系树。这时间推移荧光显微镜方法不仅可以用于研究单个细胞的生长，分裂和分化，而且对特定的细胞行为分析细胞的历史和祖先的效果。此外，时间推移显微镜非常适合在细菌细胞周期研究的基因表达的动态和蛋白定位。该方法说明如何准备的细菌细胞和构造的显微镜幻灯片单个细胞的生长，使microcolony。总之，发现单核细胞在半固体表面辅以琼脂糖上，他们的成长和荧光显微镜下隔膜内的温度控制环境室的生长介质组成。拍摄的图像在特定的时间间隔，以后分析使用开放源码软件ImageJ。

Watch this Scientific Journal Video about Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy at JoVE.com

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

该协议提供了一步一步的程序，以监控不同细菌的单​​细胞在使用自动荧光时间推移显微镜的行为。此外，我们提供指引如何分析显微镜图像。

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 ...

live-cell-imaging-bacillus-subtilis-streptococcus-pneumoniae-using

Research

JoVE Journal

Immunology and Infection

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

42.0K 次观看.  University of Groningen. 该协议提供了一步一步的程序，以监控不同细菌的单​​细胞在使用自动荧光时间推移显微镜的行为。此外，我们提供指引如何分析显微镜图像。

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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

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 ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

完整的荧光成像定时<em&gt;秒。 venezuelae</em&gt;生命周期使用微流体设备

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.