Name: cryosectioning yeast communities for examining fluorescence patterns
Uploaded: 2012-12-26T13:00:00
Description: Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

我们要感谢在弗雷德哈钦森癌症研究中心的乔纳森·库珀实验室的许可使用低温恒温器。这项工作是由美国国立卫生研究院和WM凯克基金会的支持。 BM是一个戈登和贝蒂·摩尔的生命科学研究基金会的研究员。我们非常感谢丹尼尔Gottschling，与我们分享他的固定协议。

1。固定酵母社区<ol><li>酵母社区成长的膜过滤器（ 例如 ，Millipore公司的MF膜的过滤器， 图2A）。该协议对应于小于2×10 8个细胞的一个6毫米直径的膜过滤器上的一个典型的社区。 </li><li>使用一块的Whatman 541滤纸覆盖1厘米直径的（ 图2B）的底部。此Whatman滤纸将用作载体，以在以后的阶段转让社区。加入1毫升100％的甲醇井，离开以及-20°C的温度达到平衡。 ...

<ol>
	<li>Tolker-Nielsen, T., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Microbial+Biofilm+Communities.">Spatial Organization of Microbial Biofilm Communities.</a> Microbial Ecology. 40 (2), 75-84 (2000).</li><li>Moller, S., 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=+In+Situ+Gene+Expression+in+Mixed-Culture+Biofilms:+Evidence+of+Metabolic+Interactions+between+Community+Members."> In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members.</a> Appl. Environ. Microbiol. 64 (2), 721-732 (1998).</li><li>Lepp, P. W., 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=Methanogenic+Archaea+and+human+periodontal+disease.">Methanogenic Archaea and human periodontal disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6176-6181 (2004).</li><li>V&#225;chov&#225;, 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=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environmental Microbiology. 11 (7), 1866-1877 (2009).</li><li>Min&#225;rikov&#225;, 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=Differentiated+Gene+Expression+in+Cells+within+Yeast+Colonies.">Differentiated Gene Expression in Cells within Yeast Colonies.</a> Experimental Cell Research. 271 (2), 296-304 (2001).</li><li>Shou, W., 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=Synthetic+cooperation+in+engineered+yeast+populations.">Synthetic cooperation in engineered yeast populations.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1877-1882 (2007).</li><li>Momeni, B., 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=Cooperation+generates+a+unique+spatial+signature+in+microbial+communities.">Cooperation generates a unique spatial signature in microbial communities.</a> , (2012).</li><li>Kiernan, J., ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histological and Histochemical Methods: Theory and Practice. , 4 (2008).</li><li>Piccirillo, S., 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=The+Rim101p/PacC+Pathway+and+Alkaline+pH+Regulate+Pattern+Formation+in+Yeast+Colonies.">The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies.</a> Genetics. 184 (3), 707-716 (2010).</li></ol>

这里介绍的过程提供了一种方法来检查的内部结构的酵母社区。甲醇定影步骤使得酵母菌落刚性8并保留的菌落的完整性，但是，它也导致收缩8，应考虑在解释结果。我们通常会看到30％左右的收缩高的酵母菌菌落，菌落直接嵌入到OCT无固定9。 为了避免收缩为cryosectioning，另一种方法是直接嵌入在华侨城社区，并冻结他们。荧光是保存在方法基于直接?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 100％甲醇</td><td> JT贝克</td><td> 9070-01 </td><td></td></tr><tr><td>组织康OCT化合物</td><td> Andwin科学</td><td> 4583 </td><td></td></tr><tr><td> Whatman公司级541号定量滤纸</td><td> Whatman公司</td><td> 1541-047 </td><td></td></tr><tr><td>微孔-MF膜过滤器</td><td>密理博</td><td> HAWP04700 </td><td></td></tr><tr><td>低温恒温器</td><td>徕卡</td><td> CM 1510小号</td><td></td></tr></tbody></table>

cryosectioning yeast communities for examining fluorescence patterns

微生物通常生活在社区。社区空间组织的细胞内被认为是影响社区1的生存和功能。光学切片技术，包括共聚焦和双光子显微镜，已被证明可用于观察细菌和古细菌的社区空间组织的2,3。共聚焦成像和物理切片的酵母菌菌落的组合显示内部组织单元4。然而，直接用激光共聚焦或双光子显微镜的光学切片只能达到几个细胞层深入酵母菌菌落。这种限制可能是因为强散射光从酵母细胞中4。 在这里，我们提出了一种基于固定和cryosectioning获得的荧光细胞内的空间分布酿酒酵母（Saccharomyces cerevisiae）社区。我们使用甲醇作为定香剂保存细胞的空间分布。固定社区的渗透与OCT化合物，冷冻，和cryosectioned在低温恒温器。荧光成像的部分揭示了内部组织社区内的荧光细胞。 酵母表达红色和绿色荧光蛋白的菌株组成的社区的例子演示的cryosectioning方法来揭示的空间分布的荧光细胞，以及酵母菌落内的基因表达2,3的电位。虽然我们的重点是在酿酒酵母（Saccharomyces cerevisiae）社区，同样的方法也可能适用于研究其他的微生物群落。

在图3中示出的荧光图像的垂直截面包含红色和绿色标记的竞争种群6酵母社区。这些社区包括两个原养型菌株的共享资源，包括空间的酵母和其竞争，导致，显示在它们的垂直的横截面的柱状图形7。分辨率下降到一个单一的单元，可以解决在横截面， 图3比较复制社区获得的（底部）和无（顶部）甲醇-定影后，在图1中的协议的横截面。不固?...

Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

我们提出了一个协议冻结和cryosectioning的酵母菌社区观察荧光细胞的内部模式。该方法依赖于甲醇固定，OCT-嵌入社区内的荧光蛋白失活的细胞没有保留的空间分布。

检查荧光模式Cryosectioning酵母社区

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

Research

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