Name: characterization of aquatic biofilms with flow cytometry
Uploaded: 2018-06-06T13:30:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Characterization of Aquatic Biofilms with Flow Cytometry at JoVE.com

所提出的工作得到了 SNF Ambizione 奖学金 (PZ00P2_142533) 和 Velux 研究补助金 (Amplebig) 的支持。我们要感谢贝蒂娜瓦格纳对实验工作的帮助。

1. 生态系统选择和生物膜取样
<ol><li>选择一个感兴趣的水生生态系统, 找到生物膜生长的取样点。一条小溪的浅部, 水流缓慢, 中等水流和石河床为生物膜附件是适当的17,18。</li>
	<li>可选: 如果该站点没有足够的表面用于生物膜附着, 或者为了减少站点内的生物膜变异性, 请将人工基质用于生物膜附件 (例如玻璃滑梯、瓷砖), 同时确保它们被放置在与水流相同的方向上, 并且在类似的深度和光照强度下为19。</li>
	<li>为了确定取样点的环境条件, 使用便携式仪器测量表面生物膜上的光强度、水温、电导率、氧和 pH 值。在取样过程中, 采取重复措施 (3–5), 以计算平均值。</li>
	<li>可选: 取水样品, 以确定相关的水化学参数 (溶解有机碳 (DOC), K+, Na+, Ca2 +, Mg2 +, Cl-, F, 所以42-, PO42-, NO32-, 4 4-)。</li>
	<li>使用防护手套, 从每个站点 (或人造基底) 收集可比较的石头 (大小, 形状和类型)。石头应暴露在类似的流量和光线条件下18。</li>
	<li>从该站点过滤足够的流水, 通过0.22 微米过滤器, 以便为所有收集的样品 (例如,每样15毫升) 做好准备。</li>
	<li>使用软牙刷将生物膜从基体中移除到瓶中 (刷直到所有生物膜被去除) 与15毫升过滤流水20。</li>
	<li>将样本固定在0.01% 多聚甲醛和0.1% 戊二醛21 中。</li>
</ol>2. 最佳流细胞 (FC) 参数的确定
<ol><li>将标本转移到光显微镜 (使用 40 x/0.75 目标; 如果需要, 请使用 100 x 1.3 石油目标), 并确定样本中存在的物种22,23。</li>
	<li>从适当的文化集合中确定单个物种 (例如在格廷根大学 [EPSAG] 的海藻的实验性心理学和文化收集或海藻的文化收集 [管理委员会] 如果监测是在中欧生态系统中进行的)。</li>
	<li>在类似的环境条件 (例如,光、温度、pH 值) 中连续生长单个物种, 从而将生物膜样品从环境中抽取出来。建议在1摄氏度和 0.5 pH 点的环境样品中停留。</li>
	<li>要分离/分散细胞, 将15毫升的单种样品放入离心管中, 将管子放在水浴的中心并浸没, 使试样的液面与浴缸的表面处于同一水平。油脂实验在45赫24、25上的采样值为1分钟.</li>
	<li>用平板阅读器记录单个物种的吸光度和荧光光谱。请参阅板阅读器选择的说明。在这个例子中, 96 井平底透明 polystyrol 板加载200µL 样品量和吸光度测量。(使用的设置: (a) 荧光扫描模式; 发射波长步长 10 nm, 发射扫描数 30, 带宽 20 nm, 增益 100, 25 闪烁, 20 美国集成时间或 (b) 吸光度扫描模式; 25 闪烁, 带宽 9 nm)。</li>
	<li>设置流式细胞仪与激光器, 分色分配器和过滤器, 组合覆盖所有的荧光范围, 其中不同的物种具有特定的性质。对于中欧溪流, 405 毫微米, 488 毫微米和 638 nm 激光产生有用的结果。</li>
	<li>调整光电倍增管的电压设置 (不适用于所有流 cytometers) 和所测量的 fluorescences 的范围, 以包括样本中至少99% 的事件。</li>
	<li>或者, 如果在生物膜中存在物种的知识可用, 请从步骤2.2 开始。如果已知荧光属性, 请从步骤2.6 开始。</li>
</ol>3. 可选: 编制流动细胞参考数据库
注意: FC 不允许对生物膜中存在的细胞进行分类鉴定。一个已知存在于生物膜中的单一物种 FC 测量的参考数据库, 在与生物膜相似的条件下生长, 可以帮助解释数据。
<ol><li>
参考: 单种
	<ol>
<li>使用在2.5–7中设置的优化 FC 参数, 测量单个物种 (固定在多聚甲醛和戊二醛中) 的光学和荧光特性, 并通过适当大小的过滤器对流式细胞仪进行过滤。</li>
		<li>以与生物膜中的数据相同的方式正常化来自单一物种的数据 (见步骤 6.1.2)。</li>
	</ol>
</li>
	<li>
参考: 损坏的和腐烂的细胞 生物膜健康的一个最重要的指标是生物膜中存在的受损/死亡细胞的比例。为了量化这些细胞, 可以准备一个腐烂细胞的参考。<ol>
<li>采取1毫升标本稀释生物膜和离心机在室温下在 8000 x g 10 分钟的台式离心机。将产生的颗粒悬浮在1毫升90% 乙醇中, 并将悬浮液隔夜存放在4摄氏度。第二天重复。</li>
		<li>使用优化的 FC 设置, 测量受损和衰减细胞的光学和荧光特性 (固定和过滤)。</li>
	</ol>
</li>
	<li>
参考: microplastics 注意:如果有兴趣监测生物膜中的 microplastic 粒子, 请检查它们是否与光学和荧光特性中的生物粒子有足够的鉴别。<ol>
<li>根据 microplastics 生产者的指示暂停 microplastic 粒子, 并测量它们的光学和荧光特性 (使用 FC 参数从 2.7)。</li>
		<li>将 microplastic 粒子添加到生物膜样品中, 并在4摄氏度的晚上离开。</li>
		<li>用上面的 FC 参数过滤悬浮和测量。</li>
	</ol>
</li>
</ol>4. 样品准备和贮存
<ol><li>一旦 fc 的设置和 fc 参考数据库准备就绪, 你可以进行评估新鲜生物膜样品, 收集根据步骤1.1–7。分离/分散生物膜, 从烧瓶中转移15毫升的样品 (见步骤 1.7) 进入离心管。</li>
	<li>把每管在水浴的中心和浸没, 使样品的液体表面与浴缸的表面相同的水平。在45赫油脂实验样品1分钟。</li>
	<li>将样品固定在0.01% 多聚甲醛和0.1% 戊二醛 (nanopure 水中的股票)。存储在4°c, 直到准备分析。</li>
	<li>重要: 使用一半的样本进行流式细胞分析, 并保持一半的存储安全和可选验证与荧光活化细胞分类和/或光显微镜 (见 6)。 注意:存储在冰箱中, 固定生物膜样品应保持其光学和荧光性能长达三周。</li>
</ol>5. 通过 FC 运行示例
<ol><li>如果样品储存在冰箱里, 油脂实验他们快速或吸管几次, 以分离颗粒。在测量前, 用50微米过滤器过滤标本 (过滤器大小取决于 FC 毛细管的宽度)。</li>
	<li>装载各自的样品 (, 这取决于流动细胞仪使用) 入流式细胞仪和测量每个微粒的光学和荧光特性。重复测量每一个生物复制三次 (三技术复制)。</li>
	<li>以. csv 格式导出 FC 中的数据。</li>
</ol>6. 数据准备和相关分析
FC 可以分析多个参数 (例如, 信号区域, 高度, 宽度) 为每个测量的光学属性和荧光范围。对实测的变量进行相关分析, 只保留那些不太相关的度量值。这通常会移除一个 FC 参数 (例如、信号区域), 并且还可以删除高度相关的荧光测量 (通常是从相同的激光器和相邻滤波器中产生的)。
<ol><li>
在 Matlab 中运行 CYT 作为工具箱:
	<ol>
<li>从要分析到 CYT 软件16的所有示例中导入. csv 格式的缩减 FC 数据, 包括所有技术和生物复制。(单击 +, 文件筛选 ** csv, 选择要导入的文件)。</li>
		<li>使用双曲反正弦值变换变换所有样品的荧光通道。需要选择余子的价值;150工作的大多数 phototrophic 生物膜和流 cytometers, 但优化可能是必要的特定实验设置和异养生物膜。(右键单击/转换/输入余子 150)</li>
		<li>为了质量控制, 视觉上比较 (在 CYT) 单个样品和单独的光学和荧光通道的直方图, 以确保在复制的技术和生物水平上没有异常点。异常点将在复制之间显式地转移荧光/光分布。(选择数据, 选择频道, 绘图)<ol>
<li>替代方法: 对荧光分布的中值进行主成分分析 (PCA), 确保技术和生物复制聚集在一起。</li>
		</ol>
</li>
		<li>在每种生物复制中合并技术复制, 并亚组生物复制, 使每一个由相同数量的粒子 (细胞, 细胞片段和非生物粒子) 来表示, 从而使粒子的总数量被分析巴恩斯-小屋随机邻域嵌入 (bh-SNE) 大约 15万 (为最佳的可视化结果26)。(选择数据, 右键单击, 合并/亚组)</li>
		<li>为了比较样品, 重要的是只选择那些要比较的样品。选择示例并运行 bh27。算法完成后, 就会出现新的通道, 命名为 bh-SNE1 和 bh-SNE2。这些是所有分析粒子的 SNE 坐标, 可用于在散点图 (viSNE 图) 中可视化数据。(选择数据, 选择频道, 右键单击, bh, 是规范化数据)</li>
		<li>在 viSNE 图中, 粒子根据相似性定位, 并分为可视可分离簇。检查簇的光学/荧光特性, 并通过使用 CYT 中可用的绘图工具标记和命名那些分离良好并具有不同光学/荧光性质的产品。(选择通道 bh-SNE1, bh-SNE2)</li>
		<li>可选: 如果单个微生物物种的参考数据库 (用相同的流式细胞仪设置和生长在类似条件下) 可用, 将 viSNE 映射导出到 Matlab 中, 并将参考数据库投射到地图上。这样, 特定的集群可以连接到特定的物种/分类群 (脚本可在 https://github.com/anzezupanic/FC_analysis 下载)。(会话/保存)</li>
		<li>疑难解答: 如果 bh-sne 的分析不返回视觉上可分离的簇, 但一个单一的大, 太多的粒子被用于在 bh-sne。请再试一次, 以减少每个样本的粒子。如果没有簇, 而是稀疏的单个点, 则增加使用的粒子数。</li>
	</ol>
</li>
	<li>可选: 可以使用其他聚类方法 (例如、分层、基于质心或基于密度的聚类), 而不是依赖于 viSNE 映射 (https://github.com/anzezupanic/FC_analysis)。</li>
	<li>一旦确定了簇, 请计算每个样本中属于每个生物复制的每个簇中的粒子数。评估样本之间的差异, 使用方差分析和 Tukey 的测试适当 (例如, 福尔摩斯) 更正多个比较。</li>
</ol>7. 可选: 利用荧光活化细胞分类验证聚类解释
<ol><li>一旦确定了簇, 使用荧光活化细胞分选 (外地资产管制), 如果需要的话, 将它们从样本中分类, 前提是该探测器具有类似 (或相同) 的激光器和荧光过滤器的配置。</li>
	<li>对于每个已识别的群集, CYT 提供了用于定义群集的光学和荧光门。使用这些门来整理每个集群中的粒子, 然后用光镜识别被排序的细胞。</li>
</ol>

<ol>
	<li>Battin, T. J., Besemer, K., Bengtsson, M. M., Romani, A. M., Packmann, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+and+biogeochemistry+of+stream+biofilms.">The ecology and biogeochemistry of stream biofilms.</a> Nat Rev Microbiol. 14 (4), 251-263 (2016).</li><li>Rotter, S., Heilmeier, H., Altenburger, R., Schmitt-Jansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+stressors+in+periphyton+-+comparison+of+observed+and+predicted+tolerance+responses+to+high+ionic+loads+and+herbicide+exposure.">Multiple stressors in periphyton - comparison of observed and predicted tolerance responses to high ionic loads and herbicide exposure.</a> J Appl Ecol. 50 (6), 1459-1468 (2013).</li><li>Tlili, A., 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=Pollution-induced+community+tolerance+(PICT):+towards+an+ecologically+relevant+risk+assessment+of+chemicals+in+aquatic+systems.">Pollution-induced community tolerance (PICT): towards an ecologically relevant risk assessment of chemicals in aquatic systems.</a> Freshw Biol. 61 (12), 2141-2151 (2016).</li><li>Lavoie, I., Lavoie, M., Fortin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mine+of+information:+Benthic+algal+communities+as+biomonitors+of+metal+contamination+from+abandoned+tailings.">A mine of information: Benthic algal communities as biomonitors of metal contamination from abandoned tailings.</a> Science of the Total Environment. 425, 231-241 (2012).</li><li>Tlili, A., Montuelle, B., Berard, A., Bouchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+chronic+and+acute+pesticide+exposures+on+periphyton+communities.">Impact of chronic and acute pesticide exposures on periphyton communities.</a> Sci Total Environ. 409 (11), 2102-2113 (2011).</li><li>Dorigo, U., 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=Lotic+biofilm+community+structure+and+pesticide+tolerance+along+a+contamination+gradient+in+a+vineyard+area.">Lotic biofilm community structure and pesticide tolerance along a contamination gradient in a vineyard area.</a> Aquat Microb Ecol. 50 (1), 91-102 (2007).</li><li>Pesce, 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=Evaluation+of+single+and+joint+toxic+effects+of+diuron+and+its+main+metabolites+on+natural+phototrophic+biofilms+using+a+pollution-induced+community+tolerance+(PICT)+approach.">Evaluation of single and joint toxic effects of diuron and its main metabolites on natural phototrophic biofilms using a pollution-induced community tolerance (PICT) approach.</a> Aquat Toxicol. 99 (4), 492-499 (2010).</li><li>Ricart, 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=Effects+of+low+concentrations+of+the+phenylurea+herbicide+diuron+on+biofilm+algae+and+bacteria.">Effects of low concentrations of the phenylurea herbicide diuron on biofilm algae and bacteria.</a> Chemosphere. 76 (10), 1392-1401 (2009).</li><li>Martinez, A., Kominoski, J. S., Larranaga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf-litter+leachate+concentration+promotes+heterotrophy+in+freshwater+biofilms:+Understanding+consequences+of+water+scarcity.">Leaf-litter leachate concentration promotes heterotrophy in freshwater biofilms: Understanding consequences of water scarcity.</a> Sci Total Environ. , 1677-1684 (2017).</li><li>Corcoll, 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=Comparison+of+four+DNA+extraction+methods+for+comprehensive+assessment+of+16S+rRNA+bacterial+diversity+in+marine+biofilms+using+high-throughput+sequencing.">Comparison of four DNA extraction methods for comprehensive assessment of 16S rRNA bacterial diversity in marine biofilms using high-throughput sequencing.</a> Fems Microbiol Lett. 364 (14), (2017).</li><li>Welsh, A. K., McLean, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+bacteria+in+mixed+biofilm+communities+using+denaturing+gradient+gel+electrophoresis+(DGGE).">Characterization of bacteria in mixed biofilm communities using denaturing gradient gel electrophoresis (DGGE).</a> Curr Protoc Microbiol. , (2007).</li><li>Ranjard, 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=Characterization+of+bacterial+and+fungal+soil+communities+by+automated+ribosomal+intergenic+spacer+analysis+fingerprints:+Biological+and+methodological+variability.">Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: Biological and methodological variability.</a> Appl Environm Microbiol. 67 (10), 4479-4487 (2001).</li><li>Stewart, T. J., Traber, J., Kroll, A., Behra, R., Sigg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+extracellular+polymeric+substances+(EPS)+from+periphyton+using+liquid+chromatography-organic+carbon+detection-organic+nitrogen+detection+(LC-OCD-OND).">Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND).</a> Environ Sci Pollut R. 20 (5), 3214-3223 (2013).</li><li>Kroll, A., 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=Mixed+messages+from+benthic+microbial+communities+exposed+to+nanoparticulate+and+ionic+silver:+3D+structure+picks+up+nano-specific+effects,+while+EPS+and+traditional+endpoints+indicate+a+concentration-dependent+impact+of+silver+ions.">Mixed messages from benthic microbial communities exposed to nanoparticulate and ionic silver: 3D structure picks up nano-specific effects, while EPS and traditional endpoints indicate a concentration-dependent impact of silver ions.</a> Environ Sci Pollut R. 23 (5), 4218-4234 (2016).</li><li>Sgier, L., Freimann, R., Zupanic, A., Kroll, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+combined+with+viSNE+for+the+analysis+of+microbial+biofilms+and+detection+of+microplastics.">Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics.</a> Nat Commun. 7, 11587 (2016).</li><li>Amir el, A. D., 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=viSNE+enables+visualization+of+high+dimensional+single-cell+data+and+reveals+phenotypic+heterogeneity+of+leukemia.">viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.</a> Nat Biotechnol. 31 (6), 545-552 (2013).</li><li>Mora-Gomez, J., Freixa, A., Perujo, N., Barral-Fraga, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limits+of+the+biofilm+concept+and+types+of+aquatic+biofilms.">Limits of the biofilm concept and types of aquatic biofilms.</a> Aquatic Biofilms: Ecology, Water Quality and Wastewater Treatment. , 3-27 (2016).</li><li>Matthaei, C. D., Guggelberger, C., Huber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+disturbance+history+affects+patchiness+of+benthic+river+algae.">Local disturbance history affects patchiness of benthic river algae.</a> Freshw Biol. 48 (9), 1514-1526 (2003).</li><li>Tlili, A., Hollender, J., Kienle, C., Behra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropollutant-induced+tolerance+of+in+situ+periphyton:+Establishing+causality+in+wastewater-impacted+streams.">Micropollutant-induced tolerance of in situ periphyton: Establishing causality in wastewater-impacted streams.</a> Water Res. 111, 185-194 (2017).</li><li>Robinson, C. T., Rushforth, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+physical+disturbance+and+canopy+cover+on+attached+diatom+community+structure+in+an+idaho+stream.">Effects of physical disturbance and canopy cover on attached diatom community structure in an idaho stream.</a> Hydrobiologia. 154, 49-59 (1987).</li><li>Pomati, F., Nizzetto, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+triclosan-induced+ecological+and+trans-generational+effects+in+natural+phytoplankton+communities:+A+trait-based+field+method.">Assessing triclosan-induced ecological and trans-generational effects in natural phytoplankton communities: A trait-based field method.</a> Ecotoxicology. 22 (5), 779-794 (2013).</li><li>Hulliger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methoden zur Untersuchung und Beurteilung der Fliessgew&#228;sser. , (2007).</li><li>Tlili, A., 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+spatio-temporal+changes+in+pollution-induced+community+tolerance+to+zinc+in+autotrophic+and+heterotrophic+biofilm+communities.">In situ spatio-temporal changes in pollution-induced community tolerance to zinc in autotrophic and heterotrophic biofilm communities.</a> Ecotoxicology. 20 (8), 1823-1839 (2011).</li><li>Foladori, P., Laura, B., Gianni, A., Giuliano, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sonication+on+bacteria+viability+in+wastewater+treatment+plants+evaluated+by+flow+cytometry+-+Fecal+indicators,+wastewater+and+activated+sludge.">Effects of sonication on bacteria viability in wastewater treatment plants evaluated by flow cytometry - Fecal indicators, wastewater and activated sludge.</a> Water Res. 41 (1), 235-243 (2007).</li><li>Buesing, N., Gessner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+detachment+procedures+for+direct+counts+of+bacteria+associated+with+sediment+particles,+plant+litter+and+epiphytic+biofilms.">Comparison of detachment procedures for direct counts of bacteria associated with sediment particles, plant litter and epiphytic biofilms.</a> Aquat Microb Ecol. 27 (1), 29-36 (2002).</li><li>van der Maaten, L., Hinton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Data+using+t-SNE.">Visualizing Data using t-SNE.</a> J Mach Learn Res. 9, 2579-2605 (2008).</li><li>van der Maaten, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+t-SNE+using+Tree-Based+Algorithms.">Accelerating t-SNE using Tree-Based Algorithms.</a> J Mach Learn Res. 15, 3221-3245 (2014).</li></ol>

作者没有什么可透露的。

上面描述的协议相对简单, 可以实现。然而, 虽然所提出的默认设置已被证明适用于迄今为止所测试的所有 phototrophic 生物膜, 但优化 (如协议中所描述的) 对于最大化从该方法获得的信息是必要的。事实上, 根据环境条件 (季节、温度、水的化学成分)1, 生物膜的光学和荧光特性会有所不同。因此, 在设置 FC 分析时, 必须考虑到这种可变性。这样做的一个方法是, 从取样点采集生物膜?...

生物膜是微生物的动态联合体, 在淡水生态系统中发挥关键作用, 从初级生产、养分循环和净水到影响微生物在生态系统中的分布及其生物多样性。1. 当生物膜暴露在变化的环境条件或压力源 (如化学物质) 中时, 它们的社区结构迅速转向更容忍的物种2,3。它们的高灵敏度使生物膜成为环境监测的有吸引力的模型系统4, 但是目前的方法都不完全适合于以快速和简便的方式实际跟踪生物膜群落的动态。
通常使用的一套方法来表征生物膜, 包括测量功能和结构端点。在整个社区的水平上, 光合和呼吸活动以及胞外酶活性提供有关生物膜的功能状态的信息5,6,7,8 ,9。生物量应计被用作整体生物膜生长的指标。目前的结构变化是通过使用光学显微镜或基于核苷酸技术的传统物种鉴定 (例如、变性梯度凝胶电泳 (DGGE)、自动核糖体基因间间隔) 来测量的。分析 (ARISA), metagenomics)10,11,12。这些方法提供了信息, 但对于执行或需要特定的知识或仍在开发中是非常耗时的。最后, 评估胞外高分子物质 (EPS)13、14和生物膜结构1的新方法虽然敏感, 但吞吐量低, 尚未发展到监视目的。
很明显, 对于淡水生物膜的完整描述, 有必要结合几种不同的方法, 这就提供了对生物膜功能, 组成和结构的洞察。另一方面, 为了进行环境监测, 需要一种快速而灵敏的方法, 能够检测生物膜的变化, 并对功能和结构层面上的转变进行基本的生物解释。
我们开发了一种新的方法, 微生物群落的 phototrophic 成分的流生物膜 (periphyton), 这是足够快的监测目的, 并同时提供充分的信息, 生物膜群落允许生物解释15的结构。它是基于单细胞流式细胞术 (FC) 的生物膜样品, 并结合计算可视化, 并提供信息的光和荧光性质的生物膜在单细胞水平。
生物膜取样后的工作流包括样品的制备, 以超声波、固定和大小为基础的过滤, 然后通过流式细胞仪对样品进行评估。所获得的数据提供了一个单一的测量中的几个细胞特性的信息: 颗粒大小, 密度, 颜料含量, 非生物含量 (例如microplastics) 在生物膜。这组数据比通过可视化随机邻域嵌入 (viSNE)16的计算可视化进行分析, 从而能够快速、方便地解释数据。虽然需要几个星期的时间来建立和优化方法, 一旦建立, 它只需要几个小时收集生物膜样本, 以解释结果。
所提出方法的主要优点是分析速度快, 信息内容高。此外, 样品可在收集后数周储存, 而不会丧失其光学和荧光特性。当需要对大量样本进行描述时, 例如大型取样研究或生物监测技术程序, 但也可以在较小的探索性研究中提供大量信息, 这一点非常有用。
提出的协议是基于流细胞分析的 phototrophic 生物膜 (periphyton) 收集从不同地点的溪流。协议的许多步骤,例如选择适合于监视目的的站点, 取决于研究的目标, 因此不能规定。另一些人则允许较少的自由, 并要求严格遵守该议定书, 下面的详细议定书将对此作出明确说明。
该议定书的出发点是选择以生物膜为基础的环境监测地点。下一步是建立流式细胞仪 (FC), 使其测量能够使在受监测环境中生活在生物膜中的不同 phototrophic 生物体之间存在歧视。这是通过收集来自这些地点的生物膜样本, 确定在生物膜中的不同物种的荧光特性, 并设置流式细胞仪与激光和过滤器, 使测量在适当的光学波长。一旦建立了 FC, 可以周期性地收集生物膜, 用流式细胞仪测量生物膜中的单个粒子的光学和荧光特性, 以及通过视觉聚类分析的数据。为了更好地解释结果, 有可能建立一个 fc 参考数据库的地方生物膜生物物种及其表型, 并利用数据库, 以确定不同分类法的 fc 数据。验证是可能的, 通过荧光分类的鉴定的单一细胞簇使用的外地观察团和显微镜下的分类鉴定目前的物种。协议的示意图在图 1中给出。

<table><tbody><tr><td>Multimeter</td><td>WTW</td><td>MultiLine 3620 IDS</td><td>For measuring temperature, pH, dissolved oxygen</td></tr><tr><td>Ultrasonic cleaner</td><td>VWR International</td><td>97043-986</td><td>Tank dimesions: 15*14*10 cm</td></tr><tr><td>Flow cytometer</td><td>Beckman Coulter</td><td>Gallios</td><td>Lasers: 405, 488 and 638 nm. Filters bands in Supplementary Table 11. Sgier et, Nat Comm, 2016.&amp;nbsp;</td></tr><tr><td>Plate reader</td><td>Tecan</td><td>Infinite M200</td><td>used for selecting appropriate setting of the FC</td></tr><tr><td>Cell sorter</td><td>Beckman Coulter</td><td>MoFlo Astrios</td><td>Settings in Supplementary Table 12. Sgier et, Nat Comm, 2016.&amp;nbsp;</td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axiovert 135</td><td>Zeiss EC Plan-Neofluar 40x/0.75 objective</td></tr><tr><td>SOFTWARE</td><td></td><td></td><td></td></tr><tr><td>Matlab</td><td>MathWorks</td><td>R2013a</td><td>software for numerical computing</td></tr><tr><td>CYT</td><td>Dana Pe&#039;er Lab</td><td>Version 1.1</td><td>free interactive visualization tool for analysis of cytometry data</td></tr></tbody></table>

characterization of aquatic biofilms with flow cytometry

生物膜是微生物的动态联合体, 在淡水生态系统中起着关键作用。通过改变其群落结构, 生物膜对环境变化反应迅速, 因而可以用作水质指标。目前, 生物膜评估主要基于综合和功能端点, 如光合或呼吸活动, 不提供关于生物膜群落结构的信息。流式细胞术和计算可视化提供了一种替代性的、敏感的和易于使用的方法来评估群落组成, 特别是淡水生物膜的 photoautotrophic 部分。它只需要基本的样品准备, 然后整个样品通过流式细胞仪运行。单细胞光学和荧光信息用于计算可视化和生物解释。它的主要优势比其他方法是速度的分析和高信息内容的性质。流式细胞仪在单个测量中提供了几种细胞和生物膜特性的信息: 颗粒大小、密度、颜料含量、生物膜中的非生物含量以及粗分类学信息。但是, 它没有提供关于物种水平上的生物膜组成的信息。我们认为, 使用水生生态系统环境监测方法的潜力很大, 作为初步的生物膜评估步骤, 通过补充和更详细的方法向下游进行详细调查。

使用此处介绍的过程 (图 1), 分析了从瑞士本地流的多个站点抽取的样本。在每个地点, 三个相似大小的石头 (10–12 cm) 被采取了, 并且生物膜被刷掉石头。然后用流式细胞仪对样品进行微气泡、固定、过滤和后期分析。流式细胞仪的设置和所使用的参考数据库与前一篇论文15中所描述的相同: 三激光器使用了 (405、488和 638 nm)、七个分?...

Watch this Scientific Journal Video about Characterization of Aquatic Biofilms with Flow Cytometry at JoVE.com

Characterization of Aquatic Biofilms with Flow Cytometry

流式细胞仪对水生生物膜的表征

流式细胞术与视觉聚类结合, 为研究水生生物膜提供了一种简便、快速的方法。它可用于生物膜的表征, 检测生物膜群落结构的变化, 以及检测埋在生物膜中的非生物微粒。

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

characterization-of-aquatic-biofilms-with-flow-cytometry

Research

JoVE Journal

Environment

9.0K 次观看.  Eawag - Swiss Federal Institute for Aquatic Science and Technology. 流式细胞术与视觉聚类结合, 为研究水生生物膜提供了一种简便、快速的方法。它可用于生物膜的表征, 检测生物膜群落结构的变化, 以及检测埋在生物膜中的非生物微粒。

视频: Characterization of Aquatic Biofilms with Flow Cytometry

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

科研

环境科学

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

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 ...

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

生物工程

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

分子探针的优化，以确定细胞死亡的光合生物（<em&gt;铜绿微囊藻</em&gt;）使用流式细胞仪

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

医学

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...