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>

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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的代表性样本和/或采取单一物种, 并在不同的条件下生长, 并使用不同的设置来测量它们。生长单一物种对于构建可用于解释 FC 结果的荧光属性数据库也很有用。然而, 要做到这一点, 重要的是, 单一物种的测量, 在类似的条件, 对那些下生物膜将被取样。例如, 一个由25摄氏度生长的单种群的 FC 测量数据库将很少了解生物膜生长在15摄氏度的成分。但是, 建立一个参考数据库来有效地使用此方法并不是绝对必要的。已经使用了基本的 FC 协议, 有可能获得信息的大小, 粒度和存在的不同色素在细胞中存在的生物膜。可视化聚类允许将单元格分类为在测量的属性中类似的簇, 并确定哪些属性在样本之间发生了最大的变化。但是, 在将分类标识分配给不同的群集时, 参考数据库变得非常重要。一旦检测到生物膜特性的变化, 就有可能将它们与取样点上的水的物理和化学测量相关联。这允许确定哪个环境因子对被监测的生态系统中的生物膜群落影响最大。
该方法还有一些限制。例如, FC 不允许在物种层次上识别生物膜组成。此外, 当检测到生物膜特性的变化时, FC 不能归咎于负责任的原因: 转向更宽容的物种, 或朝向更宽容的表型 (物种保持不变)。还需要其他补充方法, 如 metagenomics, 以解决这一问题。该方法的另一个局限性是, 对具有不同荧光性质的颜料的生物膜的 phototrophic 成分进行了分析。虽然可以用这种方法进行异养生物膜的表征, 但尚不清楚是否有足够的信息可通过无污点分析获得。由于细菌和真菌的天然荧光性较低, 必须使用不同的设置, 因此不可能在单个 FC 运行中对生物膜的 phototrophic 和异养成分进行评估。相反, 需要将样本分离, 并用 phototrophic 和一个部分与异养设置进行评估。最后, 利用 viSNE 对生物膜的可视化具有局限性。最好的结果是与 ca 15万细胞一起成像。这对可以分析和与方法进行比较的样本数设置了限制, 而不计算过多地稀释样品。例如, 如果要比较150个样本, 则每个样本只能由 ca. 1000 个单元格表示。围绕这一限制的一个方法是, 使用 "移动窗口" 方法分析样本, 通过 viSNE 可以单独分析重叠的样本组, 然后将结果汇集在一起, 然而, 这种解决方案尚未实现。
我们认为, 基于流式细胞仪的生物膜评价可以有效地应用于水生生态系统的监测。它可用于比较在不同地点采集的时间序列样品或样品, 而且, 除了取样本身, 还具有完全自动化的潜力。该方法可以被认为是一个初始生物膜评估步骤, 为下游详细调查提供信息, 补充和更详细的方法。最后, 它可以扩展到更具体的应用, 如检测生物膜的非生物污染, 我们已经表明, microplastic 的情况下。

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 border="0" cellpadding="0" cellspacing="0" width="1095">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Multimeter</td>
			<td style="width:123px;">WTW</td>
			<td style="width:344px;">MultiLine 3620 IDS</td>
			<td style="width:413px;">For measuring temperature, pH, dissolved oxygen</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ultrasonic cleaner</td>
			<td>VWR International</td>
			<td>97043-986</td>
			<td>Tank dimesions: 15*14*10 cm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>Gallios</td>
			<td style="width:413px;">Lasers: 405, 488 and 638 nm. Filters bands in Supplementary Table 11. Sgier et, Nat Comm, 2016.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td>used for selecting appropriate setting of the FC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cell sorter</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:344px;">MoFlo Astrios</td>
			<td style="width:413px;">Settings in Supplementary Table 12. Sgier et, Nat Comm, 2016.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fluorescence microscope</td>
			<td style="width:123px;">Zeiss</td>
			<td style="width:344px;">Axiovert 135</td>
			<td style="width:413px;">Zeiss EC Plan-Neofluar 40x/0.75 objective</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SOFTWARE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Matlab</td>
			<td>MathWorks</td>
			<td>R2013a</td>
			<td>software for numerical computing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CYT</td>
			<td>Dana Pe&#39;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)、七个分色分光器和十个包含从425到 > 755 nm 的荧光辐射的滤镜。和参考数据库, 包括 > 30 种覆盖蓝藻、绿藻、硅藻和红藻类的植物, 在瑞士溪流的生物膜中发现。用于创建 viSNE 映射的粒子总数约为 15万 (图 2A)。使用描述的方法, 可以区分不同的站点 (图 2B), 将 viSNE 映射的不同部分分类为具有粗分类信息的单元格 (图 3), 并确定哪些样本之间的亚群不同 (图 4)。
一种类似的方法被用来量化 microplastic 粒子在生物膜。与淡水生物膜中细胞相似大小的 microplastic 颗粒的纯样品 (约10µm), 在实验室中生长在 microplastic 颗粒存在或不存在的生物膜样本 (使用 Tlili et中描述的生物膜生长协议)。20115) 进行了测量和比较 (图 5)。确定了具有已知光学和荧光特性的 microplastic 粒子在 1000 ppm 以上的浓度下可以得到稳健的检测。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57655/57655fig1.jpg"> 图 1: 实验性协议的架构.收集环境生物膜样品之后, 确定生物膜中存在的物种, 测定其光学和荧光特性, 并将流式细胞仪与适当的激光器和过滤器配合使用, 以测量这些性能。对于流细胞测量, 样品微气泡, 固定和储存。当采集到足够的样本时, 用流式细胞仪测量每个测量细胞, 利用视觉聚类方法获得和分析了许多光学和荧光特性。可选 (虚线箭头), 可以建立一个 FC 数据库的物种存在于监测的生物膜和使用数据库来解释结果。还可以使用进一步的验证方法,例如基于荧光显微镜的细胞分类和分类学分析<a href="/files/ftp_upload/57655/57655fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57655/57655fig2.jpg"> 图 2: 使用 viSNE 地图比较环境生物膜。(A)从太多单元格 (30万) 获得的 viSNE 映射有一个大的簇 (顶部), 太少 (1万) 单元格根本不形成簇 (底部), 而最佳细胞数 (ca 15万) 形成清晰可见的可分离簇 (中间)。属于六个不同示例的 viSNE 映射的(B)子集在 viSNE 映射的不同部分中具有不同的密度。<a href="/files/ftp_upload/57655/57655fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57655/57655fig3.jpg"> 图 3: 生物膜中粒子亚群的聚类。(A) viSNE 图的不同部分由具有不同光学和荧光特性的粒子填充, 这些微粒给出了粒子的生物/非物质起源的线索, 以及生物粒子 (细胞) 所属的分类群 (波长从上到下: 525 毫微米, 695 毫微米, 660 毫微米)。对于每个波长, 颜色刻度从最高测量的荧光 (红色) 到最低测量 (蓝色) 正常化。(B)将参考数据库 (红色点) 投影到 viSNE 地图上可以帮助解释地图的哪些部分是由哪些分类和/或非生物微粒填充的: 衰变细胞 (顶端)、硅藻 (中间)、蓝藻 (底部)。(C)基于 (A) 和 (B) 中的信息以及 viSNE 映射的可视可分离簇, 可以将 viSNE 映射的不同部分分配给不同的簇 (SP1-11), 并据此对簇进行分类。<a href="/files/ftp_upload/57655/57655fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57655/57655fig4.jpg"> 图 4: 对生物膜样品的统计分析。(A)量化是通过计算属于不同生物复制的每个亚群粒子的数量来执行的。A–F 代表六个不同的地点沿当地小河, 生物膜样品被采取。(B)方差分析可用于评估样本之间的统计差异, 并进行适当的多重比较校正 (p < 0.05, 对 Tukey HSD 所有站点 vs 站点 A)。<a href="/files/ftp_upload/57655/57655fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57655/57655fig5.jpg"> 图 5: 用 viSNE 跟踪 microplastic 粒子。(A) Microplastic 粒子, (B)淡水生物膜, (C)淡水生物膜与 Microplastic 粒子混合, (D)荧光粒子, 由 FC 695 nm 测量。色标从最高的被测量的荧光 (红色) 到最低的被测量的荧光 (蓝色) 在695毫微米被规范化。microplastic 颗粒与生物膜颗粒的比值约为1:1。<a href="/files/ftp_upload/57655/57655fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Environment

8.9K 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

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

科研

环境科学

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

表征和被动采样的应用在农药水监测

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Alternative Method of Removing Otoliths from Sturgeon

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a transverse vertical incision on the top of the skull with a hand or electrical saw may damage the otolith if not performed correctly. Sturgeons (Acipenseridae) are one family in particular that have a very large and thick skull. A new laboratory method entering the brain cavity from the ventral side of the fish to expose the otoliths was easier than other otolith extraction methods found in the literature. Methods reviewed in the literature are designed for the field and are more efficient at processing large quantities of fish quickly. However, this new technique was designed to be more suited for a laboratory setting when time is not pressing and successful extraction from each specimen is critical. The success of finding and removing otoliths using this technique is very high and does not compromise the structure in any manner. This alternative technique is applicable to other similar fish species for extracting the otoliths.

The goal of the protocol is to show an effective method to extract the otoliths from sturgeon carcasses.

从鲟鱼去除耳石替代方法

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

对水生生命值评价悬浮泥沙影响的流通系统曝光

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Improving Infrared Spectroscopy Characterization of Soil Organic Matter with Spectral Subtractions

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic bonds that constitute the organic component of soils. However, the relatively low organic matter content of soils (commonly &#60; 5% by mass) and absorbance overlap of mineral and organic functional groups in the mid-infrared (MIR) region (4,000-400 cm-1) engenders substantial interference by dominant mineral absorbances, challenging or even preventing interpretation of spectra for SOM characterization. Spectral subtractions, a post-hoc mathematical treatment of spectra, can reduce mineral interference and enhance resolution of spectral regions corresponding to organic functional groups by mathematically removing mineral absorbances. This requires a mineral-enriched reference spectrum, which can be empirically obtained for a given soil sample by removing SOM. The mineral-enriched reference spectrum is subtracted from the original (untreated) spectrum of the soil sample to produce a spectrum representing SOM absorbances. Common SOM removal methods include high-temperature combustion (&#39;ashing&#39;) and chemical oxidation. Selection of the SOM removal method carries two considerations: (1) the amount of SOM removed, and (2) absorbance artifacts in the mineral reference spectrum and thus the resulting subtraction spectrum. These potential issues can, and should, be identified and quantified in order to avoid fallacious or biased interpretations of spectra for organic functional group composition of SOM. Following SOM removal, the resulting mineral-enriched sample is used to collect a mineral reference spectrum. Several strategies exist to perform subtractions depending on the experimental goals and sample characteristics, most notably the determination of the subtraction factor. The resulting subtraction spectrum requires careful interpretation based on the aforementioned methodology. For many soil and other environmental samples containing substantial mineral components, subtractions have strong potential to improve FTIR spectroscopic characterization of organic matter composition.

SOM underlies many soil functions and processes, but its characterization by FTIR spectroscopy is often challenged by mineral interferences. The described method can increase the utility of SOM analysis by FTIR spectroscopy by subtracting mineral interferences in soil spectra using empirically obtained mineral reference spectra.

利用光谱减法改进土壤有机质的红外光谱表征

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only recently have microplastics in freshwater systems, such as rivers and lakes, been quantified. Microplastic sampling at the surface usually consists of deploying drift nets behind either a stationary or moving boat, which limits the sampling to environments with low levels of suspended sediments and floating or submerged debris. Previous studies that employed drift nets to collect microplastic debris typically used nets with &#8805;300 &#181;m mesh size, allowing plastic debris (particles and fibers) below this size to pass through the net and elude quantification. The protocol detailed here enables: 1) sample collection in environments with high suspended loads and floating or submerged debris and 2) the capture and quantification of microplastic particles and fibers &lt;300 &#181;m. Water samples were collected using a peristaltic pump in low-density polyethylene (PE) containers to be stored before filtering and analysis in the lab. Filtration was done with a custom-made microplastic filtration device containing detachable union joints that housed nylon mesh sieves and mixed cellulose ester membrane filters. Mesh sieves and membrane filters were examined with a stereomicroscope to quantify and separate microplastic particulates and fibers. These materials were then examined using a micro-attenuated total reflectance Fourier transform infrared spectrometer (micro ATR-FTIR) to determine microplastic polymer type. Recovery was measured by spiking samples using blue PE particulates and green nylon fibers; percent recovery was determined to be 100% for particulates and 92% for fibers. This protocol will guide similar studies on microplastics in high velocity rivers with high concentrations of sediment. With simple modifications to the peristaltic pump and filtration device, users can collect and analyze various sample volumes and particulate sizes.

Most microplastic research to date has occurred in marine systems where suspended solid levels are relatively low. Focus is now shifting to freshwater systems, which may feature high sediment loads and floating debris. This protocol addresses collecting and analyzing microplastic samples from aquatic environments that contain high suspended solid loads.

高悬浮泥沙荷载和大型漂浮碎屑水生环境中 Microplastics 的取样、分类和表征

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only ...

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 ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

将流体设备与显微镜和流式细胞学相结合，研究跨空间尺度的多孔介质中的微生物传输

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...