Name: a small volume bioassay to assess bacterial/phytoplankton co-culture using water-pulse-amplitude-modulated (water-pam) fluorometry
Uploaded: 2015-03-11T13:00:00.000+00:00
Duration: 9 min 54 s
Description: Watch this Scientific Journal Video about A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry at JoVE.com

This work was supported by Natural Sciences and Engineering Research Council of Canada (grant 402105), Canadian Foundation for Innovation (grant 129087) and Alberta Education and Training (grant AAETRCP-12-026-SEG) to RJC.

1.计算实验装置<ol><li>计算藻类和/或所需要的，这将需要为整个实验通过使用等式1控制细菌培养物的体积: <img alt="式（1）" src="/files/ftp_upload/52455/52455equation1.1.jpg" /> 其中，y等于每天和z所需的控制的数目等于的天数。 </li><li>计算是通过使用等式2所需要的共培养物的实验是藻类和/或细菌培养物的体积: <img alt="公式2" src="/files/ftp_upload/52455/52455equation1.2.jpg" /> 注:这是可能取代"联合培养"实验与任何复合屏幕;只是调整的最终化合物，溶剂和藻类的浓度，以适应实验设计。 </li><li>使用方程1和2来计算早期指数藻培养物的最终体积（通常5天〜10 4个细胞/ ml，但本应由performi确定纳克藻类生长曲线）所需的试验（该体积是需要使用公式3步骤2.3）: <img alt="公式3" src="/files/ftp_upload/52455/52455equation1.3.jpg" /></li><li>使用方程1和2计算为10 4 CFU / ml的细菌培养物（或期望的接种浓度）所需的试验（该体积所需的步骤4.2），使用等式4的最终体积: <img alt="公式4" src="/files/ftp_upload/52455/52455equation1.4.jpg" /> 注意:使用在步骤1.3和1.4考虑到移液误差的另外10 ml和所执行的任何测试（ 例如，水-PAM，流式细胞术，显微术等 ）对0 D。如果需要，以适应实验设计增加这个数量。 注:举一个例子计算一个8天的实验中看到第10章见补充表媒体食谱。 </li></ol> 2.成长的藻类细胞的实验装置<ol><li>隔离或获得积极增长的无菌藻类培养。 </li><li>无菌转移藻类培养物的最终体积至无菌藻类介质（ 例如，L1或类似的海洋藻类介质，参见材料和设备表）的10％，从而确保1:9稀释藻培养物的新鲜培养基中。使用预先确定的生长条件，菌株生长在一个昼夜培养箱稀释藻（ 例如，18℃，16:8小时亮:暗循环常用）。 </li><li>当培养物达到早期指数阶段，重新培养在相同的无菌藻类培养基的藻类（ 例如，L1或类似的海洋藻类介质中），确保了最终浓度为1:9稀释和藻类的最终体积是等于计算的V A（步骤1.3）。 注:为确保这些文化都是无菌是很重要的。由镀有20微升等分到一般的海洋细菌培养基（ 如海洋测试所有藻类媒体和藻类股票瓶污染肉汤2216补充有1.5％琼脂或类似的）。 </li><li>生长藻类培养至早期指数阶段（〜10 4个细胞/ ml）。 注:每个藻菌株具有取决于培养条件独特的生长曲线。早期指数期（〜10 4个细胞/ ml），可以通过基于细胞密度进行藻类生长曲线（即用流式细胞仪或显微镜）测定的。 </li></ol> 3.准备细菌细胞接种<ol><li>通过执行一个生长曲线中所选择的细菌培养基和生长条件（预先决定的固定相的细菌的细菌浓度（CFU / ml）和光密度（OD）， 例如，海水肉汤2216在25℃和160转，或类似）。使用这些信息来增加细胞（步骤3.3），后来正确稀释的细胞在步骤3.7。 </li><li>无菌从1.5％琼脂平板上（ 如海水肉汤2216柔顺传输一个孤立的和新鲜的成长菌落mented用1.5％的琼脂或类似的海洋细菌固体培养基）到5ml细菌液体介质（ 如的，海水肉汤2216或类似的海洋细菌培养基）。 </li><li>生长在滚动筒或振动筛的细菌，以固定相（〜12 - 36小时，这取决于细菌）。计划的实验，使得细菌到达固定相（〜8月 10日至9月 10日CFU / ml）的同时，该藻细胞已达到早期指数期（〜10 4个细胞/ ml）。 </li><li>使用移液管，冲洗附着于试管到培养基中的任何生物膜。吸管1毫升充分混合的细菌培养物进入无菌1.5毫升微型管。离心机在14000×g下1分钟。 </li><li>取下并丢弃上清液（细菌性媒体）而不破坏颗粒（细菌细胞）。加入1毫升无菌藻类介质（ 例如，L1或类似）与微管 ​​（带有粒料）。涡管以悬浮颗粒的藻类媒体。 </li><li>二洗:重复步骤3.5。 注意:因为这可能会改变的营养成分，是洗的细菌细胞与藻类媒体以彻底所有的细菌培养基，细胞碎屑，排出体外的蛋白质和小分子从细胞的接种藻类与他们之前删除关键藻类介质或引入生物活性分子到屏幕上。 </li><li>串联稀释洗涤细菌细胞中藻类介质，至最终浓度是100倍，比所期望的最终菌浓度（CFU / ml）的更浓缩。保存包含已清洗和稀释的藻类媒体步4.2细胞微管。 注:计划的基础上具有1的实验:0 d 1的比藻类到细菌。要做到这一点所需的初始细菌浓度为实验为10 4 CFU / ml的，所以在步骤3.7的初始细胞应连续稀释至10 6 CFU /毫升。 </li></ol>用于实验4.准备细菌人安装<ol><li>制备4无菌蒸压玻璃锥形瓶中，并标记它们:1）"藻类控制烧瓶&#39;，B）&#39;稀释细菌库存烧瓶&#39;，C）&#39;细菌对照烧瓶&#39;，以及d）&#39;的共培养烧瓶&#39;。 </li><li>稀释菌悬液从 步骤3.7 1:99用无菌藻类介质至最终体积= V B（步骤1.4）。使稀释烧瓶标记稀释细菌的股票（步骤4.1）。 注意:在前面的例子中（步骤4.2），这1:99稀释给出10 4 CFU / ml的终浓度。旋流烧瓶混合细胞。 </li><li>吸管V 控制从稀释细菌股票（步骤1.1），并把它放在细菌控制烧瓶中。 </li><li>吸管V 控制无菌藻类介质进入细菌对照烧瓶（这是一个1:1稀释）。旋流烧瓶混合细胞和预留步7.3。 </li><li>吸管V 共培养从稀释细菌股票烧瓶于共培养烧瓶中，设置烧瓶静置步骤6.1。 注意:当在相同的藻类进行多次共培养物一次（ 即，两个不同的细菌的共培养与一种对照菌株），有必要重新计算在V 共培养各菌株分别然后重复步骤4.5对于每个共培养分别。它也有必要提高在步骤1.3中计算出的V A为包括在公式5中所示两个共培养: <img alt="公式5" src="/files/ftp_upload/52455/52455equation4.5.jpg" /></li></ol> 5.准备藻类的实验装置<ol><li>轻轻混合初指数藻类培养（步骤2.4）与广口的枪头，直到细胞出现充分混合。 </li><li>吸管V 控制从藻类股票瓶藻类控制烧瓶中。轻轻地用移液10毫升吸管。然后返回中日孵化藻类股票瓶。 </li><li>吸管V 续无菌藻类介质向藻类控制烧瓶ROL（这是一个1:1稀释）。摇匀瓶中，发生在白天孵化器，直到需要的步7.4。 </li></ol> 6.准备实验共培养<ol><li>轻轻吸取V 共培养从藻库存烧瓶到来自步骤4.5的细菌共培养瓶中。返回共培养烧瓶中，昼夜培养箱直到需要用于步骤7.5。 注:细菌在细菌对照的浓度应等于细菌浓度在实验共培养。同样地，在藻类的藻类控制的浓度应等于藻类的浓度在实验共培养。由具有相同的初始细菌和藻类的浓度，人口密度可以在整个实验进行比较。 </li></ol> 7.建立微量滴定盘<ol><li>将一个无菌的48孔微量滴定板按照图1。标号上述含任一无菌稀释剂或非光合样品外井。 注:所述板孔的随机化可以为在同一天拍摄的样品进行。例如，象限1将在1天的实验中进行采样;应随机的井B2 - 4和C 2 - 4.保持板充满无菌溶液的周边， 如图1。 </li></ol><img alt="图1" src="/files/ftp_upload/52455/52455fig1highres.jpg" /> 。样品放置在48孔微量滴定板的孔图1的示意图是要填充如下:列1和6中，孔A到F（ <img alt="1圈" src="/files/ftp_upload/52455/circle1.jpg" /> ）填充用1ml的1×PBS（或其它无菌溶液/介质）。行A和F，水井2 - 8（ <img alt="圈2" src="/files/ftp_upload/52455/circle2.jpg" /> ）填充用1ml细菌控制; ROWS B和E井2 - 8（ <img alt="3圈" src="/files/ftp_upload/52455/circle3.jpg" /> ）填充用1ml藻类控制;行C和D，水井2 - 8（ <img alt="4圈" src="/files/ftp_upload/52455/circle4.jpg" /> ）填充用1ml共培养。该板被分成4个象限（A2，A5，D2和D5），这些象限各自特定的抽样天1 - 4，这应该被随机化在整个板和相应标记。在每一天，我们劝随机藻类控制（ <img alt="5圈" src="/files/ftp_upload/52455/circle5.jpg" /> ）和共培养（ <img alt="6圈" src="/files/ftp_upload/52455/circle6.jpg" />使用随机数生成器）的孔中。标签盖在1×PBS中和/或细菌的对照孔，以防止藻类培养物的着色。 <ol start="2"><li>吸管1毫升1X磷酸盐缓冲液（PBS，pH 7.4）中或在如图1所示的适当的孔中其它无菌溶液。执行此缓慢和小心。 PBS陈会GE的藻类和/或共同培养液的离子强度，如果飞溅在其他井小心处理。 </li><li>吸取1毫升细菌控制文化融入井标记细菌控制（ 图1）。吹打每块板，以避免细菌沉淀前晃动细菌瓶。 </li><li>吸管1毫升藻类控制培养的成孔标记用宽口移液管尖（ 图1）控制藻类。作为定期漩涡藻瓶的细菌瓶。如果藻类趋于下沉或浮动，如需要保持一个统一的视觉文化漩涡的定期。 </li><li>使用一个宽口移液管尖，吸管1毫升共培养中的适当的孔（ 图1）。作为定期漩共培养烧瓶中的藻类烧瓶中。 </li><li>密封各板用封口膜和放置在一个昼夜培养箱中在期望的温度和日光照循环（18℃，16:8小时亮:暗循环常用）。离开在昼夜孵化器，直到准备板采取PAM读数（第8）。确保所有板都在相同的方向取向，以允许持续的光曝光。 </li><li>就拿从PBS，藻媒体，藻股票和藻类控制20微升等分拖放到板的适当的非选择性培养基（ 如海水肉汤2216辅以1.5％琼脂）和孵化板在18 - 25℃，72小时测试的污染。如果增长出现在任何不应该含有细菌的溶液，然后将这些数据不应该被使用。 </li><li>使用从藻类控制和共培养瓶中剩余的样品用于实验0 D的PAM荧光读数（见步骤8.1）采取的PAM荧光读数。任何其他0 D测量也应与剩余的藻类控制，细菌对照和共培养的样品进行的。 </li></ol> 8.以PAM荧光的股票样本读数<ol><li>零WATER-PAM与服用读数8日前无菌藻媒体在干净的试管。 </li><li>吸移管将300μl来自藻类控制或共培养烧瓶到含有2.7毫升在实验中使用的相同的藻类介质的干净试管中。具有宽口移液管尖混合的样品和稀释液轻轻。 </li><li>擦去从比色杯的用组织以外的所有指纹放置反应杯在水的PAM之前。 </li><li>将试管成水-PAM。覆盖与帽的样品，并允许其暗适应3分钟。暗适应时间取决于藻类的物种，并且必须在实验中使用的特定藻来确定。由藻类昼夜孵化器16,17的黑暗周期的中间过程中采取WATER-PAM读数避免冗长暗适应时间（&gt; 20分钟）。 </li><li>暗适应后，打f 0的按钮。如果荧光读数大于3900，稀释样品1:1在藻类介质。暗适应额外3分钟，德EA新的读数。如果F 0或F 米读数仍高于3900，继续稀释样品1:1的在藻类介质直到在F 0和F 米读数都低于3900。 注意:请确认记录最终荧光时考虑到这些稀释液:例如，如果藻类样品稀释1:9从井的初始传送到稀释管中，然后在500藻类荧光读数应当由逆乘以稀释因子​​（在这种情况下，10）和该管的实际荧光然后5000。 </li><li>设置F 0后，取饱和脉冲（SAT-PULSE）通过点击按钮SAT拿F M读读书，每90秒。读数之间的时间间隔可以根据藻菌株进行调整。丢弃样本。 </li><li>重复步骤8.1 - 8.6对剩余的样品。 </li></ol> 9.以PAM荧光从微量滴定盘读数<ol><li>标签4藻类控制和井C2 - - 4细菌共培养（ 见图1板布局）&gt; 3 ml，此井B2 6无菌样品管。 </li><li>等分2.7毫升无菌藻类培养基到每个管中。 </li><li>将管在白天孵化器，让他们适应新环境，以藻类生长，在30分钟时的温度。 </li><li>之前从培养箱取出微量滴定板确保光穿透到暗适应井通过用铝箔（或类似）覆盖所述板，只取出金属箔，同时积极地从孔中转移培养到稀释管（步骤9.5限于 - 9.7）。 </li><li>无菌通过缓慢上下抽吸混合第一微量滴定板孔（井B2）具有宽口移液管尖端。 </li><li>获得从培养箱并无菌转移从井B2（ 图1）将300μl稀释管到其相应的样品管中具有宽口移液管尖（这是1:9稀释于培养基中的藻类的样品）。 </li><li>重复步骤9.5 - 9.6，其余井（B3,4和C2 - 4）。 </li><li>盖样品管用铝箔，并将其退回到周日孵化器，直到准备WATER-PAM。 </li><li> 8.7 - 按照步骤8.1中所述进行WATER-PAM读数。它返回到培养箱之前密封所述微量滴定板用封口膜。 </li><li>在计划的时间间隔为实验的持续时间重复读数。的频率和取样的长度应计划在实验的开始。 </li></ol> 10.实验样品样品的实验是细菌（Phaeobacter gallaeciensis BS107）和微藻（ -贺株（CCMP3266））的为期10天的联合培养。它包括一个控制藻类，细菌控制，以及细菌，藻类试验共培养。 <ol><li>计算所需的细菌和藻类的股票数量（步骤1。1 - 1.4） <img alt="10式" src="/files/ftp_upload/52455/52455equation10.1.jpg" /></li><li>制备藻库存，以40ml为V A，由接种36毫升培养基4毫升藻并孵育直至早期指数增长，实现具有〜10 4个细胞/ ml（步骤2.1 - 2.4）的细胞密度。 </li><li>通过稀释400μl的第3至40毫升藻媒体为V B所得的10 6 CFU / ml的细菌（细菌的最终浓度将是10 4 CFU / ml）的制备细菌库存烧瓶中。 </li><li>细菌库存转移一半（20毫升）中的细菌对照烧瓶中并添加20毫升藻类介质，使细菌的控制。转移一半（20毫升）的藻类股票藻类控制烧瓶中，加入20藻类媒体毫升，以弥补藻类控制（第5）。传输剩余20毫升细菌库存到共培养烧瓶中，并与剩余的20毫升藻股票的混合来建立共培养（第6）。 </li><li>吸管的1×PBS（pH7.4）中，对照和共培养在两个预标记的微量滴定板（ 图1）中，每孔1ml。用3毫升剩余的控制和共培养，使0 D的测量（CFU计数，水-PAM（第8节），显微镜观察的藻细胞形态，和藻类细胞固定于流式细胞仪）。 </li><li>取水-PAM读数藻类控制和共培养，每天一次，10天，并始终在同一时间作为0 D测量是（第8部分）。 </li></ol>利益11.其它参数<ol><li>确定细菌菌落浓度:执行所述细菌对照和共培养在无菌的1×PBS（或类似）的系列稀释，再滴板到海水肉汤2216补充有1.5％琼脂（或类似的），并观察菌落的生长的数量以确定细菌CFU / ml的每个孔18。 </li><li>评估藻细胞浓度:修复藻类控制与合作培养与戊二醛的0.15％的最终浓度。孵育在液氮中黑暗中，然后快速冷冻10分钟，并在-80℃存储。过程上的流式细胞仪（FACS刀魂或类似）的所有样本来计算的藻细胞。 </li><li>观察藻细胞形态:藻类培养，用显微镜（ 即光镜，萤光，或类似）的细菌，藻类共培养。 </li></ol>

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The authors have nothing to disclose.

藻类生长在一个小型化的格式。 藻培养物在微量滴定板1毫升培养物体积的小型化允许一个实验中的复制增加。以确保藻是在整个实验的健康是非常重要的;执行一个生长曲线（ 图2），使用微量滴定板形式，以评估各种藻类介质，以确保满足所述藻类的营养需求。此外，为了优化昼夜周期（亮和暗期）和温度可能是重要的。适当的优化对于给定的藻类可以允许用于?...

浮游植物生理学历来研究中尺度实验范围从20毫升锥形瓶中，以5升大玻璃瓶中1-7。这个试验规模需要二次抽样实验监测，作为牺牲重复样品对每个时间点创建一个无法控制的实验装置。 的能力，同时使用相同的昼夜培养箱空间通过小型化的实验量为藻类生理学实验将减少或消除二次抽样和伪复制的限制从大量增加的独立实验的数目。微孔板格式已经开发出用1毫升培养体积的实验操作藻可变的条件藻类生物测定。这种小规模的试验体积允许要增加重复的次数，增加实验重复性由于重复样品之间降低的变异性实验中，并且允许真复制，同时保持试验对照（ 即无菌藻类培养物）140天（ 图2）12。 这种微量滴定板形式很容易适应于多种实验问题，如:没有细菌具有与其藻宿主共生，中性或致病相互作用？是另外一种化合物刺激或有毒的藻类？这些和其它问题，可在使用这种新格式9-11快速高通量的方式加以解决。 48孔微量滴定培养皿允许每个1毫升井是一个独立的实验装置所采样的单个时间点。各种参数可以从这个1毫升体积包括被采样，但不限于:使用水-脉冲幅度调制（WATER-PAM）荧光（参见材料和设备表 ）1叶绿素荧光和光化学参数3。WATER-PAM荧光是一种快速，非侵入性的技术，可用于监测与藻类13进行的实验。它允许光合效率和PSII健康从小培养体积测量（150 - 300μl的文化在培养基中稀释到2 - 4毫升体积WATER-PAM）14,15。除了水-PAM荧光，这种设置可以用来测量各种其它参数，包括但不限于:显微镜可视化附着到的藻细胞和变化，藻细胞形态的细菌;细菌菌落形成单位（CFU）计数;和流式细胞仪对藻类细胞计数和识别的亚群。

<table><tbody><tr><td>10 cu. ft. Diurnal incubator (6012-1)</td><td>Caron Corporate</td><td>112310-6012-1-11</td><td>&lt;a href=&quot;http://www.caronproducts.com&quot;&gt;www.caronproducts.com&lt;/a&gt;</td></tr><tr><td>Nunc EasYFlask 25 cm&lt;sup&gt;2&lt;/sup&gt;, Vent/Close Cap, 7 ml working volume, 200/cs</td><td>Thermo Fisher Scientific</td><td>N156340</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>Multiwell TC Plates &amp;ndash; 48-well</td><td>BD Biosciences Discovery Labware</td><td>353078</td><td>&lt;a href=&quot;http://www.bdbiosciences.com/&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>P1000 Gilson The Pipetting Standard&amp;mdash;Gilson&#039;s Pipetman</td><td>Mandel Scientific Company Inc.</td><td>GF-F123602</td><td>&lt;a href=&quot;http://www.mandel.ca&quot;&gt;www.mandel.ca&lt;/a&gt;</td></tr><tr><td>P10mL Gilson The Pipetting Standard&amp;mdash;Gilson&#039;s Pipetman</td><td>Mandel Scientific Company Inc.</td><td>GF-F161201</td><td>&lt;a href=&quot;http://www.mandel.ca&quot;&gt;www.mandel.ca&lt;/a&gt;</td></tr><tr><td>Wide Orifice Tips nonsterile [100&amp;ndash;1,250 &amp;micro;l]</td><td>VWR International</td><td>89079-468</td><td>&lt;a href=&quot;http://www.ca.vwr.com&quot;&gt;www.ca.vwr.com&lt;/a&gt;</td></tr><tr><td>Ultrafine Tips nonsterile [100&amp;ndash;1,250 &amp;micro;l]</td><td>VWR International</td><td>89079-470</td><td>&lt;a href=&quot;http://www.ca.vwr.com&quot;&gt;www.ca.vwr.com&lt;/a&gt;</td></tr><tr><td>Finntip 10 ml [Vol: 1 - 10 ml]</td><td>Thermo Fisher Scientific</td><td>9402151</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>WATER-Pulse Amplitude Modulation (Water-ED)</td><td>Heinz Walz GmbH, Effeltrich, Germany</td><td>EDEE0232</td><td>&lt;a href=&quot;http://www.walz.com&quot;&gt;www.walz.com&lt;/a&gt;</td></tr><tr><td>15 mm diameter quartz glass cuvette (WATER-K)</td><td>Caron Corporate</td><td></td><td>&lt;a href=&quot;http://www.caronproducts.com&quot;&gt;www.caronproducts.com&lt;/a&gt;</td></tr><tr><td>Sodium chloride (crystalline/certified ACS), Fisher Chemical</td><td>Thermo Fisher Scientific</td><td>Thermo Fisher Scientific</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>BD Difco Marine Broth 2216</td><td>BD Biosciences Discovery Labware</td><td>BD Biosciences Discovery Labware</td><td>&lt;a href=&quot;http://www.bdbiosciences.com&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>BD Bacto Agar</td><td>BD Biosciences Discovery Labware</td><td>BD Biosciences Discovery Labware</td><td>&lt;a href=&quot;http://www.bdbiosciences.com&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>L1 Medium Kit, 50 L</td><td>NCMA [National Center for Marine Algae and Microbiota</td><td>NCMA [National Center for Marine Algae and Microbiota</td><td>&lt;a href=&quot;http://www.ncma.bigelow.org&quot;&gt;www.ncma.bigelow.org&lt;/a&gt;</td></tr></tbody></table>

a small volume bioassay to assess bacterial/phytoplankton co-culture using water-pulse-amplitude-modulated (water-pam) fluorometry

常规方法实验操作微藻雇用大量培养物（20毫升至5升），以使培养能在整个实验1-7进行二次取样。大容量二次抽样可能会产生问题，原因如下:1）它引起的变化的总体积与表面积:在实验过程中培养的体积比; 2）伪复制（ 即复制从相同的待遇烧瓶8个样本）经常采用而不是真正的复制（ 即从重复治疗抽样）; 3）在实验的持续时间是有限的总体积;和4）纯性培养物或通常的细菌微生物是困难的，因为污染子采样期间通常发生在长期实验来维持。 使用微量滴定板的使1毫升培养体积要用于每个复制，与内多达48个独立的处理一12.65 X 8.5×2.2厘米板，从而降低了实验体积并允许广泛的复制子采样未经任何处理。此外，这种技术可以被修改以适应多种实验格式，包括:细菌藻共培养，藻类的生理测试，以及毒素筛选9-11。各孔用藻类，细菌和/或共培养物可以被采样为众多的实验室程序，包括但不限于:水 - 脉冲幅度调制（WATER-PAM）荧光，显微镜检查，细菌菌落形成单位（cfu）的计数和流式细胞术。微量滴定板格式和水的PAM荧光的组合允许光化学产量与样品中，高再现性之间的低变异性等光化学参数的多个快速测量，并避免了子采样一个大玻璃瓶或锥形瓶中在实验的过程中的许多陷阱。

WATER-PAM荧光读数。 WATER-脉冲幅度调制（PAM）荧光是一种快速而有效的方法来确定荧光（叶绿素含量代理）和藻类培养光合产量（PSII健康）。在PAM WINCONTROL软件生成原始数据值的表格（下面是对暗适于藻类样品的基本参数）: f 0的=荧光暗适应的细胞的 F M =最大荧光饱和发光二极管后（LED）的脉冲暗?...

Watch this Scientific Journal Video about A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry at JoVE.com

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

小体积的生物测定评估细菌/浮游植物共培养使用水脉冲幅度调制（WATER-PAM）荧光

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

a-small-volume-bioassay-to-assess-bacterialphytoplankton-co-culture

Research

JoVE Journal

Environment

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

10.5K 次观看.  University of Alberta. The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

视频: A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

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 ...

环境科学

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

生物工程

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

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

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 ...

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 ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, phototrophs also require illumination. Routinely, researchers seek to provide custom light supplies, i.e., vary the light intensity and time over which it is delivered. Such flexibility is difficult with standard benchtop lights. Usually, cultivation studies also require growth comparisons between experimental treatments. Frequently, growth is assessed over an extended duration, e.g., multiple times a day over a week-long trial. Manual measurements can be time-consuming and lack data resolution. Therefore, photobioreactors (PBRs) with automatic growth monitoring and customizable light supply are useful for replicated experiments with multiple treatments. The current work presents the design, construction, and operation of laboratory PBRs. The materials are easily sourced and relatively inexpensive. The design could be duplicated with moderate skill. Each structure has a footprint of ~40 cm2 and hosts three 1 L glass bottles for triplicate replication. Bottles rest upon platforms containing magnetic stirrers and are arranged vertically within a 1 m high and 15 cm diameter polyvinyl chloride (PVC) pipe. The pipe interior is lined with light-emitting diodes (LEDs). These LEDs produce continuous light intensities from 0&#8211;2400 &#181;mol photons m-2 s-1 of photosynthetically active radiation (PAR). Users design a custom lighting program. The light intensity can be adjusted each second or held constant for longer durations. Oxygen produced from photosynthesis exits each bottle via a one-way volumetric gas sensor. Software is used to record gas sensor data. The amount of oxygen produced can be correlated to biomass growth. If biomass samples are required, a syringe can be used to extract culture. The method is suited for microalgae grown with bicarbonate as the carbon source. These PBRs are valuable to a laboratory that requires replicated experiments, light regime flexibility, and continuous high-resolution growth data.

This publication describes the design of laboratory photobioreactors (PBRs) with customizable light regimes. The growth of cyanobacteria or microalgae, using bicarbonate as their carbon source, is monitored continuously by measuring volumetric oxygen production. These PBRs facilitate rapid, replicated laboratory growth comparisons with little user intervention during experiments.

通过在线生长测量和可定制的光照模式操作实验室光生物反应器

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, ...

生物学

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.