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。

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			<td height="21" style="height:21px;width:413px;">Name of Material/ Equipment</td>
			<td style="width:328px;">Company</td>
			<td style="width:331px;">Catalog Number</td>
			<td style="width:163px;">Comments/Description</td>
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			<td height="20" style="height:20px;">10 cu. ft. Diurnal Incubator (6012-1)</td>
			<td>Caron Corporate</td>
			<td>112310-6012-1-11</td>
			<td><a href="http://www.caronproducts.com">www.caronproducts.com</a></td>
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			<td>Thermo Fisher Scientific</td>
			<td>N156340</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
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			<td height="20" style="height:20px;">Multiwell TC Plates &#8211; 48 Well</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>353078</td>
			<td><a href="http://www.bdbiosciences.com/">www.bdbiosciences.com</a></td>
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			<td height="20" style="height:20px;">P1000 Gilson The Pipetting Standard&#8212;Gilson&#39;s Pipetman</td>
			<td>Mandel Scientific Company Inc.</td>
			<td>GF-F123602</td>
			<td><a href="http://www.mandel.ca">www.mandel.ca</a></td>
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			<td height="20" style="height:20px;">P10mL Gilson The Pipetting Standard&#8212;Gilson&#39;s Pipetman</td>
			<td>Mandel Scientific Company Inc.</td>
			<td>GF-F161201</td>
			<td><a href="http://www.mandel.ca">www.mandel.ca</a></td>
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			<td height="20" style="height:20px;">Wide Orifice Tips nonsterile [100&#8211;1250 &#181;L]</td>
			<td>VWR International</td>
			<td>89079-468</td>
			<td><a href="http://www.ca.vwr.com">www.ca.vwr.com</a></td>
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			<td height="20" style="height:20px;">Ultrafine Tips nonsterile [100&#8211;1250 &#181;L]</td>
			<td>VWR International</td>
			<td>89079-470</td>
			<td><a href="http://www.ca.vwr.com">www.ca.vwr.com</a></td>
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			<td>Thermo Fisher Scientific</td>
			<td>9402151</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
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			<td height="20" style="height:20px;">WATER-Pulse Amplitude Modulation (Water-ED)</td>
			<td>Heinz Walz GmbH, Effeltrich, Germany</td>
			<td>EDEE0232</td>
			<td><a href="http://www.walz.com">www.walz.com</a></td>
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			<td height="20" style="height:20px;">15 mm diameter quartz glass cuvette (WATER-K)</td>
			<td>Caron Corporate</td>
			<td></td>
			<td><a href="http://www.caronproducts.com">www.caronproducts.com</a></td>
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			<td height="20" style="height:20px;">Sodium&#160;Chloride&#160;(Crystalline/Certified ACS), Fisher Chemical</td>
			<td>Thermo Fisher Scientific</td>
			<td>Thermo Fisher Scientific</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
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			<td height="20" style="height:20px;">BD Difco Marine Broth 2216</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>BD Biosciences Discovery Labware</td>
			<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
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			<td height="20" style="height:20px;">BD Bacto Agar</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>BD Biosciences Discovery Labware</td>
			<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
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			<td height="21" style="height:21px;">L1 Medium Kit, 50L</td>
			<td>NCMA [National Center for Marine Algae and Microbiota</td>
			<td>NCMA [National Center for Marine Algae and Microbiota</td>
			<td><a href="http://www.ncma.bigelow.org">www.ncma.bigelow.org</a></td>
		</tr>
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</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 ...

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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

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

优化负染:通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

科研

环境科学

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

小体积的方法从水中病毒浓度

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

从在土X射线计算机断层扫描数据量和表面分析:提取指标三维根系

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has been produced. Lithium chloride (LiCl) is the preferred agent used in livestock to induce CTA because it specifically stimulates the vomit center. In addition, LiCl is commercially available, and easy to prepare and administer using a drenching gun. Nevertheless, some factors have to be considered to obtain an effective long-lasting CTA, which allows small ruminants to graze during the cropping season. A key aspect is to use animals with no previous contact with the target plant (the plant chosen to be avoided; new feed). Due to their native neophobic feeding behavior, small ruminants can easily associate the negative feedback effects with the new feed, resulting in a strong and persistent CTA. The recommended doses are 200 and 225 mg LiCl/kg body weight (BW) for goats and sheep, respectively. To induce CTA, 100 g of the target plant should be individually offered for at least 30 min, and LiCl administered thereafter if the intake is greater than 10 g. Each time the animal eats the target plant without negative consequences, the CTA becomes weaker. Consequently, to minimize the risk of target plant consumption, it is essential to have sufficient palatable ground cover available. The presence of an alternative feed (of quality and quantity) prevents the accidental consumption of the target plant. A close monitoring of the flock is recommended to remove and re-dose any animal consuming more than 4 bites or 10 g of the target plant. At the beginning of each grazing season, check the CTA status of each animal before moving them to the crop.

Feeding behavior preferences in livestock can be modified to implement a grazing management plan in woody crops. Here, we present a protocol to show a lithium chloride dose after eating a new plant that induces conditioned taste aversion.

如何创建条件性味觉厌恶放牧在地面木本作物与小反刍动物封面

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

Ø的底栖交易所<sub&gt; 2</sub&gt;，N<sub&gt; 2</sub&gt;和营养盐使用小的核心孵化

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

利用低成本、小型、易于建造的Quercivorus测量豚草甲虫、鸭嘴兽的飞行能力 (村山富市)

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.