笔者想感谢6月米德尔顿供给E.粪肠球菌细菌培养和指导。C.线虫提供了CGC，这是由美国国立卫生研究院办公室研究基础设施建设项目（P40 OD010440）。

1）制备线虫生长培养基（NGM） <ol><li>为了使50板，加1.5克氯化钠，8.5克超纯琼脂和1.25克蛋白胨1 Lflask的。 </li><li> DH 2 O添加487.5毫升的烧瓶。轻轻摇晃混合覆盖开幕烧瓶用一块铝箔。 </li><li>消毒的解决方案，通过在标准条件下高压灭菌（121磅，120℃，20分钟）。 </li><li>让溶液冷却至45℃的水浴中。 </li><li>要解决方案，添加下面的顺序:500微升胆固醇5毫克/毫升原液（溶解在乙醇中），500微升1 2 M的氯化钙，500微升1 M 硫酸镁和12.5毫升KPO 4缓冲液（54.15克KH 2 PO 4和17.8克K 2 HPO 4 500毫升蒸馏水2 O）中所描述的18。 </li><li>使用无菌移液器，加10毫升琼脂溶液各60毫米的聚苯乙烯培养皿。允许介质在室温下固化overn洞察力。 </li></ol> 2）制备的B肉汤E.大肠杆菌媒体<ol><li>五个100毫升文化，加2.5克酵母提取物，蛋白胨，5.0克氯化钠5.0克到烧杯中。 </li><li>添加无菌蒸馏水至终体积为500ml。在热板上加热溶液，边搅拌，直至溶解溶质。 </li><li>分为五个玻璃瓶（至少200毫升）倒入100毫升的B肉汤。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>允许乙肉汤冷却至室温后再使用。 </li></ol> 3）播种C.线虫维护板<ol><li> NGM板在室温下干燥至少一个星期后，一个100毫升乙肉汤培养物接种的大肠杆菌菌落有几大肠杆菌 （OP50）。 </li><li> E.成长菌在37℃过夜。 </li><li>吸取1滴（约10081，L）OP50文化到每个NGM板的中心。片通常叠盖侧。移液时（播种），开始在堆栈的底部吹打文化上板。您的工作方式，每个堆栈。尽量不要移动的板，因为它是重要的文化太靠近板的边缘后播种。 </li><li>允许种子板在室温下干燥，在使用前至少一个星期。 </li><li>种子板块在密封的塑料容器中，在室温下保存（倒）。 </li></ol> 4）维护C.线虫菌株<ol><li>为了维持精心喂养C.线虫股票，使用蠕虫挑L4S或妊娠成人移动到一个新的种子NGM板每3-4天。 </li><li>商店板（倒）里面最好的孵化器在15 - 22°C。所描述的测定中的培养物储存在20℃下下线虫的发展是温度相关的。由于maintenaNCE温度的升高，发育阶段之间的时间减少。 </li></ol> 5）胰酶大豆琼脂（TSA）E.粪文化传媒<ol><li> 50板，测量一瓶500毫升的无菌蒸馏水，把煮沸，一边搅拌，在电炉上。 </li><li>粉状TSA琼脂20克加入烧瓶，并允许溶液溶解。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>让溶液冷却至45℃的水浴中。 </li><li>使用无菌移液器，加10毫升琼脂溶液各60毫米的聚苯乙烯培养皿。允许介质在室温下固化过夜。 </li></ol> 6）胰酶大豆肉汤（TSB）E.粪文化传媒<ol><li>为了使250个培养管，测量到一个烧瓶和温暖的烤盘上，而500毫升的无菌蒸馏水搅拌。 </lI&gt; <li> TSB琼脂粉15克加入到烧瓶中，使粉末溶解。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>让溶液冷却至室温，然后移液管2毫升，放入灭菌的5毫升试管。 </li></ol> 7）脑心浸液（BHI）媒体，链霉素，E.粪文化<ol><li>测量无菌蒸馏水500毫升的烧瓶中，并把煮沸，一边搅拌，在电炉上。 </li><li> 26克BHI培养基加入到烧瓶中，使该溶液溶解。 </li><li>在标准条件下高压灭菌（121磅，120°C，20分钟）消毒解决方案。 </li><li>让溶液冷却至45℃的水浴中。 </li><li>使用无菌技术，加入0.5毫升10毫克/毫升链霉素的原液和涡流混合。 </li><li>加入10毫升的溶液，以每60毫米的聚苯乙烯培养皿中。 </li><l&gt;允许媒体在室温下固化过夜。商店板（倒）在4℃，直到几个小时后再使用。 </li></ol> 8）维护E.粪肠球菌 <ol><li>为了维持E。粪股市连胜单独的胰酶大豆琼脂（TSA）平板上的单个菌落，用无菌环。 </li><li>文化生长在37℃过夜。店板（倒）了几个星期在室温下在密封的袋子或盒子中。 </li></ol> 9）准备E.粪板EIW含量的<ol><li>接种2毫升TSB文化的殖民地E.粪肠球菌和孵育过夜，在37°C。 </li><li>移液管加入20μl 大肠杆菌粪到准备好的BHI链霉素板的中心文化，孵育过夜，在37°C。 </li></ol> 10）准备E.大肠杆菌板EIW含量（控制板） <ol><li>接种了100毫升的B-BRO次文化的殖民地E.大肠杆菌 OP50在37℃孵育过夜</li><li>吸取20μl的的OP50培养的到非种子选手NGM板的中心和允许种子板在室温下干燥过夜。 </li></ol> 11）蛋蠕虫含量<ol><li>挑选15 - 20上演L4 C.线虫 （ 图1）上制备的BHI-链球菌， 大肠杆菌到草坪的细菌的中心粪板。要小心不转让了很多OP50的BHI板的。接送L4S相同数量的到控制NGM-OP50板。 </li><li> 40小时，在20°C孵育板</li><li>准备20％的漂白粉溶液。的商业漂白剂（6.0％次氯酸钠）混合，用适当体积的蒸馏水。 </li><li>加入10微升滴漂白剂溶液，以10个不同的位置上的塑料盖（一个蠕虫病毒或细菌板）。 </li><li>使用蠕虫挑，一个蠕虫传输到每个漂白下降。轻轻旋转的尖端挑中的漂白剂液滴，，确保蠕虫洗掉挑年底（确认在解剖显微镜下观察液滴）。 </li><li>使角质层溶解约10分钟或直到蠕虫爆裂开来，驱逐鸡蛋（ 图2）。要小心，不要将鸡蛋从板块。 </li><li>使用解剖显微镜每一滴漂白剂，算上鸡蛋。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

成功地执行这个实验中最关键的步骤是:1）使用精心喂养C.股票线虫 ，2）单一类型的细菌培养测定板，3）准确吞吐量上演L4蠕虫暴露于E。粪 ，4）保持曝光时间E.粪一致的，所有试验和5），漂白时间不应超过10分钟，以防止鸡蛋解体。 这个实验，重要的是要挑选健康，良好的喂养蠕虫。产妇饥饿会影响后代的繁殖力和生长19,20，因此重要的是，蠕虫已经吃得饱饱的，在进行这个实验之前的几代人。很容易保持良好的美联储库存蠕虫通过简单的几个成虫转移到一个新的种子NGM板每2-3天。检测前两天，成虫应放置在新鲜草坪OP50，以便有足够的L4子代可用于检测。 &lt;P类=的"jove_content"&gt; EIW测定板同样重要的是促进只有一个单一类型的细菌的生长。E.粪肠球菌菌株生长上BHI板。链霉素板选择对大肠杆菌大肠杆菌 OP50 L4蠕虫，这是转移，因为他们转移到维护NGM板的检测板。 OP50还生长上BHI板。如果不反对，C.选择线虫两种细菌（ 大肠杆菌和粪肠球菌 ）将饲料而BHI板。 大肠杆菌菌株在该测定中所使用的粪肠球菌对链霉素耐药。如果不同类型的细菌被用于这个实验中，不同的抗生素，也许是不同的培养基，应使用的，用于选择给出的细菌和对大肠杆菌大肠杆菌 OP50。 准确地选择这个实验上演虫是非常重要的有以下几个原因。首先，重要的是要算鸡蛋保持在t<html>他的时间在自我施肥C.鸡蛋生产/铺设线虫 。禽蛋产量仅发生在第一五天C.线虫成年（峰值约40个小时后，L4），此后迅速下降4,21。成虫平均两到三个星期，所以很多成虫生活在至少一个星期，而不是​​产生受精卵。因此，重要的是确保EIW法不执行unstaged的成虫年龄不详。 上演的第二个原因，重要的是使用动物在EIW检测是这样的时间发展，在该蠕虫已暴露于细菌，毒素或药物被严密控制。 C.文化线虫通常是鸡蛋，L1或L4阶段同步。用于该测定的L4阶段的关注，因为较长的曝光为E。粪会导致杀伤力而后虫生成为妊娠成人。 帐篷"&gt;持续时间蠕虫留下的漂白剂溶液中也是重要的监测。的蠕虫通常应溶于下10分钟，虽然这个蠕虫蠕虫的时间也有所不同。受精卵最终解散，漂白剂，但过程是慢于角质层由于存在保护蛋壳，蛋壳将开始溶解的漂白剂，如果留在溶液中的时间超过10-15分钟。 蛋保留反映产蛋量和产蛋之间的平衡。 EIW单独检测，不区分，数量是否保留子宫的鸡蛋由于鸡蛋生产或产蛋改变。这尤其是一个问题，如果蠕虫的细菌菌株或毒素治疗保留较少的蛋比对照组的。尤其是在这种情况下，后续的育雏规模试验应该做的。育雏大小分析量化产卵的数量超过蠕虫的生殖寿命4 </sup&gt;。如果在实验组和对照组中的蠕虫卵的大小是相同的，然后在卵性差异可以归因于不同的产蛋行为。因为它是不可能的，轻度致病性大肠杆菌菌株，如粪将采取行动，增加产蛋量，它是合理的假设，增加蛋保留曝光后E.粪肠球菌 （ 如图3所示），减少产卵行为的结果。 EIW检测也没有确定的机制，可能会改变细菌蛋保留。这可能是C。线虫可保留鸡蛋，因为致病细菌殖民和阻塞张口或影响喂养，因为它是一个贫穷的食物来源。另外，也可以细菌定植和阻止外阴部的开口，或在产蛋系统，影响细胞的功能。需要进一步分析，以确定机制WHICH蛋保留被改变。 此EIW法可以很容易地修改，以确定影响的许多类型的化合物对蛋保留。另外，该测定是很简单的，它可以被纳入画面中，在主机（ 秀丽隐杆线虫 ），或病原体（ 粪肠球菌 ），所需的病原体的影响，以确定基因。 EIW测定，也可用于筛选调节产蛋本身所需的基因。C.线虫产卵的行为是由神经递质，如5 -羟色胺和乙酰胆碱的5-9调制，一些神经元和肌肉群12需要的协调的活动。 EIW检测一直继续被使用，以确定基因重要的神经递质的信号和/或肌肉活动。 EIW检测提供一个定量的，而不是定性分析中的一个重要C.线虫的行为。

一个微小的，自由生活的蛔虫， 线虫 ，是一种模式生物，传统上用来研究人类发育和细胞的信令流程，因为透明解剖特征的发展，全基因组测序，短代时，基因的同源性。最近，C.线虫已经成为一种模式生物在该领域的环境毒理学和先天免疫10，11。 这些自我施肥雌雄同体蠕虫从卵孵化后的两到三天内性成熟。在它的生命周期，C.线虫通过四个幼虫阶段（L1-L4），达到成年之前。可以产生一个孤立的雌雄同体，平均300个后代在三天之内的峰值繁殖力。在生殖成熟C.线虫雌雄同体，受精卵在子宫内保留几个小时前被解雇。该存储在子宫中的蛋在任何一个时间的正常数量的（在繁忙繁殖力）是在10和15之间12。鸡蛋在子宫产蛋率和产蛋率的函数。受精卵从子宫排出，外阴13日开幕的周围布置的16个外阴部肌肉的收缩。的雌雄同体特定运动神经元（HSN）和VC影响肌肉收缩阴部肌肉的运动神经元突触上，从而鸡蛋产蛋行为5,7,13,14。驱逐鸡蛋从子宫的发生是由于神经元和肌肉的协调活动。 C.实验室培养线虫通常提出非致病性大肠杆菌 OP50的饮食。在自然环境中，C。线虫接触到的各种食物来源，如致病性细菌，也可以有潜在危害。当暴露于有害物质的的环境中，C。线虫保留鸡蛋，直到环境变得更加有利。据推测，这鸡蛋保留是为了保护他们的后代。 在这个蛋虫（EIW）测定，C.线虫暴露于潜在的致病细菌， 粪肠球菌 ，这是存在于环境中。 大肠杆菌致病形式曝光粪可引起持续性肠道感染，甚至死亡，在C线虫 15。暴露于其他形式的致病性细菌已被证明影响蛋保留16,17，但是没有量 ​​化的效果。此外，轻度致病性菌株的大肠杆菌的效果肠球菌 ，菌株不会立即致命，产蛋行为还没有被研究。 EIW检测涉及计数鸡蛋的数量保留在子宫C.线虫 4。尽管C。线虫是透明的，积聚在子宫的卵可以是难以量化，在一个完整的动物。 EIW检测涉及漂白妊娠成人C.线虫暴露于细菌一段固定的时间。漂白粉溶液溶解身后留下的鸡蛋外被角质层。鸡蛋是折射的漂白剂，由于存在保护蛋壳的效果。漂白后，能够非常容易地发布从子宫的蠕虫后漂白的鸡蛋的数量来计算。 检测是一种简单，廉价和快速的方法来量化在子宫内一次鸡蛋的数量，从而量化的影响E.粪蛋保留。本试剂盒可用于其他类型的细菌，环境毒素或药物的效果进行量化的蛋性。本试剂盒也有可能被用作细菌致病性的屏幕。

<table><tbody><tr><td>Agar, ultrapure</td><td>Affymetrix</td><td>10906</td><td></td></tr><tr><td>Bacto Peptone</td><td>Becton Dickinson</td><td>211677</td><td></td></tr><tr><td>Bacto Tryptone</td><td>Becton Dickinson</td><td>211705</td><td></td></tr><tr><td>Brain Heart Infusion dehydrated medium</td><td>Carolina Biological Supply</td><td>781781</td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt;, N2 strain</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;C. elegans&lt;/em&gt;</td><td>Tritech Research Inc.</td><td>T3308</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;E. faecalis&lt;/em&gt;</td><td>Fisher Scientific-Fisherbrand</td><td>875713</td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;(OP50)</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>&lt;em&gt;E. faecalis&lt;/em&gt; strains</td><td></td><td></td><td>provided by J. Middleton. All isolates were confirmed as enterococci</td></tr><tr><td colspan="4">by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td></tr><tr><td colspan="4"> &lt;p&gt;all strains grew at 44.5 &amp;ordm;C and were catalase negative and hydrolyzed esculin. A simplified&amp;nbsp;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;dichotomous key based on pigmentation and fermentation reactions for six sugars&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;(arabinose, mannitol, methyl-&amp;alpha;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;presumptive identification of all &lt;em&gt;E&lt;/em&gt;. &lt;em&gt;faecalis&lt;/em&gt; strains (Efs lacks pigmentation and is arabinose, MGP&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;were confirmed using the API 20 STREP system (Biomerieux).&lt;/span&gt;&lt;/p&gt; </td></tr><tr><td>Microscope</td><td>Motic</td><td>SMZ 168B</td><td>any microscope with transmitted illumination and 50X magnification should be sufficient</td></tr><tr><td>Streptomycin sulfate</td><td>Fisher BioReagents</td><td>BP910-50</td><td></td></tr><tr><td>Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td><td>Becton Dickinson</td><td>236950</td><td></td></tr><tr><td>Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td><td>Becton Dickinson</td><td>211768</td><td></td></tr><tr><td>Yeast extract</td><td>Acros</td><td>61180-1000</td><td></td></tr></tbody></table>

measuring the effects of bacteria on c. elegans behavior using an egg retention assay

线虫产卵行为是受环境因素如渗透压和振动2。在完全没有食物C.线虫也停止产蛋，并保留受精卵在子宫3。然而，不同来源的食品，特别是病原细菌，特别是粪肠球菌 ，对产卵行为的影响没有得到很好的特征。蛋在蜗杆（EIW）法是一种有用的工具，不同类型的细菌的影响量化，在这种情况下， 大肠杆菌粪，产蛋行为。 EIW检测涉及计数鸡蛋的数量保留在子宫C.线虫 4。 EIW检测涉及漂白上演，妊娠成人C.线虫去除角质层，从动物中分离保留的鸡蛋。在漂白之前，蠕虫暴露于细菌（或任何不同的环境线索）一段固定的时间。漂白后，一个是很容易能够指望鸡蛋保留子宫内蠕虫的数量。在这个实验中，一个量化的增长后，在鸡蛋保留E.粪曝光可以很容易地测量。的的EIW测定试验，可用于筛选潜在致病细菌或环境毒素的存在是一种行为。此外，EIW检测可能是一个工具，屏幕药物，影响神经递质的信号，因为产卵的行为是由神经递质，如5 -羟色胺和乙酰胆碱的5-9调制。

此法可以让一个量化C.内保留的鸡蛋数量线虫暴露于E.后粪 。 L4上演蠕虫（特点是透明开放的空间高于其外阴， 图1）暴露于E.粪到成年。经过40个小时的接触E.粪 ，漂白。由于角质层崩解在漂白剂液滴，蛋变得更加明显（ 图2）。保留鸡蛋的数量很容易量化计算，而解剖显微镜下观看。 存在的大肠杆菌中保留更多的蛋测试粪肠球菌菌株相比，为E。大肠杆菌 OP50对照组（ 图3）。中任意一项的效果的大肠杆菌菌株中存在着某些差异个别虫粪 ，从错误的酒吧。然而，其效果的大肠杆菌的菌株的粪蛋内滞留从控制人口明显不相同的蠕虫。鸡蛋滞留引起程度不同的大肠杆菌菌株粪测试也可能有所不同。这种多样的效果可能是由于不同的毒力因子的表达，在每一株。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/51203/51203fig1highres.jpg" src="/files/ftp_upload/51203/51203fig1.jpg" /> 图1。查明L4上演C.线虫 。 L4阶段C.线虫幼虫板仅略短的长度比成虫幼虫目前是最大的。 L4S比成人为好，由于没有保留鸡蛋（这使成虫稍宽），B更薄。在确定这些幼虫比成人略短，L4上演C.线虫进一步确定了在较轻的的半月形点，位于自己的身体中（黑色箭头）。此点是在较小的幼虫（L2和L3阶段）可见，但在成人不存在。 NGM板的蠕虫采取所有图像，摄像头安装在解剖显微镜（40X和50X的放大倍率为A和B，分别） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51203/51203fig1highres.jpg">点击此处查看大图</a> 。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/51203/51203fig2highres.jpg" src="/files/ftp_upload/51203/51203fig2.jpg" /> 图2。漂白粉溶液溶解C.线虫角质层随着时间的推移分阶段成虫的漂白剂溶液的液滴被放置在（A）。在五到十分钟，角质层的蠕虫开始溶解（BF）。由于大多数的角质层被溶解后，将蛋变得可见（F黑色箭头）。发商店里面的蠕虫ALS<html> Ø慢慢溶解，漂白剂，仍然可见（F，灰色箭头）。 NGM板的蠕虫采取所有图像，摄像头安装在解剖显微镜（40X） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51203/51203fig2highres.jpg">点击此处查看大图</a> 。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/51203/51203fig3.jpg" src="/files/ftp_upload/51203/51203fig3.jpg" /> 图3。有些E.粪肠球菌引起提高产蛋蠕虫暴露两个六E. 保留40小时后曝光。 粪肠球菌 （橙色）表明增加的水平相比，蠕虫喂E.蛋保留大肠杆菌 （OP50）控制株（蓝色条）。 10蠕虫喂每次试验中的每个菌株。试验一式三份，在不同的日子。 *表示P &lt;0.05（学生t-检验）。"&gt;点击这里查看大图。

Watch this Scientific Journal Video about Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay at JoVE.com

使用卵保留测定测量细菌对线虫行为的影响生物学 |视频

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

一个鸡蛋在蠕虫（EIW）法是一种有用的方法来量化产蛋行为。产蛋的改变可以是一个模型有机体的行为反应<em&gt;线虫</em到潜在的有害环境的物质，例如由致病细菌产生的。

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

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17.0K 次观看.  Fairleigh Dickinson University. 一个鸡蛋在蠕虫（EIW）法是一种有用的方法来量化产蛋行为。产蛋的改变可以是一个模型有机体的行为反应线虫

视频: Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

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遗传学

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

行为学

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

测量乙醇的影响上的步态速度的测定<em&gt;秀丽隐杆线虫</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

神经科学

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

一个<em秀丽隐杆线虫</em基于营养状态的铜反转测定

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

测定化学物质对线虫线虫生长和繁殖的影响

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

医学

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

生物学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

使用多聚甲醛对 秀丽隐杆 线虫进行细菌代谢灭活

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a diet of live bacteria, the metabolic activity of their food source can confound experiments looking for the direct effects of various interventions on the worm. To avoid the confounding effects of bacterial metabolism, C. elegans researchers have used multiple methods to metabolically inactivate bacteria, including ultraviolet (UV)-irradiation, heat-killing, and antibiotics. UV treatment is relatively low-throughput and cannot be used in liquid culture because each plate must be examined for successful bacterial killing. A second treatment method, heat-killing, negatively affects the texture and nutritional quality of the bacteria, leading to the developmental arrest of C. elegans. Finally, antibiotic treatment can directly alter C. elegans physiology in addition to preventing bacterial growth. This manuscript describes an alternative method to metabolically inactivate bacteria using paraformaldehyde (PFA). PFA treatment cross-links proteins within bacterial cells to prevent metabolic activity while preserving cellular structure and nutritional content. This method is high-throughput and can be used in liquid culture or solid plates, as testing one plate of PFA-treated bacteria for growth validates the whole batch. Metabolic inactivation through PFA treatment can be used to eliminate the confounding effects of bacterial metabolism on studies of drug or metabolite supplementation, stress resistance, metabolomics, and behavior in C. elegans.

作者聚焦:使用多聚甲醛处理的细菌推进秀丽隐杆线虫研究 

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

秀丽隐杆线虫研究的代谢灭活细菌的方法

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

测量细菌的影响

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线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

基于线虫人群的化学感应优选测定的自动分析

测量乙醇的影响上的步态速度的测定

一个

测定化学物质对线虫线虫生长和繁殖的影响

线虫趋含量的

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

使用卡诺哈布迪炎埃莱甘斯筛选组织特异性查佩龙相互作用

秀丽隐杆线虫研究的代谢灭活细菌的方法

秀丽隐杆线虫研究的代谢灭活细菌的方法

测量细菌的影响

摘要

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此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

基于线虫人群的化学感应优选测定的自动分析

测量乙醇的影响上的步态速度的测定

一个

测定化学物质对线虫线虫生长和繁殖的影响

线虫趋含量的

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

秀丽隐杆线虫研究的代谢灭活细菌的方法

秀丽隐杆线虫研究的代谢灭活细菌的方法

使用卡诺哈布迪炎埃莱甘斯筛选组织特异性查佩龙相互作用