Name: the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions
Uploaded: 2017-10-18T14:00:00.000+00:00
Duration: 11 min 58 s
Description: Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

这项工作是在伍斯特理工学院的支持下进行的。

1. 制备线虫生长培养基 (NGM) <ol> <li> 用于 1 l 介质, 结合 20 g 琼脂、2.5 g 有机氮源 ( 如 、bacto-蛋白胨) 和3克氯化钠在 2 l 烧瓶中。加入975毫升的无菌水. 
	<ol> <li> 在无菌搅拌栏中添加。如果使用自动介质倒, 高压釜管和介质为15分钟;如果有更高的音量, 介质应更长的时间进行蒸压. </li>
	</ol> </li> <li> 在搅拌板上设置介质, 并允许冷却。
	<ol> <li> 一旦媒体被热触碰 (大约60和 #176; C) 菌增加1毫升的1米 MgSO 4 和 #9679; H 2 O, 1 毫升的 1M CaCl 2 , 25 毫升的 KPO 4 , 和1毫升的0.5% 的胆固醇在乙醇中. </li>
		<li> 将介质 (手动或自动泵) 倒入 35 mm x 10 mm 无菌培养皿中。允许在使用前1-2 天晾干引擎盖. 
		注: 板材应存储在4和 #176; C, 以防止污染. </li>
	</ol> </li> </ol> 2。制作蠕虫选择 <ol> <li> 将一根1.5 厘米的铝线切下。小心地将大约0.5 厘米的长度插入到巴斯德吸管的尖端. 
	<ol> <li> 使用本生燃烧器或酒精灯, 将玻璃吸管尖端慢慢地旋转到火焰中, 直到导线牢固地附着在吸管上, 而不会损害吸管尖端的原始形状. </li>
	</ol> </li> </ol> 3。 大肠杆菌 培养基的制备 <ol> <li> 使用无菌循环, 在200毫升的 Luria 汤中接种单一菌落的 大肠杆菌 菌株 OP50。在37和 #176 摇晃过夜; C. </li>
	<li> 液体养殖已准备就绪, 可在次日使用, 并可存储在4和 #176; C 时不使用. 
	注意: OP50 的 大肠杆菌 菌株被用作 C. 线虫的食物来源 此区域性可在4和 #176 存储时使用长达几个月; C. </li>
</ol> 4。基本的 线虫 维护 <ol> <li> 种子准备 NGM 琼脂板与 大肠杆菌 , 通过发现大约 50-100 和 #181; L 准备 OP50 液体文化到板材的中心. </li>
	<li> 盖板, 使其在室温下干燥24小时。一旦干燥, 放置在37和 #176 的盘子; C 隔夜为迅速成长或保持在室温下, 如果增长是渴望的. </li>
	<li> 通过任意和 #34 将蠕虫添加到板中; 分块、#34; (请参阅协议步骤5和 #34; 将蠕虫和 #34;), 逐个选取蠕虫, 或将蠕虫卵直接放到板上 (参见协议步骤6和 #34; 蛋准备和 #34;). </li>
</ol> 5。分块和拾取蠕虫 注意: 和 #34; #34. 是指在 C. 线虫 维护中常见的一种做法。这涉及切割含有蠕虫的 NGM 琼脂板的一部分, 并将其转移到一个新的板块, 从而也转移了大量的蠕虫在这个过程中。污染也可以用这种方式从盘子里切出来。当采摘蠕虫时, 重要的是要保持琼脂的完整性, 因为蠕虫可以在琼脂的孔中丢失。此外, 重要的是, 让采摘冷却前触摸板, 以防止融化的琼脂或燃烧的蠕虫. 
<ol> <li> 为了快速将大量的蠕虫转移到新的板上, 请用刮刀将含有所选蠕虫的一块 NGM 琼脂切开。删除切割件, 并将其面朝下的 OP50 草坪的边缘上的一个新的板块. </li>
	<li> 在协议步骤8.2、#34、生存分析和 #34 中分别传输蠕虫; 使用蠕虫拾取。在蠕虫的选择中火焰, 直到它是红色的。从火焰中取出, 让它冷却几秒钟. </li>
	<li> 用铁丝在镐上, 轻轻刮去 OP50 文化的草坪上生长的新盘子, 盖上铁丝上的镐. </li>
	<li> 在导线尖端的粘性培养物会使蠕虫粘在拾取的尖端, 从而用滑动运动轻轻地拾取选定的蠕虫. </li>
	<li> 一旦选定的蠕虫被收集, 将它们放到一个新的盘子上, 轻轻地在琼脂上滑动区域性。将导线放入火焰中, 以去除拾取中的剩余区域性. </li>
</ol> 6。鸡蛋准备 <ol> <li> 将大约20成年动物 (大约 2-3 天后孵化时, 生长在20和 #176; C) 按协议步骤 5, #34; 分块和采摘蠕虫, 和 #34; 在一个有50个种子的盘子上和 #181 大肠杆菌菌株, OP50. </li>
	<li> 收集鸡蛋, 在1-2 天后用10毫升的 M9 缓冲液淹没盘子。使用玻璃血清吸管, 旋流, 并轻轻地搅动琼脂板上的内容, 以释放鸡蛋粘在 OP50 或成年动物。收集所有含有卵和成虫的液体. </li>
	<li> 将 M9 和 OP50 溶液转换为15毫升锥形管。离心机15毫升锥管在 2100 x g 2 min. </li>
	<li> 吸气约9毫升的上清, 不干扰 OP50 颗粒. </li>
	<li> 重2毫升无菌水中的颗粒, 1 毫升的漂白剂和1毫升的0.25 米氢氧化钠. </li>
	<li> 通过反转轻轻混合, 直到成年动物的大多数 (大约 70%) 出现裂解; 通常情况下, 由于蛋壳的原因, 鸡蛋在漂白过程中保持完好。离心锥管在 990 x g 为 2 min. </li>
	<li> 吸入上清液而不干扰颗粒。重新悬浮在10毫升的 M9 缓冲颗粒. </li>
	<li> 离心锥管 990 x g, 2 分钟, 无干扰颗粒。200、#181 的 M9 缓冲颗粒重新悬浮. 
	注: 卵子准备可能需要多次试验。如果鸡蛋暴露在漂白剂中太久, 它们可能会被破坏。如果鸡蛋不孵化, 要么减少溶液中漂白剂的浓度, 要么减少接触漂白剂的时间. </li>
</ol> 7。感染板设置 <ol> <li> 将3-5 毫升的 YPD 分配到一个无菌试管中, 并使用一个无菌循环将它接种到一个单一菌落的白色念珠菌 中. 
	<ol> <li> 将导管放置在旋转鼓上大约16-18 小时, 在30和 #176; C. </li>
	</ol> </li> <li> 标记一个1.5 毫升离心管, 以包含 C. 白色念珠菌 , 另一个包含 OP50。记录每个空管的重量. 
	<ol> <li> 将500和 #181; 过夜的 c. 白色念珠菌 培养成管标为 白色念珠菌 和1500和 #181; 夜间 OP50 培养 (从步骤 3.1) 到标有 OP50 的管子. </li>
		<li> 离心10分钟, 16100 x g. 吸上清液而不干扰颗粒. </li>
		<li> 重新挂起500和 #181 的每个颗粒; 无菌水的 L。离心机为5分钟, 在 16100 x g. </li>
		<li> 吸入上清液而不干扰颗粒。记录离心管的最终重量. </li><li> 通过从最终重量中减去离心管的初始重量来确定每个颗粒的重量. </li>
	</ol> </li> <li> 使用无菌水, 将 re-suspend 为10毫克/毫升, 并将 OP50 颗粒至200毫克/毫升。通过结合10和 #181; 50 毫克/毫升链霉素, 2.5 和 #181; l 的 OP50 培养, 0.5 和 #181; l. 白色念珠菌 培养, 7 和 #181; 无菌水的 l. 
	<ol> <li> 对于控制板, 通过用无菌水替换 C. 白色念珠菌 的体积来创建主混合。种子20和 #181; L 感染混合或 OP50 控制解决方案, NGM 板的中心使用微. 
		注: 在分析过程中, 需要大约100蠕虫来确定统计意义。这种化验通常是三份的。因此, 一个3.2x 的感染混合以及3.2x 控制解决方案作出。这些混合被制成略超过3x 的原始, 以确保一个完整的20和 #181; 我被播种在每一个盘子上, 允许有错误的空间。此主组合是创建的, 因为在初始幼体阶段 (L1-L3) 中, 蠕虫的咽太小, 无法使用 C. 白念珠菌 。对于接触药物, 如氟康唑 ( 图 3B ), 该剂被引入到感染混合物。药剂的集中是经验主义地坚定的。例如, 在 图 3B 中, 50 和 #181; m 氟康唑的代表, 但 0, 12.5, 25, 50 和100和 #181; m 氟康唑也使用相同的协议进行了测试. </li>
	</ol> </li> </ol> 8。变形肛门区 (dar) 表型与生存试验分析 <ol> <li> 可视化 dar 表型 注: dar 在 L3 阶段和更远处是最好的可视化。Dar 在蠕虫的后肛门区域 ( 图 2A ) 中显示为一个小突起, 随着时间的推移, 它变得更加突出。
	<ol> <li> 通过在解剖显微镜的舞台上快速敲击板来确认动物是否有 dar; 没有 dar 的动物将立即向后移动, 而与 dar 的动物将需要反复的敲击来反转它们的方向, 或者不会这样做. </li>
	</ol> </li> <li> 生存分析 <ol> <li> 完整的蛋准备, 如协议步骤6中所述, #34; 鸡蛋准备和 #34;。计算下解剖范围内的卵, 用 M9 稀释卵子溶液, 直到每 #181 约5-6 健康卵的浓度; 我成功了使用吸管, 免除20和 #181; 样品中含有约30鸡蛋在准备的 NGM 板上, 在细菌培养和侧板之间的区域 (卵和蠕虫食物, OP50, 是在两个不同的斑点). </li>
		<li> 在20和 #176 孵育板; C 为48小时。在解剖显微镜下, 统计每个板块上的活的和死的成虫数, 以及与 Dar 的蠕虫数量。用 Dar 记录百分比. </li>
		<li> 如果它不响应由铝线敲击头部或在板上敲击, 则认为是动物死亡. </li>
		<li> 每隔一天, 按协议步骤5中所述的方法选择转移剩余的成虫, 并 #34; 分块和拾取蠕虫、#34; 进入新的感染或控制板。在这一点上, 如果需要, 通过跟踪每天的活、死和缺失蠕虫的数量来进行生存试验. 
		注: 此化验可运行两周, 从准备卵子开始。此外, 卵子的解决方案不应被分配到细菌培养, 因为解决方案可能含有漂白剂的痕迹, 可以杀死 OP50。该解决方案也应免除约0.5 厘米远离板块的边缘, 因为鸡蛋可以成为卡在两侧的板块和琼脂。此外, 由于蠕虫的迅速扩散, 将成人转移到新的板块上是将其与后代分离的关键. </li>
	</ol> </li> <li> 分析生存 <ol> <li> 使用生存曲线分析软件分析结果 (参阅材料列表). </li>
		<li> 使用 Grehan-Breslow-魏氏方法比较生存曲线 (假设早期生存时间的数据比以后的时间更准确, 相应地对数据进行加权) 以及 Logrank 统计工具 45 . 
		注意: 例如, 在 图 4B -c 中, 使用变种人 c. 白色念珠菌治疗的蠕虫的生存率与基因野生型控制或 complementarystrains 治疗相比. 
	</ol>
	</li>
</ol>

作者没有什么可透露的。

用于检测线虫感染和生存期的方法对我们所描述的c. 白色念珠菌的影响可以通过修改来测试另一种病原体。另一种细菌或真菌的液体培养物可以用类似的方式制成并喂给C. 线虫。此外, 串行感染可以通过首先暴露幼虫到一个病原体, 然后转移到一个新的板块, 包含一个独立的病原体后, 成年。
在进行这种化验时, 注意时间是很重要的。首先, 当完成鸡蛋准备, ...

线虫已被用作一个强大的模型有机体超过50年。在二十世纪六十年代, 南非生物学家悉尼. 博纳率先使用了线虫来研究神经元的发育, 为长期的科学家们研究线虫的细胞和动物生物学的各个方面铺平了道路。这一血统包括诺贝尔奖得主克雷格. 梅洛和安德鲁火为他们的 rna 干扰工作1, 罗伯特 Horvitz 并且约翰 Sulston 为他们的工作器官发展和细胞凋亡2,3,4, 和马丁查尔菲为他的工作绿色荧光蛋白5。尽管这种模型生物体已经被传统用于研究分子和发育生物学, 但在过去15年中, 研究人员已经开始使用线虫来调查各种人类病原体的生物学, 包括假单胞菌铜绿假单胞菌,金黄色葡萄球菌,沙门氏菌 enterica, 和沙雷菌6,7,89,10。这些研究表明, 许多涉及人与病原体相互作用的机制都是在线虫中保存的, 但也有一些免疫机制是独特的, 这种模式生物体11,12。在自然界中,线虫遇到了来自土壤中的受感染病原体的各种威胁, 这为进化和维持一个复杂的先天免疫系统在其肠道腔内提供了强烈的选择性压力。许多涉及保护肠道腔的基因和机制都是由高度保守的元素组成的, 它们也存在于高哺乳动物中11,13。因此,线虫是研究胃肠病原体的一个很好的模型, 如沙门氏菌 enterica14,痢疾 boydii15, 或霍乱弧菌16。
在这里, 我们强调了显着的多功能性的c. 线虫作为一个模型的宿主, 以研究传染性药物, 如C. 白色念珠菌。C. 线虫作为模型宿主, 可以对毒性进行高通量筛选, 这比小鼠模型更便宜、更耗时, 通常用于研究念珠菌病42。
在这项研究中, 我们表明, 该模型和 assosiated 生存分析可以可靠地用于研究宿主先天免疫效应的重要性, 以抵消感染, 病原体的决定因素驱动毒力, 和药理化合物, 可以干预发病机制。与先前所描述的化验方法不同, 此法提供了一种研究动物生命周期中的病原体, 从幼虫阶段到成年, 而不是仅成年到死亡的手段43,44。总之, 我们的线虫--白色念珠菌模型是一种多功能、功能强大的工具, 不仅可以用于研究驱动感染和免疫的遗传基础, 还可用于识别治疗干预的新化合物。

<table><tbody><tr><td>Agar (granulated, bacterilogical grade)</td><td>Apex BioResearch Products</td><td>20-248</td><td></td></tr><tr><td>Aluminum Wire (95% Pt, 32 Gauge)</td><td>Genesee Scientific</td><td>59-1M32P</td><td></td></tr><tr><td>Axiovision Zeiss Inverted Microscope</td><td>Axiovision Zeiss</td><td></td><td></td></tr><tr><td>Bacto-Peptone</td><td>Fisher BioReagants</td><td>BP1420-500</td><td></td></tr><tr><td>C. elegans strain Bli-3</td><td>Caenorhabditis Genetics Center</td><td>Bli-3(e767) CB767</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>BP51-250</td><td></td></tr><tr><td>Cholesterol, Sigma Grade, minimum 99%</td><td>Sigma</td><td>C8667-25G</td><td></td></tr><tr><td>Disposable Culture Tubes (20 x 150 mm)</td><td>FIsherBrand</td><td>14-961-33</td><td></td></tr><tr><td>Dissection Microscope (NI-150 High Intensity Illuminator)</td><td>Nikon Instrument Inc.</td><td></td><td></td></tr><tr><td>E. coli</td><td>Caenorhabditis Genetics Center</td><td>OP50</td><td></td></tr><tr><td>GraphPad Prism (Survival Curve Analysis Software)</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>LB Broth (Miller&#039;s)</td><td>Apex BioResearch Products</td><td>11-120</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher Scientific</td><td>10034-99-8</td><td></td></tr><tr><td>Medium Petri Dishes (35 X 10 mm)</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Potassium Phosphate monobasic</td><td>Sigma</td><td>P0662-500G</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Wildtype C. albicans SC5314</td><td>ATCC</td><td>SC5314</td><td></td></tr><tr><td>Wildtype C. elegans</td><td>Caenorhabditis Genetics Center</td><td>N2</td><td></td></tr></tbody></table>

the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions

我们演示了一种使用线虫线虫作为模型宿主来研究微生物相互作用的方法。通过饮食引入微生物, 使肠道成为疾病的主要部位。线虫肠道结构和功能模仿哺乳动物的肠道, 是透明的, 使其适于对殖民的微观研究。在这里, 我们表明病原体可以导致疾病和死亡。我们能够识别出具有改变毒性的微生物突变体。它对生物胁迫的固有反应使线虫成为一个极好的系统, 可以探测宿主先天免疫相互作用的方方面面。我们发现, 双氧化酶基因突变的寄主不能产生活性氧, 无法抵抗微生物的侮辱。我们进一步证明了所提出的生存试验的通用性, 表明它可以用于研究微生物生长抑制剂的影响。这种方法也可用于发现真菌致病因子作为发展新的抗真菌药物的目标, 以及提供一个机会, 进一步揭示宿主-微生物相互作用。这种检测方法的设计很好地适应了高通量全基因组的屏幕, 而低温保存蠕虫以备将来使用的能力使得它成为一个 cost-effective 和有吸引力的整体动物模型来研究。

病机检测 (图 1) 使用c. 白色念珠菌和c. 线虫以前曾被我们的实验室描述17,18和其他实验室19,,。我们演示了使用可研究 白色念珠菌的毒力, 该病毒显示了c. 白念珠菌的细胞迅速被蠕虫吸收并积聚在肠道内, 导致运动迟缓, 肛门畸形?...

Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

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

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

在这里, 我们提出线虫线虫线虫作为一个多用途的寄主模型来研究微生物的相互作用。

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

the-nematode-caenorhabditis-elegans-versatile-vivo-model-to-study

Research

JoVE Journal

Genetics

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

9.3K 次观看.  Worcester Polytechnic Institute. 在这里, 我们提出线虫线虫线虫作为一个多用途的寄主模型来研究微生物的相互作用。

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

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

科研

遗传学

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

免疫与感染

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

线虫模型中气单胞菌感染的毒力及发病机制评价

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

秀 丽隐杆线虫 感染模型中的遗传免疫研究

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

生物学

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

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

摘要

探索更多视频

此视频中的章节

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

研究微生物群落

线虫模型中气单胞菌感染的毒力及发病机制评价

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

秀丽隐杆线虫感染模型中的遗传免疫研究

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

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

摘要

探索更多视频

此视频中的章节

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

研究微生物群落

线虫模型中气单胞菌感染的毒力及发病机制评价

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

秀 丽隐杆线虫 感染模型中的遗传免疫研究

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

秀丽隐杆线虫感染模型中的遗传免疫研究

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用