Name: using single-worm data to quantify heterogeneity in caenorhabditis elegans-bacterial interactions
Uploaded: 2022-07-22T14:20:00
Description: Watch this Scientific Journal Video about Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions at JoVE.com

作者要感谢H.舒伦伯格和C.拉洛克在这些实验中使用的细菌菌株的慷慨分享。这项工作得到了埃默里大学和NSF（PHY2014173）的资助。

这些实验中使用的蠕虫是从 Caenorhabditis 遗传中心获得的，该中心由NIH研究基础设施计划办公室（P40 OD010440）资助。布里斯托尔N2是野生型。DAF-2/IGF 突变体 daf-16（mu86） I （CGC CF1038） 和 DAF-2（e1370） III （CGC CB1370） 用于说明肠道细菌负荷的差异。
携带 pos-1 RNAi 载体的大 肠杆菌 HT115 （DE3） 来自阿林格文库21。 秀丽隐杆线?...

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这里提供了关于 秀丽隐杆线虫中细菌负荷的单蠕虫定量的优势的数据，以及96孔破坏方案，以允许快速和一致地获取这种类型的大型数据集。与现有方法33相比，这些方案允许对蠕虫中的肠道微生物群落进行更高通量的测量。
这种方法将电镀作为速率限制步骤，并不是真正的“高吞吐量”。大物体流式细胞术（图3C，D</stron...

宿主 - 微生物关联的异质性无处不在，个体之间的变异越来越被认为是从竞争和共存1到疾病传播2，3，4的人群水平过程的一个促成因素。在秀丽隐杆线虫中，反复观察到等基因群体内的“隐藏的异质性”，个体亚群在热休克反应5，6，衰老和寿命7，8，9，10，11以及生理学和发育的许多其他方面显示出不同的表型12.大多数试图确定亚种群结构的分析为等基因同步蠕虫实验种群中的两个亚种群提供了证据5，7，8，尽管其他数据表明性状的种群内分布的可能性，而不是不同的群体7，12，13.与此相关的是，即使在从共享的微生物来源定植的蠕虫的等基因群体中，也观察到肠道群体中的大量异质性13，14，15，16，并且这种异质性可以通过批量消化测量来掩盖，该测量广泛用于蠕虫中的细菌定量17，18，19，20。
这项工作提供的数据表明，在宿主 - 微生物关联中需要更多地依赖单蠕虫测量，以及提高单蠕虫破坏准确性和通量的方案。这些方案旨在促进对大量单个 秀丽隐杆线虫 的机械破坏，以定量活的细菌负荷，同时提供比基于杵的单个蠕虫更好的可重复性和更低的每个样品的努力。包括推荐的肠道清除步骤，其中允许蠕虫在准备破坏之前以热杀死 的大肠杆菌 为食，以尽量减少最近摄入的细菌和其他瞬时（非粘附）细菌的贡献。这些方案包括用低浓度表面漂白处理清洁角质层的冷麻痹方法;表面漂白可用作单一蠕虫破坏的准备步骤，或作为制备活的、外部无菌蠕虫的方法。这种表面漂白方法足以去除广泛的外部微生物，冷处理提供了常规左旋咪唑类麻痹的替代方案;虽然左旋咪唑将首选用于冷敏感实验，但冷麻痹可最大限度地减少对危险废物流的贡献，并允许迅速恢复正常活动。虽然完整的方案描述了一个实验室实验，其中蠕虫被已知细菌定植，但清洁蠕虫和单蠕虫破坏的程序可以很容易地应用于从野生样本中分离出或在微观实验中定植的蠕虫。这里描述的方案产生从蠕虫肠中提取的活细菌，适用于单个蠕虫中菌落形成单元（CFU）的电镀和定量;对于基于测序的肠道群落分析，应在这些方案中添加随后的细胞裂解和核酸提取步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well flat-bottom polypropylene plates, 300 uL</td>
			<td>Evergreen Labware</td>
			<td>290-8350-03F</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate sealing mat, silicon, square wells (AxyMat)</td>
			<td>Axygen</td>
			<td>AM-2ML-SQ</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, 2 mL, square wells</td>
			<td>Axygen</td>
			<td>P-2ML-SQ-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well polypropylene plate lids</td>
			<td>Evergreen Labware</td>
			<td>290-8020-03L</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>443570050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead mill adapter set for 96-well plates</td>
			<td>QIAGEN</td>
			<td>119900</td>
			<td>Adapter plates for use with two 96-well plates on the TissueLyser II</td>
		</tr>
		<tr>
			<td>Bead mill tissue homogenizer (TissueLyser II)</td>
			<td>QIAGEN</td>
			<td>85300</td>
			<td>Mechanical homogenizer for medium to high-throughput sample disruption</td>
		</tr>
		<tr>
			<td>BioSorter</td>
			<td>Union Biometrica</td>
			<td>By quotation</td>
			<td>Large object sorter equipped with a 250 micron focus for C. elegans</td>
		</tr>
		<tr>
			<td>Bleach, commercial, 8.25% sodium hypochlorite</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-Easy 96-well gas permeable sealing membrane</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td>Multiwell plate gas permeable polyurethane membranes. Thin sealing film is permeable to O2, CO2, and water vapors and is UV transparent down to 300 nm. Sterile, 100/box.</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientific</td>
			<td>AC423525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>VWR</td>
			<td>AAA11470-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Fisher Scientific</td>
			<td>AC124910010</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) sulfate pentahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC197722500</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 6765 LSE Mini Microcentrifuge</td>
			<td>Corning</td>
			<td>&#160;COR-6765</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium EDTA</td>
			<td>Fisher Scientific</td>
			<td>409971000</td>
			<td></td>
		</tr>
		<tr>
			<td>DL 1,4 Dithiothreitol, 99+%, for mol biology, DNAse, RNAse and Protease free, ACROS Organics</td>
			<td>Fisher Scientific</td>
			<td>327190010</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 1.5 mL microcentrifuge tubes, natural</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5406000640</td>
			<td>24-place refrigerated benchtop microcentrifuge</td>
		</tr>
		<tr>
			<td>Eppendorf 5810R centrifuge with rotor S-4-104</td>
			<td>Eppendorf</td>
			<td>22627040</td>
			<td>3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates</td>
		</tr>
		<tr>
			<td>Eppendorf plate bucket (x2), for Rotor S-4-104</td>
			<td>Eppendorf</td>
			<td>22638930</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, 2.7 mm</td>
			<td>Life Science Products</td>
			<td>LS-79127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, acid-washed, 425-600 &#181;m</td>
			<td>Sigma</td>
			<td>G877-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass plating beads</td>
			<td>VWR</td>
			<td>76005-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>VWR</td>
			<td>BDH7204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>423731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes pellet pestle motor</td>
			<td>DWK Life Sciences</td>
			<td>749540-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes polypropylene pellet pestles and microtubes, 0.5 mL</td>
			<td>DWK Life Sciences</td>
			<td>749520-0590</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMi8 motorized inverted microscope with motorized stage</td>
			<td>Leica</td>
			<td>11889113</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica LAS X Premium software</td>
			<td>Leica</td>
			<td>11640687</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC124900010</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese(II) chloride tetrahydrate</td>
			<td>VWR</td>
			<td>470301-706</td>
			<td></td>
		</tr>
		<tr>
			<td>PARAFILM M flexible laboratory sealing film</td>
			<td>Amcor</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, round, 10 cm</td>
			<td>VWR</td>
			<td>25384-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, round, 6 cm</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, square, 10 x 10 cm</td>
			<td>VWR</td>
			<td>10799-140</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-buffered saline (1X PBS)</td>
			<td>Gold Bio</td>
			<td>P-271-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene autoclave tray, shallow</td>
			<td>Fisher Scientific</td>
			<td>13-361-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>AC134062500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-1</td>
			<td></td>
		</tr>
		<tr>
			<td>R 4.1.3/RStudio 2022.02.0 build 443</td>
			<td>R Foundation</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Scoop-type laboratory spatula, metal</td>
			<td>VWR</td>
			<td>470149-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon carbide 36 grit</td>
			<td>MJR Tumblers</td>
			<td>n/a</td>
			<td>Black extra coarse silicon carbide grit. Available in 0.5-5 lb sizes from this vendor.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP166-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>VWR</td>
			<td>BDH7247-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodum chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>AC419760010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-potassium citrate monohydrate</td>
			<td>Fisher Scientific</td>
			<td>AC611755000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC205982500</td>
			<td></td>
		</tr>
	</tbody>
</table>

using single-worm data to quantify heterogeneity in caenorhabditis elegans-bacterial interactions

线虫 秀丽隐杆线虫 是宿主-微生物和宿主-微生物组相互作用的模型系统。迄今为止，许多研究使用间歇消化物而不是单个蠕虫样本来量化该生物体中的细菌负荷。这里认为，在 秀丽隐杆线虫 肠道的细菌定植中看到的较大的个体间变异性是信息性的，并且批量消化方法丢弃了对跨条件准确比较很重要的信息。由于描述这些样品固有的变异需要大量的个体，因此建立了一种方便的96孔板方案，用于单个蠕虫的破坏和菌落电镀。

活虫的漂白灭菌 表面漂白的蠕虫实际上没有外部细菌，直到运动恢复并恢复排泄。在这里使用的条件下，观察到缓冲液中细菌的快速灭绝（图1A-C，补充图2，视频1），而不会干扰冷麻痹蠕虫中的肠道相关细菌（图1D-F，视频2）。这些数据表明，表面漂白可以有效地用于外部消毒蠕虫，而...

Watch this Scientific Journal Video about Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions at JoVE.com

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

该协议描述了在冷麻痹和表面漂白以去除外部细菌后，对单个细菌定植 的秀丽隐杆线虫炎的 96孔破坏。将得到的悬浮液接种在琼脂平板上，以便对大量单个蠕虫中的细菌负荷进行准确的中等通量定量。

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 ...

该协议展示了秀丽隐杆线虫的机械破坏。采用 96 孔格式，允许中等通量定量单个蠕虫中的细菌负荷。与基于 Pasel 的方法相比，该技术提高了机械破碎的速度和均匀性，使研究人员能够轻松生成更大的高质量单蜗杆测量数据集。这种技术涉及许多活动部件，很容易失去您的位置。在开始之前，请确保已设置所有材料、标记的试管、缓冲液和板。演示该程序的将是我实验室的研究专家梅根泰勒。首先将蠕虫样品重新悬浮在微量离心管中的一毫升M nine TX zero one中。通过离心收集成虫并弃去上清液。使用相同的离心参数。用一毫升M9TX01冲洗蠕虫两次，用一毫升M9蠕虫缓冲液冲洗一次，以尽量减少外部细菌。然后，将每个样品重新悬浮在一毫升S培养基中，并在培养管中将两个x热杀死OP50。将试管在 25 摄氏度下孵育 20 至 30 分钟，以使非 AD 粘附的细菌从肠道中排出。孵育后，用一毫升冷M9TX01冲洗吹扫的蠕虫两次，并丢弃超级清液。将试管放在冰上 10 分钟以麻痹蠕虫。然后在每个试管中加入一毫升冰冷的 M nine 蠕虫缓冲液和无味漂白剂。将试管在冰上孵育至少 10 分钟以杀死外部细菌。丢弃漂白缓冲液并将管子放回冰中以防止蠕虫泵送。接下来，向每个试管中加入一毫升冷的M9TX01并离心五秒钟。在小型离心机中。将试管放回冰上并除去上清液。重复此步骤一次。用于蠕虫角质层的化学透化。将含有蠕虫的管在20微升缓冲液中转移到室温管架中。然后向每个温热的样品中加入100微升SDS / DTT溶液。将试管在工作台上孵育长达八分钟，以部分分解成虫的角质层。此时，蠕虫将死亡并沉淀在管子底部。孵育后，小心地弃去SDS / DTT上清液。然后，向每个试管中加入一毫升M9TX01，并短暂离心以沉淀蠕虫。弃去上清液，将蠕虫重新悬浮在含有0.1%Triton x-100的1毫升M九蠕虫缓冲液中。为了准备蠕虫的机械破碎，请获得无菌的两毫升深井96孔板和硅胶板盖。使用无菌勺刮刀向每个孔添加少量无菌36网格碳化硅，使网格几乎覆盖孔底部。向每个孔中加入180微升M nine蠕虫缓冲液。标记列和行，然后用硅胶板盖松散地覆盖板。接下来，将透化的蠕虫转移到一个小培养皿中，将M9TX01填充到一厘米的深度。使用解剖显微镜移液出20微升体积的单个蠕虫，并将它们转移到96孔板的各个孔中以弹出粘在移液器上的任何蠕虫，从培养皿的透明区域吸出20微升M9TX01并将其释放回培养皿中。转移所有蠕虫后，用一张柔性天花板膜覆盖 96 孔板，纸背面朝下放在样品孔上。将硅胶天花板垫轻轻放在柔性天花板膜的顶部，不要将盖子向下压入井中。将板在 4 摄氏度下放置 30 到 60 分钟，以防止在中断期间过热。冷却板后，将硅胶天花板垫牢固地压入井中以密封它们。然后将板固定在组织破坏器中。以 30 赫兹摇动盘子一分钟。然后将板旋转 180 度并重复摇晃一分钟。将板子牢牢地敲击在长凳上两到三次，以清除柔性天花板薄膜上的任何砂砾。将板组2， 400倍G离心两分钟，以收集孔底的样品。然后取下硅胶盖并拉下天花板薄膜。为了制备蠕虫消化物的十倍连续稀释，在96孔板的B至D行中填充180微升一个X PBS。使用设置为200微升的多孔移液器，通过移液缓慢混合蠕虫消化物。将最大量的液体转移到含有PBS的96孔板的A行。使用多通道移液器，从顶排取出20微升，分配到B行中，然后混合。对B行到C重复此步骤，然后对C行到D重复此步骤以获得1000倍稀释度，用于单定植蠕虫的细菌定量。在固体琼脂平板上平板每次稀释10至20微升。对于多物种定植，将每次稀释液100微升平板在10厘米琼脂平板上。使用该协议，在冷麻痹蠕虫的表面漂白后观察到有效的外部消毒，如缓冲液中外部细菌的减少所见。不影响肠道相关细菌。手动破坏蠕虫导致更多的异质性。碳化硅砂砾是带或不带三安达斯的破碎的理想选择。在96孔法中，大玻璃珠不适合96井技术。小玻璃珠产生一致的结果，但在尖端堵塞了管道。在同一细菌池上定植的蠕虫在观察单个细菌和多物种细菌群落时显示出肠道负荷的高度变化。用表达GFP的细菌定植的蠕虫在单个蠕虫测量中显示出异质性。对于这种表达GFP的细菌菌株比较野生型布里斯托尔伦两种蠕虫的定植。对DAF的两个IGF突变体表明，DAF 16突变体支持更多的细菌种群，而DAF两个对定植具有抗性。这种异质性是特征性的，并且在同一实验的不同运行中保持一致。与单个蠕虫数据相比，发现重新采样数据以模拟批次摘要会改变分布。每条蠕虫批量外推CFU以导致生物变异损失的单个数据的算术平均值为中心。代替继续消化活表面漂白剂和冲洗蠕虫可用于进一步的实验。一旦蠕虫从寒冷中移开，运动将迅速恢复。

Research

JoVE Journal

Biology

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

科研

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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

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 ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Techniques to Induce and Quantify Cellular Senescence

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

鼻电位差量化小鼠跨上皮离子转运

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

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, ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

微殖器的高通量实时成像，测量生长和基因表达中的异质性

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

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

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