Name: 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 (视频) | JoVE Journal
Uploaded: 2022-07-22T14:20:00.000+00:00
Duration: 9 min 54 s
Description: 了解利用96孔板机械破碎技术定量分析单个秀丽隐杆线虫细菌负荷的中通量方法。该技术通过冷麻痹和表面漂白去除外部细菌，同时保留肠道相关菌群，为微生物定植异质性研究提供可靠数据。

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作者感谢 H. Schulenberg 和 C. LaRock 慷慨提供了本实验所用的细菌菌株。本研究工作获得了埃默里大学以及美国国家科学基金会（NSF，项目编号 PHY2014173）的经费支持。

<ol><li>Armitage, D. W., Jones, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+sample+heterogeneity+can+obscure+the+signal+of+microbial+interactions%5BTitle%5D)+AND+%22The+ISME+Journal%22%5BJournal%5D)">How sample heterogeneity can obscure the signal of microbial interactions.</a> The ISME Journal. 13 (11), 2639-2646 (2019).</li>
<li>Stephenson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+heterogeneity+affects+both+parasite+transmission+to+and+fitness+on+subsequent+hosts%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+B%3A+Biological+Sciences%22%5BJournal%5D)">Host heterogeneity affects both parasite transmission to and fitness on subsequent hosts.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 372 (1719), 20160093(2017).</li>
<li>VanderWaal, K. L., Ezenwa, V. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+in+pathogen+transmission%3A+mechanisms+and+methodology%5BTitle%5D)+AND+%22Functional+Ecology%22%5BJournal%5D)">Heterogeneity in pathogen transmission: mechanisms and methodology.</a> Functional Ecology. 30 (10), 1606-1622 (2016).</li>
<li>Dwyer, G., Elkinton, J. S., Buonaccorsi, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+heterogeneity+in+susceptibility+and+disease+dynamics%3A+tests+of+a+mathematical+model%5BTitle%5D)+AND+%22The+American+Naturalist%22%5BJournal%5D)">Host heterogeneity in susceptibility and disease dynamics: tests of a mathematical model.</a> The American Naturalist. 150 (6), 685-707 (1997).</li>
<li>Wu, D., Rea, S. L., Yashin, A. I., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+hidden+heterogeneity+in+isogenic+populations+of+C.+elegans%5BTitle%5D)+AND+%22Experimental+Gerontology%22%5BJournal%5D)">Visualizing hidden heterogeneity in isogenic populations of C. elegans.</a> Experimental Gerontology. 41 (3), 261-270 (2006).</li>
<li>Yashin, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heat+shock+changes+the+heterogeneity+distribution+in+populations+of+Caenorhabditis+elegans+does+it+tell+us+anything+about+the+biological+mechanism+of+stress+response%5BTitle%5D)+AND+%22The+Journals+of+Gerontology+Series+A%3A+Biological+Sciences+and+Medical+Sciences%22%5BJournal%5D)">Heat shock changes the heterogeneity distribution in populations of Caenorhabditis elegans does it tell us anything about the biological mechanism of stress response.</a> The Journals of Gerontology Series A: Biological Sciences and Medical Sciences. 57 (3), 83-92 (2002).</li>
<li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+forms+of+death+in+ageing+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Two forms of death in ageing Caenorhabditis elegans.</a> Nature Communications. 8 (1), 1-8 (2017).</li>
<li>Eckley, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+characterization+of+the+transition+to+mid-life+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22AGE%22%5BJournal%5D)">Molecular characterization of the transition to mid-life in Caenorhabditis elegans.</a> AGE. 35 (3), 689-703 (2012).</li>
<li>Rea, S. L., Wu, D., Cypser, J. R., Vaupel, J. W., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+stress-sensitive+reporter+predicts+longevity+in+isogenic+populations+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans.</a> Nature Genetics. 37 (8), 894-898 (2005).</li>
<li>Kinser, H. E., Mosley, M. C., Plutzer, I. B., Pincus, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global%2C+cell+non-autonomous+gene+regulation+drives+individual+lifespan+among+isogenic+C.+elegans%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Global, cell non-autonomous gene regulation drives individual lifespan among isogenic C. elegans.</a> eLife. , (2021).</li>
<li>Churgin, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longitudinal+imaging+of+Caenorhabditis+elegans+in+a+microfabricated+device+reveals+variation+in+behavioral+decline+during+aging%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging.</a> eLife. 6, 26652(2017).</li>
<li>Perez, M. F., Francesconi, M., Hidalgo-Carcedo, C., Lehner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+age+generates+phenotypic+variation+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Maternal age generates phenotypic variation in Caenorhabditis elegans.</a> Nature. 552 (7683), 106-109 (2017).</li>
<li>Baeriswyl, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+aging+profiles+in+isogenic+populations+of+Caenorhabditis+elegans+by+bacteria+causing+different+extrinsic+mortality+rates%5BTitle%5D)+AND+%22Biogerontology%22%5BJournal%5D)">Modulation of aging profiles in isogenic populations of Caenorhabditis elegans by bacteria causing different extrinsic mortality rates.</a> Biogerontology. 11 (1), 53(2009).</li>
<li>Taylor, M., Vega, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+immunity+alters+community+ecology+and+stability+of+the+microbiome+in+a+Caenorhabditis+elegans+model%5BTitle%5D)+AND+%22mSystems%22%5BJournal%5D)">Host immunity alters community ecology and stability of the microbiome in a Caenorhabditis elegans model.</a> mSystems. 6 (2), 00608-00620 (2021).</li>
<li>Diaz, S. A., Restif, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spread+and+transmission+of+bacterial+pathogens+in+experimental+populations+of+the+nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Spread and transmission of bacterial pathogens in experimental populations of the nematode Caenorhabditis elegans.</a> Applied and Environmental Microbiology. 80 (17), 5411-5418 (2014).</li>
<li>Twumasi-Boateng, K., Berg, M., Shapira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+separation+of+C.+elegans+variably+colonized+by+a+bacterial+pathogen%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Automated separation of C. elegans variably colonized by a bacterial pathogen.</a> Journal of Visualized Experiments: JoVE. (85), e51090(2014).</li>
<li>Ortiz, A., Vega, N. M., Ratzke, C., Gore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interspecies+bacterial+competition+regulates+community+assembly+in+the+C.+elegans+intestine%5BTitle%5D)+AND+%22The+ISME+Journal%22%5BJournal%5D)">Interspecies bacterial competition regulates community assembly in the C. elegans intestine.</a> The ISME Journal. 15 (7), 2131-2145 (2021).</li>
<li>Berg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF%CE%B2%2FBMP+immune+signaling+affects+abundance+and+function+of+C.+elegans+gut+commensals%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">TGFβ/BMP immune signaling affects abundance and function of C. elegans gut commensals.</a> Nature Communications. 10 (1), 604(2019).</li>
<li>Portal-Celhay, C., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Competition+and+resilience+between+founder+and+introduced+bacteria+in+the+Caenorhabditis+elegans+gut%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Competition and resilience between founder and introduced bacteria in the Caenorhabditis elegans gut.</a> Infection and Immunity. 80 (3), 1288-1299 (2012).</li>
<li>Scott, E., Holden-Dye, L., O'Connor, V., Wand, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intra+strain+variation+of+the+effects+of+gram-negative+ESKAPE+pathogens+on+intestinal+colonization%2C+host+viability%2C+and+host+response+in+the+model+organism+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Intra strain variation of the effects of gram-negative ESKAPE pathogens on intestinal colonization, host viability, and host response in the model organism Caenorhabditis elegans.</a> Frontiers in Microbiology. 10, 3113(2020).</li>
<li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome Biology. 2 (1), (2001).</li>
<li>Dirksen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+native+microbiome+of+the+nematode+Caenorhabditis+elegans%3A+gateway+to+a+new+host-microbiome+model%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model.</a> BMC Biology. 14, 38(2016).</li>
<li>Vega, N. M., Allison, K. R., Samuels, A. N., Klempner, M. S., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salmonella+typhimurium+intercepts+Escherichia+coli+signaling+to+enhance+antibiotic+tolerance%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance.</a> Proceedings of the National Academy of Sciences. 110 (35), 14420-14425 (2013).</li>
<li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+C.+elegans%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Maintenance of C. elegans.</a> WormBook. , (2006).</li>
<li>Tabara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rde-1+gene%2C+RNA+interference%2C+and+transposon+silencing+in+C.+elegans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The rde-1 gene, RNA interference, and transposon silencing in C. elegans.</a> Cell. 99 (2), 123-132 (1999).</li>
<li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse+genetics%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Reverse genetics.</a> WormBook. , (2006).</li>
<li>Rual, J. -F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+improving+Caenorhabditis+elegans+phenome+mapping+with+an+ORFeome-based+RNAi+library%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library.</a> Genome Research. 14 (10), 2162-2168 (2004).</li>
<li>Revtovich, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+characterization+of+high-throughput+Caenorhabditis+elegans+-+Enterococcus+faecium+infection+model%5BTitle%5D)+AND+%22Frontiers+in+Cellular+and+Infection+Microbiology%22%5BJournal%5D)">Development and characterization of high-throughput Caenorhabditis elegans - Enterococcus faecium infection model.</a> Frontiers in Cellular and Infection Microbiology. 11, 667327(2021).</li>
<li>Anderson, Q. L., Revtovich, A. V., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput%2C+high-content%2C+liquid-based+C.+elegans+pathosystem%5BTitle%5D)+AND+%22JoVE+%28Journal+of+Visualized+Experiments%22%5BJournal%5D)">A high-throughput, high-content, liquid-based C. elegans pathosystem.</a> JoVE (Journal of Visualized Experiments. (137), e58068(2018).</li>
<li>Scholz, M., Dinner, A. R., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stochastic+feeding+dynamics+arise+from+the+need+for+information+and+energy%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Stochastic feeding dynamics arise from the need for information and energy.</a> Proceedings of the National Academy of Sciences. 114 (35), 9261-9266 (2017).</li>
<li>Wu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pheromones+modulate+learning+by+regulating+the+balanced+signals+of+two+insulin-like+peptides%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Pheromones modulate learning by regulating the balanced signals of two insulin-like peptides.</a> Neuron. 104 (6), 1095-1109 (2019).</li>
<li>Ching, T. -T., Hsu, A. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solid+plate-based+dietary+restriction+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Solid plate-based dietary restriction in Caenorhabditis elegans.</a> Journal of Visualized Experiments: JoVE. (51), e2701(2011).</li>
<li>Walker, A. C., Bhargava, R., Vaziriyan-Sani, A. S., Brust, A. S., Czyz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+bacterial+loads+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Bio-protocol%22%5BJournal%5D)">Quantification of bacterial loads in Caenorhabditis elegans.</a> Bio-protocol. 12 (2), 4291-4291 (2022).</li>
<li>Manjarrez, J. R., Mailler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stress+and+timing+associated+with+Caenorhabditis+elegans+immobilization+methods%5BTitle%5D)+AND+%22Heliyon%22%5BJournal%5D)">Stress and timing associated with Caenorhabditis elegans immobilization methods.</a> Heliyon. 6 (7), 04263(2020).</li>
<li>Zhang, S., Banerjee, D., Kuhn, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+larval+cells+from+C.+elegans%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Isolation and culture of larval cells from C. elegans.</a> PLoS ONE. 6 (4), 0019505(2011).</li>
<li>Garsin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-lived+C.+elegans+daf-2+mutants+are+resistant+to+bacterial+pathogens%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens.</a> Science. 300 (5627), New York, N.Y. 1921(2003).</li>
<li>Thutupalli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Farming+and+public+goods+production+in+Caenorhabditis+elegans+populations%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Farming and public goods production in Caenorhabditis elegans populations.</a> Proceedings of the National Academy of Sciences. 114 (9), 2289-2294 (2017).</li>
<li>Ly, K., Reid, S. J., Snell, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+RNA+analysis+of+individual+Caenorhabditis+elegans%5BTitle%5D)+AND+%22MethodsX%22%5BJournal%5D)">Rapid RNA analysis of individual Caenorhabditis elegans.</a> MethodsX. 2, 59-63 (2015).</li>
<li>Johnke, J., Dirksen, P., Schulenburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Community+assembly+of+the+native+C.+elegans+microbiome+is+influenced+by+time%2C+substrate%2C+and+individual+bacterial+taxa%5BTitle%5D)+AND+%22Environmental+Microbiology%22%5BJournal%5D)">Community assembly of the native C. elegans microbiome is influenced by time, substrate, and individual bacterial taxa.</a> Environmental Microbiology. 22 (4), 1265-1279 (2020).</li>
<li>Vega, N. M., Gore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stochastic+assembly+produces+heterogeneous+communities+in+the+Caenorhabditis+elegans+intestine%5BTitle%5D)+AND+%22PLOS+Biology%22%5BJournal%5D)">Stochastic assembly produces heterogeneous communities in the Caenorhabditis elegans intestine.</a> PLOS Biology. 15 (3), 2000633(2017).</li>
<li>Gulyas, L., Powell, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cold+shock+induces+a+terminal+investment+reproductive+response+in+C.+elegans%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Cold shock induces a terminal investment reproductive response in C. elegans.</a> Scientific Reports. 12 (1), 1338(2022).</li>
<li>Jiang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetic+program+mediates+cold-warming+response+and+promotes+stress-induced+phenoptosis+in+C.+elegans%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans.</a> eLife. 7, 35037(2018).</li>
<li>Robinson, J. D., Powell, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+recovery+from+acute+cold+shock+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22BMC+Cell+Biology%22%5BJournal%5D)">Long-term recovery from acute cold shock in Caenorhabditis elegans.</a> BMC Cell Biology. 17 (1), 2(2016).</li>
</ol>

作者无利益冲突。

本文展示了在线虫 C. elegans 中进行单条线虫细菌载量定量的优势，并提供了一种96孔破碎方案，可快速、一致地获取大量此类数据。与现有方法33相比，该方案能够实现对线虫肠道微生物群落更高通量的检测。
该方法以平板接种为限速步骤，实际上并非&#34;高通量&#34;。大颗粒流式细胞术（图3C、D）是一种可用于定量单个线虫体内荧光标记细菌的有用高通量方法16，但在多物种群落中，同时检测的荧光染料数量受到限制。将多孔板裂解与群落测序相结合是提高通量的另一种方法；然而，本文所述的96孔板裂解程序经过专门优化，旨在保持细菌细胞完整。基于测序的分析需要充分裂解细胞，因此需额外增加核酸提取步骤，或对拍打程序（方案3.10–3.11）进行修改，以提取细胞内容物而非活菌。针对单条线虫裂解及核酸提取的实验方案已在其他文献中发表38,<...

宿主与微生物关联的异质性普遍存在，个体间的变异正日益被视为影响种群水平过程（从竞争与共存1到疾病传播2,3,4）的重要因素。在 C. elegans中，同基因型种群内反复观察到&#34;隐性异质性&#34;现象，表现为不同亚群个体在热激反应5,6、衰老和寿命7,8,9,10,11以及生理和发育的许多其他方面表现出明显不同的表型12。大多数试图识别亚群结构的分析均提供了实验证据，表明在同基因型、同步发育的线虫实验种群中存在两个亚群5,7,8，尽管其他数据提示种群内性状可能呈连续分布而非离散分组7,12,13。与此相关的是，即使在由共同微生物来源定植的同基因型线虫种群内部，其肠道微生物数量也表现出显著异质性13,14,15,16，而这种异质性可能被广泛用于线虫体内细菌定量的批量消化检测方法所掩盖17,18,19,20。
本研究提供的数据表明，在宿主-微生物互作研究中应更加依赖单条线虫的测量方法，同时本文还提供了提高单条线虫破碎准确性和通量的操作方案。这些方案旨在通过机械破碎大量单个 C. elegans 来定量可存活的细菌载量，相比使用研杵对单条线虫进行破碎的方法，本方案具有更高的重复性和更低的单位样本操作强度。方案中包含推荐的肠道清除步骤：在线虫破碎前使其摄食经热灭活的 E. coli，以尽量减少近期摄入及其他短暂性（非黏附性）细菌的干扰。本方案还包括一种冷诱导麻痹法，结合低浓度表面漂白处理以清洁线虫角质层；表面漂白既可作为单条线虫破碎前的预处理步骤，也可用于制备活体且体表无菌的线虫。该表面漂白方法足以去除多种体表微生物，而冷处理则可替代传统的左旋咪唑（levamisole）诱导麻痹；尽管在对低温敏感的实验中仍优先使用左旋咪唑，但冷麻痹法可减少有害废弃物的产生，并使线虫在处理后迅速恢复正常的活动能力。虽然完整方案描述的是用已知细菌定植线虫的实验室实验，但其中的线虫清洗和单条线虫破碎步骤也可直接应用于从野外样本中分离的线虫或在微宇宙实验中定植的线虫。本文所述方案可从线虫肠道中提取出活菌，适用于对单条线虫进行平板培养并定量菌落形成单位（CFUs）；若需进行基于测序的肠道菌群分析，则应在本方案基础上增加细胞裂解和核酸提取步骤。

<table><tbody><tr><td>96孔平底聚丙烯板，300 µL</td><td>Evergreen Labware</td><td>290-8350-03F</td><td></td></tr><tr><td>96孔板硅胶密封垫，方形孔（AxyMat）</td><td>Axygen</td><td>AM-2ML-SQ</td><td></td></tr><tr><td>96孔板，2 mL，方形孔</td><td>Axygen</td><td>P-2ML-SQ-C-S</td><td></td></tr><tr><td>96孔聚丙烯板盖</td><td>Evergreen Labware</td><td>290-8020-03L</td><td></td></tr><tr><td>琼脂</td><td>Fisher Scientific</td><td>443570050</td><td></td></tr><tr><td>用于96孔板的珠磨适配器套装</td><td>QIAGEN</td><td>119900</td><td>与TissueLyser II配合使用的适配板，可同时处理两个96孔板</td></tr><tr><td>珠磨组织匀浆仪（TissueLyser II）</td><td>QIAGEN</td><td>85300</td><td>适用于中高通量样本破碎的机械匀浆设备</td></tr><tr><td>BioSorter</td><td>Union Biometrica</td><td>By quotation</td><td>大型物体分选仪，配备250微米聚焦装置，用于&lt;em&gt;C. elegans&lt;/em&gt;</td></tr><tr><td>市售漂白剂，8.25%次氯酸钠</td><td>Clorox</td><td></td><td></td></tr><tr><td>96孔透气密封膜（Breathe-Easy）</td><td>Diversified Biotech</td><td>BEM-1</td><td>多孔板用透气型聚氨酯膜。薄膜可透过O₂、CO₂和水蒸气，且在波长低至300 nm时仍具有紫外透光性。无菌，每盒100片。</td></tr><tr><td>二水合氯化钙</td><td>Fisher Scientific</td><td>AC423525000</td><td></td></tr><tr><td>胆固醇</td><td>VWR</td><td>AAA11470-30</td><td></td></tr><tr><td>一水合柠檬酸</td><td>Fisher Scientific</td><td>AC124910010</td><td></td></tr><tr><td>五水合硫酸铜（II）</td><td>Fisher Scientific</td><td>AC197722500</td><td></td></tr><tr><td>Corning 6765 LSE迷你微量离心机</td><td>Corning</td><td>&amp;nbsp;COR-6765</td><td></td></tr><tr><td>乙二胺四乙酸二钠</td><td>Fisher Scientific</td><td>409971000</td><td></td></tr><tr><td>DL-1,4-二硫苏糖醇，纯度≥99%，分子生物学级，无DNA酶、RNA酶和蛋白酶，ACROS Organics</td><td>Fisher Scientific</td><td>327190010</td><td></td></tr><tr><td>Eppendorf 1.5 mL微量离心管，自然色</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Eppendorf 5424R微量离心机</td><td>Eppendorf</td><td>5406000640</td><td>24位制冷台式微量离心机</td></tr><tr><td>Eppendorf 5810R离心机，配S-4-104转子</td><td>Eppendorf</td><td>22627040</td><td>3L台式离心机，配有适用于15–50 mL管和板的适配器</td></tr><tr><td>Eppendorf板用吊篮（x2），适用于S-4-104转子</td><td>Eppendorf</td><td>22638930</td><td></td></tr><tr><td>乙醇，100%</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>玻璃珠，2.7 mm</td><td>Life Science Products</td><td>LS-79127</td><td></td></tr><tr><td>酸洗玻璃珠，425–600 &amp;micro;m</td><td>Sigma</td><td>G877-500G</td><td></td></tr><tr><td>玻璃铺板珠</td><td>VWR</td><td>76005-124</td><td></td></tr><tr><td>盐酸</td><td>VWR</td><td>BDH7204-1</td><td></td></tr><tr><td>七水合硫酸亚铁（II）</td><td>Fisher Scientific</td><td>423731000</td><td></td></tr><tr><td>Kimble Kontes颗粒研钵电动杵</td><td>DWK Life Sciences</td><td>749540-0000</td><td></td></tr><tr><td>Kimble Kontes聚丙烯颗粒研钵和0.5 mL微量管</td><td>DWK Life Sciences</td><td>749520-0590</td><td></td></tr><tr><td>Leica DMi8电动倒置显微镜，带电动载物台</td><td>Leica</td><td>11889113</td><td></td></tr><tr><td>Leica LAS X Premium软件</td><td>Leica</td><td>11640687</td><td></td></tr><tr><td>七水合硫酸镁</td><td>Fisher Scientific</td><td>AC124900010</td><td></td></tr><tr><td>四水合氯化锰（II）</td><td>VWR</td><td>470301-706</td><td></td></tr><tr><td>PARAFILM M柔性实验室密封膜</td><td>Amcor</td><td>PM996</td><td></td></tr><tr><td>蛋白胨</td><td>Fisher Scientific</td><td>BP1420-500</td><td></td></tr><tr><td>培养皿，圆形，10 cm</td><td>VWR</td><td>25384-094</td><td></td></tr><tr><td>培养皿，圆形，6 cm</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>培养皿，方形，10 × 10 cm</td><td>VWR</td><td>10799-140</td><td></td></tr><tr><td>磷酸盐缓冲液（1× PBS）</td><td>Gold Bio</td><td>P-271-200</td><td></td></tr><tr><td>聚丙烯灭菌托盘，浅型</td><td>Fisher Scientific</td><td>13-361-10</td><td></td></tr><tr><td>氢氧化钾</td><td>Fisher Scientific</td><td>AC134062500</td><td></td></tr><tr><td>磷酸氢二钾</td><td>Fisher Scientific</td><td>BP363-1</td><td></td></tr><tr><td>磷酸二氢钾</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>金属勺形实验室刮铲</td><td>VWR</td><td>470149-438</td><td></td></tr><tr><td>碳化硅，36目</td><td>MJR Tumblers</td><td>n/a</td><td>黑色超粗粒度碳化硅。该供应商提供0.5–5磅不同规格。</td></tr><tr><td>十二烷基硫酸钠</td><td>Fisher Scientific</td><td>BP166-100</td><td></td></tr><tr><td>氢氧化钠</td><td>VWR</td><td>BDH7247-1</td><td></td></tr><tr><td>无水磷酸氢二钠</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>氯化钠</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>蔗糖</td><td>Fisher Scientific</td><td>AC419760010</td><td></td></tr><tr><td>一水合三柠檬酸钾</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>七水合硫酸锌</td><td>Fisher Scientific</td><td>AC205982500</td><td></td></tr></tbody></table>

利用单条线虫数据量化异质性 秀丽隐杆线虫- 细菌相互作用

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

线虫Caenorhabditis elegans是研究宿主与微生物及宿主与微生物组相互作用的模式系统。迄今为止，许多研究采用批量消化而非单条线虫样本的方法来定量该生物体内的细菌载量。本文认为，C. elegans肠道中细菌定植所表现出的个体间高度变异性具有重要信息价值，而批量消化方法会丢失对不同条件下准确比较至关重要的信息。由于描述这些样本内在变异需要大量个体，本文建立了一种便捷的96孔板操作流程，用于单条线虫的裂解及菌落平板培养。

活体线虫的漂白灭菌 
表面漂白后的线虫在运动能力恢复和排泄重新开始前，其体外细菌基本被彻底清除。在本实验所用条件下，可观察到缓冲液中细菌的快速灭活。图1A-C，补充图2，视频1）而不干扰低温麻痹线虫中与肠道相关的细菌图1D-F，视频 2）。这些数据表明，表面漂白可有效对外部蠕虫进行消毒，同时不破坏其肠道内容物（表面漂白组与未漂白组的蠕虫相关菌落形成单位计数比较无显著差异，Wilcoxon 秩和检验 p &gt; 0.05).
多样本机械破碎方法的优化 
用于线虫机械破碎的96孔板技术对所用具体材料具有较强的耐受性，实际操作中的考虑因素决定了研磨材料的选择。与先前报道类似33，手动破碎（<strong cl...

本方案描述了一种在低温麻痹和表面漂白&#160;以去除外部细菌后，对单个细菌定植的Caenorhabditis elegans进行96孔板裂解的方法。所得悬液接种于琼脂平板上，可实现对大量个体线虫中细菌载量的准确、中等通量定量分析。

线虫秀丽隐杆线虫（Caenorhabditis elegans）是研究宿主与微生物及宿主与微生物组相互作用的模式系统。迄今为止，许多研究采用批量消化方法而非……

本方案展示了对秀丽隐杆线虫进行机械破碎的方法，采用96孔板格式，可对单个线虫中的细菌载量进行中等通量的定量分析。与基于Pasel的方法相比，该技术提高了机械破碎的速度和均一性，使研究人员能够轻松获得高质量的单虫测量数据集。该技术涉及多个动态步骤，容易遗漏操作环节。务必在开始前准备好所有材料、标记好的离心管、缓冲液和培养板。本次实验操作将由我实验室的研究专家负责人 Megan Taylor 进行示范。首先，在微量离心管中用一毫升 M 九 TX 零一重新悬浮线虫样品。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 缓冲液清洗线虫两次，再用一毫升 M9 线虫缓冲液清洗一次，以尽量减少外部细菌。然后，将每个样品重悬于含有两倍量热灭活 OP50 的一毫升 S 培养基中，置于培养管内。将培养管在 25°C 下孵育 20 至 30 分钟，以排出肠道中未黏附的细菌。孵育后，用一毫升冰冷的M9TX01缓冲液将虫体清洗两次，并弃去上清液。将离心管置于冰上10分钟，使线虫麻痹。然后向每根离心管中加入一毫升含无香型漂白剂的冰冻M九线虫缓冲液。将离心管置于冰上孵育至少10分钟，以杀死外部细菌。弃去次氯酸钠缓冲液，并将离心管放回冰上，防止线虫蠕动。随后，向每根离心管中加入1毫升冰冷的M9TX01，短暂离心5秒。在微型离心机中离心。将离心管放回冰上，弃去上清液。重复此步骤一次。用于线虫角质层的化学通透化。将含有线虫的试管（管中缓冲液体积为20微升）转移至室温下的试管架中。然后向每份温热的样品中加入100微升SDS/DTT溶液。将试管在台面上孵育最多八分钟，以部分降解成虫的角质层。此时，线虫将死亡并沉降至试管底部。孵育结束后，小心弃去 SDS/DTT 上清液。然后，向每根离心管中加入 1 毫升 M9TX01，短暂离心以沉淀线虫。弃去上清液，并将线虫重悬于 1 毫升含有 0.1% Triton X-100 的 M9 线虫缓冲液中。为进行线虫的机械破碎，准备一个无菌的 2 毫升深孔 96 孔板和一个硅胶板盖。使用无菌药匙将少量无菌的36目碳化硅颗粒加入每个孔中，使颗粒恰好覆盖孔底。向每孔加入180 µL M9线虫缓冲液。标记列和行，然后用硅胶板盖轻轻覆盖酶标板。接下来，将已透化的线虫转移至一个盛有M9TX01的小培养皿中，液体深度约为1厘米。在解剖显微镜下，用移液器吸取单个线虫，每份20微升，转移至96孔板的各个孔中。为排出可能粘附在移液器尖端的线虫，需从培养皿中无虫区域吸取20微升M9TX01，再将其释放回培养皿中。待所有线虫转移完毕后，用一张柔性的封板膜覆盖96孔板，确保有纸背衬的一面朝下，紧密贴合样品孔。将硅胶封板垫轻轻放置在柔性封板膜上方，不要将盖子压入孔中。将板置于4°C环境中冷却30至60分钟，以防止在裂解过程中过热。冷却后，用力将硅胶封板垫压入各孔中以密封。然后将培养板固定在组织破碎仪中。以30赫兹的频率振荡培养板1分钟。随后将培养板旋转180度，再次振荡1分钟。将培养板在实验台面上用力敲击两到三次，以震落柔性封板膜上的任何颗粒。将整板在2,400 ×g条件下离心2分钟，使样品完全沉降至孔底。随后取下硅胶盖，撕去封板膜。为制备线虫消化液的十倍系列稀释液，向96孔板的B至D行各孔中加入180微升1×PBS。使用设定为200微升的多通道移液器，通过反复吸打缓慢混匀线虫消化液。将尽可能多的液体转移至含有PBS的96孔板A行各孔中。使用多通道移液器，从最上方一行吸取 20 微升液体，加入到 B 行中，随后进行混匀。重复此步骤，将 B 行液体转移至 C 行，再将 C 行液体转移至 D 行，以获得 1000 倍稀释液，用于单菌定植线虫的细菌定量。取各稀释液 10 至 20 微升，涂布于固体琼脂平板上。对于多物种定植，将每种稀释液各取 100 微升接种于 10 厘米的琼脂平板上。采用本方案，在对冷麻痹的线虫进行表面漂白后，缓冲液中外部细菌数量减少，表明实现了有效的外部消毒，且未影响与肠道相关的细菌。人工破碎线虫导致更高的异质性。无论是否使用Tri Andex，碳化硅颗粒均是破碎的理想选择。在96孔板方法中，大尺寸玻璃珠不适用于96孔板技术。小玻璃珠产生的结果较为一致，但会堵塞尖端管道。在观察单一种类细菌和多物种细菌群落时，定植于相同细菌池中的线虫肠道载量表现出高度变异。定植了表达GFP细菌的线虫，在个体线虫的测量中显示出异质性。对于表达GFP的这种细菌菌株，比较了野生型Bristol Len两型蠕虫与DAF两型IGF突变体的定植情况。结果显示，DAF 16突变体支持更多的细菌群体，而DAF两型对定植具有抗性。这种异质性在同一实验的不同重复中均表现出典型且一致的特征。对数据进行重采样以模拟批次消化，结果发现与单条线虫数据相比，其分布发生了改变。通过批次外推得到的每条线虫CFU集中在个体数据的算术平均值附近，导致生物变异性的丢失。与其进行消化，不如使用经表面漂白和冲洗后的活线虫进行后续实验。一旦将线虫从低温环境中移除，其运动将迅速恢复。

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生物学

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

来源： Taylor, M. N., 等。 利用单条线虫数据量化异质性 秀丽隐杆线虫- 细菌相互作用 视频实验杂志 (2022)本视频展示了将无菌且年龄匹配的Caenorhabditis...

评估无菌 C. elegans 中的肠道细菌定植

来源： Taylor, M. N. 等 利用单条线虫数据量化异质性 秀丽隐杆线虫- 细菌相互作用 视频实验杂志 (2022)本视频展示了通过机械裂解法对单个秀丽隐杆线虫（Caenorhabditis...

机械破碎单条线虫以评估宿主-细菌相互作用的变异性

蠕虫分选仪通过根据荧光报告基因的表达对秀丽隐杆线虫（Caenorhabditis...

由细菌病原体不同程度定植的 C. elegans 的自动化分离

本文中，我们介绍线虫Caenorhabditis...

秀丽隐杆线虫——一种多功能的模式生物 体内 研究宿主-微生物相互作用的模型

本文介绍了一种高通量、快速且可靠的基因表达水平检测方法，适用于单个或批量的 C. elegans...

利用纳米流体技术在单个及批量 C. elegans 样本中进行高通量定量 RT-PCR

为了研究分子伴侣与分子伴侣之间以及分子伴侣与底物之间的相互作用，我们在秀丽隐杆线虫（Caenorhabditis...

利用 Caenorhabditis elegans 筛选组织特异性分子伴侣相互作用

肠道微生物可能对宿主的健康产生正面或负面的影响 通过 特异性或保守性机制 秀丽隐杆线虫 是一种用于筛选此类微生物的便捷平台。本实验方案描述了对48种细菌分离株进行高通量筛选，以评估其对线虫应激抗性的影响，该指标被用作线虫健康状况的替代参数。...

高通量筛选影响微生物分离物 秀丽隐杆线虫 健康

Caenorhabditis elegans 是生物学中的主要模式物种之一，然而几乎所有研究都是在缺乏其天然共生微生物的情况下进行的。本文所述方法将有助于增进我们对这些共生微生物多样性的理解，为未来开展功能性的 C. elegans...

模式线虫天然微生物群的分离与表征 秀丽隐杆线虫

堆肥微宇宙实验将自然界中存在的微生物多样性引入实验室，以促进对秀丽隐杆线虫（Caenorhabditis...

堆肥微宇宙作为微生物组研究中微生物多样性高且接近自然环境的实验体系 秀丽隐杆线虫

Caenorhabditis elegans 是研究驱动宿主-微生物组相互作用的分子决定因素的有力模型。我们提出了一种高通量分析流程，可在单个动物水平上同时表征肠道微生物组定植情况以及 C. elegans...

秀丽隐杆线虫肠道微生物组定量与分析的流式蠕虫检测技术应用

了解利用96孔板机械破碎技术定量分析单个秀丽隐杆线虫细菌负荷的中通量方法。该技术通过冷麻痹和表面漂白去除外部细菌，同时保留肠道相关菌群，为微生物定植异质性研究提供可靠数据。

利用单条线虫数据量化异质性秀丽隐杆线虫- 细菌相互作用

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利用单条线虫数据量化异质性 秀丽隐杆线虫- 细菌相互作用

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利用单条线虫数据量化异质性秀丽隐杆线虫- 细菌相互作用