저자는 사용이 프로토콜과 도구의 발전에 기여 Eugenio Vivas, 커트 Heungens, 에릭 Martens, 찰스 Cowles, 다비 설탕, 에릭 Stabb, 그리고 토드 Ciche 감사하고 싶습니다. KEM 및 JMC는 건강의 국립 연구소 (NIH) 국립 연구 서비스 상 T32 (AI55397 &quot;건강과 질병에 미생물&quot;)에 의해 지원되었다. JMC는 국립 과학 재단 (National Science Foundation) (NSF) 대학원의 연구 활동에 의해 지원되었다. 이 작품은 국립 과학 재단 (National Science Foundation) (IOS-0920631 및 IOS-0950873)의 보조금에 의해 지원되었다.

<ol>
	<li>Holterman, M., van der Wurff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylum-wide+analysis+of+SSU+rDNA+reveals+deep+phylogenetic+relationships+among+nematodes+and+accelerated+evolution+toward+crown+clades.">Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades.</a> Mol. Biol. Evol. 23, 1792-1800 (2006).</li><li>Lambshead, P. J. D., Boucher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+nematode+deep-sea+biodiversity+-+hyperdiverse+or+hype.">Marine nematode deep-sea biodiversity - hyperdiverse or hype.</a> J. Biogeogr. 30, 475-485 (2003).</li><li>Poinar, G. O. J., Thomas, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+Achromobacter+nematophilus+Poinar+and+Thomas+(Achromobacteraceae:+Eubacteriales)+in+the+development+of+the+nematode,+DD-136+(Neoaplectana+sp.+Steinernematidae).">Significance of Achromobacter nematophilus Poinar and Thomas (Achromobacteraceae: Eubacteriales) in the development of the nematode, DD-136 (Neoaplectana sp. Steinernematidae).</a> Parasitol. 56, 385-390 (1966).</li><li>Herbert, E. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friend+and+foe:+the+two+faces+of+Xenorhabdus+nematophila.">Friend and foe: the two faces of Xenorhabdus nematophila.</a> Nat. Rev. Microbiol. 5, 634-646 (2007).</li><li>Kaya, H. K., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+nematodes.">Entomopathogenic nematodes.</a> Annu. Rev. Entomol. 38, 181-206 (1993).</li><li>Bird, A. F., Akhurst, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+the+intestinal+vesicle+in+nematodes+of+the+family+Steinernematidae.">The nature of the intestinal vesicle in nematodes of the family Steinernematidae.</a> Int. J. Parasitol. 13, 599-606 (1983).</li><li>Martens, E. C., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Steinernema+carpocapsae+intestinal+vesicle+contains+a+subcellular+structure+with+which+Xenorhabdus+nematophila+associates+during+colonization+initiation.">The Steinernema carpocapsae intestinal vesicle contains a subcellular structure with which Xenorhabdus nematophila associates during colonization initiation.</a> Cell. Microbiol. 7, 1723-1735 (2005).</li><li>Snyder, H. A., Stock, S. P., Kim, S. K., Flores-Lara, Y., Forst, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+colonization+and+release+process+of+Xenorhabdus+nematophila+and+the+morphology+and+ultrastructure+of+the+bacterial+receptacle+of+its+nematode+host,+Steinernema+carpocapsae.">New insights into the colonization and release process of Xenorhabdus nematophila and the morphology and ultrastructure of the bacterial receptacle of its nematode host, Steinernema carpocapsae.</a> Appl. Environ. Microbiol. 73, 5338-5346 (2007).</li><li>Abebe, E., Abebe-Akele, F., Morrison, J., Cooper, V., Thomas, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insect+pathogenic+symbiosis+between+a+Caenorhabditis+and+Serratia.">An insect pathogenic symbiosis between a Caenorhabditis and Serratia.</a> Virulence. 2, 158-161 (2011).</li><li>Abebe, E., Jumba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+entomopathogenic+Caenorhabditis+briggsae.">An entomopathogenic Caenorhabditis briggsae.</a> J. Exp. Biol. 213, 3223-3229 (2010).</li><li>Torres-Barragan, A., Suazo, A., Buhler, W. G., Cardoza, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+entomopathogenicity+and+bacterial+associates+of+the+nematode+Oscheius+carolinensis.">Studies on the entomopathogenicity and bacterial associates of the nematode Oscheius carolinensis.</a> Biol. Control. 59, 123-129 (2011).</li><li>Ye, W. M., Torres-Barragan, A., Cardoza, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscheius+carolinensis+n.+sp+(Nematoda:+Rhabditidae),+a+potential+entomopathogenic+nematode+from+vermicompost.">Oscheius carolinensis n. sp (Nematoda: Rhabditidae), a potential entomopathogenic nematode from vermicompost.</a> Nematology. 12, 121-135 (2010).</li><li>Zhang, C., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterorhabditidoides+chongmingensis+gen.+nov.,+sp.+nov.+(Rhabditida:+Rhabditidae),+a+novel+member+of+the+entomopathogenic+nematodes.">Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes.</a> J. Invertebr. Pathol. 98, 153-168 (2008).</li><li>Martens, E. C., Heungens, K., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+colonization+events+in+the+mutualistic+association+between+Steinernema+carpocapsae+nematodes+and+Xenorhabdus+nematophila+bacteria.">Early colonization events in the mutualistic association between Steinernema carpocapsae nematodes and Xenorhabdus nematophila bacteria.</a> J. Bacteriol. 185, 3147-3154 (2003).</li><li>Ciche, T. A., Ensign, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+the+insect+pathogen,+Photorhabdus+luminescens,+which+end+of+a+nematode+is+out.">For the insect pathogen, Photorhabdus luminescens, which end of a nematode is out.</a> Appl. Environ. Microbiol. 69, 1890-1897 (2003).</li><li>Ciche, T. A., Kim, K. S., Kaufmann-Daszczuk, B., Nguyen, K. C., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+invasion+and+matricide+during+Photorhabdus+luminescens+transmission+by+Heterorhabditis+bacteriophora+nematodes.">Cell invasion and matricide during Photorhabdus luminescens transmission by Heterorhabditis bacteriophora nematodes.</a> Appl. Environ. Microbiol. 74, 2275-2287 (2008).</li><li>Easom, C. A., Joyce, S. A., Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+involved+in+the+mutualistic+colonization+of+the+nematode+Heterorhabditis+bacteriophora+by+the+bacterium+Photorhabdus+luminescens.">Identification of genes involved in the mutualistic colonization of the nematode Heterorhabditis bacteriophora by the bacterium Photorhabdus luminescens.</a> BMC Microbiol. 10, 45 (2010).</li><li>Somvanshi, V. S., Kaufmann-Daszczuk, B., Kim, K. S., Mallon, S., Ciche, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photorhabdus+phase+variants+express+a+novel+fimbrial+locus,+mad,+essential+for+symbiosis.">Photorhabdus phase variants express a novel fimbrial locus, mad, essential for symbiosis.</a> Mol. Microbiol. 77, 1021-1038 (2010).</li><li>Martens, E. C., Russell, F. M., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Xenorhabdus+nematophila+metabolic+mutants+yields+insight+into+stages+of+Steinernema+carpocapsae+nematode+intestinal+colonization.">Analysis of Xenorhabdus nematophila metabolic mutants yields insight into stages of Steinernema carpocapsae nematode intestinal colonization.</a> Mol. Microbiol. 51, 28-45 (2005).</li><li>Sugar, D. R., Murfin, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variation+and+host+interactions+of+Xenorhabdus+bovienii+SS-2004,+the+entomopathogenic+symbiont+of+Steinernema+jollieti+nematodes.">Phenotypic variation and host interactions of Xenorhabdus bovienii SS-2004, the entomopathogenic symbiont of Steinernema jollieti nematodes.</a> Env. Microbiol. 14, 924-939 (2012).</li><li>Cowles, C. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenorhabdus+nematophila+nilABC+genes+confer+the+ability+of+Xenorhabdus+spp.+to+colonize+Steinernema+carpocapsae+nematodes.">The Xenorhabdus nematophila nilABC genes confer the ability of Xenorhabdus spp. to colonize Steinernema carpocapsae nematodes.</a> J. Bacteriol. 190, 4121-4128 (2008).</li><li>Heungens, K., Cowles, C. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Xenorhabdus+nematophila+genes+required+for+mutualistic+colonization+of+Steinernema+carpocapsae+nematodes.">Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes.</a> Mol. Microbiol. 45, 1337-1353 (2002).</li><li>Goetsch, M., Owen, H., Goldman, B., Forst, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+PixA+inclusion+body+protein+of+Xenorhabdus+nematophila.">Analysis of the PixA inclusion body protein of Xenorhabdus nematophila.</a> J. Bacteriol. 188, 2706-2710 (2006).</li><li>Bhasin, A., Chaston, J. M., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+Analyses+Reveal+Overall+Topology+and+Functional+Regions+of+NilB,+a+Bacterial+Outer+Membrane+Protein+Required+for+Host+Association+in+a+Model+of+Animal-Microbe+Mutualism.">Mutational Analyses Reveal Overall Topology and Functional Regions of NilB, a Bacterial Outer Membrane Protein Required for Host Association in a Model of Animal-Microbe Mutualism.</a> J. Bacteriol. 194, 1763-1776 (2012).</li><li>Ciche, T. A., Goffredi, S. K., Reddy, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in General and Molecular Microbiology. , 394-419 (2007).</li><li>Goodrich-Blair, H., Clarke, D., Grewal, P. S., Ciche, T. A., Stock, S. P., Boemare, N., Vandenberg, J., Glazar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Pathogens, Molecular Approaches and Techniques. , 239-269 (2009).</li><li>Stock, S. P., Goodrich-Blair, H., Lacey, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Techniques in Invertebrate Pathology. , 375-425 (2012).</li><li>Martens, E. C., Gawronski-Salerno, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenorhabdus+nematophila+requires+an+intact+iscRSUA-hscBA-fdx+locus+to+colonize+Steinernema+carpocapsae+nematodes.">Xenorhabdus nematophila requires an intact iscRSUA-hscBA-fdx locus to colonize Steinernema carpocapsae nematodes.</a> J. Bacteriol. 185, 3678-3682 (2003).</li><li>Vivas, E. I., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenorhabdus+nematophilus+as+a+model+for+host-bacterium+interactions:+rpoS+is+necessary+for+mutualism+with+nematodes.">Xenorhabdus nematophilus as a model for host-bacterium interactions: rpoS is necessary for mutualism with nematodes.</a> J. Bacteriol. 183, 4687-4693 (2001).</li><li>White, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+obtaining+infective+nematode+larvae+from+cultures.">A method for obtaining infective nematode larvae from cultures.</a> Science. 66, 302-303 (1927).</li><li>Ciche, T. A., Ensign, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+the+insect+pathogen+Photorhabdus+luminescens,+which+end+of+a+nematode+is+out.">For the insect pathogen Photorhabdus luminescens, which end of a nematode is out.</a> Appl. Environ. Microbiol. 69, 1890-1897 (2003).</li><li>Sicard, M., Brun, N. L. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+native+Xenorhabdus+on+the+fitness+of+their+Steinernema+hosts:+contrasting+types+of+interaction.">Effect of native Xenorhabdus on the fitness of their Steinernema hosts: contrasting types of interaction.</a> Parasitol. Res. 91, 520-524 (2003).</li><li>Lambertsen, L., Sternberg, C., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn7+transposons+for+site-specific+tagging+of+bacteria+with+fluorescent+proteins.">Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins.</a> Environ. Microbiol. 6, 726-732 (2004).</li><li>Miller, W. G., Leveau, J. H., Lindow, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+gfp+and+inaZ+broad-host-range+promoter-probe+vectors.">Improved gfp and inaZ broad-host-range promoter-probe vectors.</a> Mol. Plant Microbe Int. 13, 1243-1250 (2000).</li><li>Teal, T. K., Lies, D. P., Wold, B. J., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiometabolic+stratification+of+Shewanella+oneidensis+biofilms.">Spatiometabolic stratification of Shewanella oneidensis biofilms.</a> Appl. Environ. Microbiol. 72, 7324-7330 (2006).</li><li>Bao, Y., Lies, D. P., Fu, H., Roberts, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+Tn7-based+system+for+the+single-copy+insertion+of+cloned+genes+into+chromosomes+of+Gram-negative+bacteria.">An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of Gram-negative bacteria.</a> Gene. 109, 167-168 (1991).</li><li>Woodring, J. L., Kaya, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steinernematid+and+Heterorhabditid+Nematodes:+A+Handbook+of+Biology+and+Techniques.">Steinernematid and Heterorhabditid Nematodes: A Handbook of Biology and Techniques.</a> Southern Cooperative Series Bulletin 331. , (1988).</li></ol>

관심 없음 충돌이 선언 없습니다.

여기에 설명 된 프로토콜은 선충류 호스트 (그림 1) 내 세균의 광학 감지하는 방법을 제공합니다. 이 방법은 선충류 호스트 (그림 3)에서 박테리아의 생체 분석 있도록 nematodes의 광학 투명성과 휘황 라벨 박테리아 할 수있는 능력을 활용합니다. 특히,이 방법은 호스트 내에서 세균 현지화를 식별합니다. 박테리아의 존재에 대한 선충류 인구 및 점수를 계산함으로써,...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 지질 한천 (무균) </td><td></td><td></td><td> 8g 영양 국물, 15g의 한천, 5g 효모 추출물, 890 ML 물 10 ML 0.2 g / ML MgCl 2. 6H 2 0, 96 ML 옥수수 시럽 솔루션 * 4 ML 옥수수 기름 * 번호판을 붓는 동안 미디어를 저어 * autoclaving 후 살균 성분을 추가 </td></tr><tr><td> 옥수수 시럽 솔루션 (무균) </td><td></td><td></td><td> 7 ML 옥수수 시럽, 89 ML 물 믹스와 압력솥 </td></tr><tr><td> 달걀 솔루션 </td><td></td><td></td><td> 16.6 ML 12 % 차아 염소산 나트륨, 5 ML 5M 코, 80 ML 물 </td></tr><tr><td> Lysogeny 국물 (무균) </td><td></td><td></td><td> 5g 효모 추출물, 10g의 tryptone, 5g 소금, 1 L의 물 믹스와 압력솥 </td></tr><tr><td> Microfuge </td><td> 어부 </td><td> 13-100-675 </td><td> microfuge 튜브를 가지고 모든 microfuge는 작동합니다 </td></tr><tr><td> 원심 분리기 </td><td> Beckman </td><td> 366802 </td><td> 15 ML 50 ML 원뿔 튜브를 가지고 대형 테이블 탑 원심 분리기 </td></tr><tr><td> 멸균 60mm X 15mm 페트리 접시 </td><td> 어부 </td><td> 0875713 </td><td></td></tr><tr><td> 50 ML의 원심 분리기 튜브 </td><td> 어부 </td><td> 05-539-6 </td><td></td></tr><tr><td> 15 ML의 원심 분리기 튜브 </td><td> 어부 </td><td> 05-531-6 </td><td></td></tr><tr><td> 멸균 100mm X 20mm 페트리 접시 </td><td> 어부 </td><td> 0875711Z </td><td> 표준 배양 접시보다 더 </td></tr><tr><td> 24 잘 판 </td><td> Greiner 바이오 한 &lt;/ TD&gt; <td> 662000-06 </td><td></td></tr><tr><td> 현미경 </td><td></td><td></td><td> 현미경은 형광과 florescent 기능이 호환 필요 </td></tr><tr><td> Paraformaldehyde </td><td> 전자 현미경 과학 </td><td> 15,710 </td><td></td></tr><tr><td> PBS (무균) </td><td></td><td></td><td> 8g NaCL 0.2 g KCL 1.44 g 그렇단 2 HPO 4 0.24 g KH 2 PO 4 1 L의 물 7.4와 물 1 L과 압력솥의 산도에 조정 </td></tr><tr><td> Microfuge 관 </td><td> 어부 </td><td> 05-408-138 </td><td> 2 ML 1.5 ML 튜브 </td></tr><tr><td> 셰이커 </td><td></td><td></td><td> 액체가 부드럽게 이동하는 원인이 모든 흔드는는 작동합니다 </td></tr><tr><td> Diaminopimelic 산 </td><td> 시그마 </td><td&gt; D-1377 </td><td> 필요한 경우 농도 또는 1 mm까지 미디어를 보완 </td></tr></tbody></table>

visualizing bacteria in nematodes using fluorescent microscopy

Symbioses은 두 개 이상의 생물의 함께 사는 삶의 모든 왕국 전역에 널리 퍼져 있습니다. 지구상에서 가장 유비쿼터스 생물의 두 바와 같이, nematodes와 세균은 다양한 종류의 유익 1-3 병원성에 공생 협회의 다양한 배열을 형성하고 있습니다. 하나의 협회는 Xenorhabdus 박테리아와 공생 4의 모델 시스템으로 떠오르고있다 Steinernema nematodes, 사이의 상호 이익 관계입니다. Steinernema의 nematodes은 곤충에게 5 죽이기 위해 세균 symbiont을 사용하여 entomopathogenic 있습니다. 곤충 호스트 사이의 전송을 위해, 박테리아가 선충류의 infective의 청소년 단계 6-8의 장을 식민지화. 최근 몇 가지 다른 선충류 종은 곤충에게 9-13를 죽이고 박테리아를 활용하는 표시되었으며, 조사는 이러한 시스템에 nematodes와 박테리아 사이의 상호 작용을 조사하기 시작했습니다 &lt;&gt; 9 논의하게 될 것입니다. 우리는 현미경으로 볼 nematodes의 광학 투명성을 활용 이내 또는 선충류 호스트에 세균 symbiont의 시각화하기위한 방법을 설명합니다. 박테리아는 형광 현미경하여 시각화를 허용하는 형광 단백질을 표현하는 설계되었습니다. 많은 plasmids는 각각 다른 파장 (예 : 녹색 또는 빨간색), 그리고받는 세균 symbiont에 기증 대장균의 변종에서 plasmids의 활용에 형광 단백질을 인코딩 유전자가 박테리아의 광범위한 성공적으로 수행하는 사용할 수 있습니다. 설명 방법은 Steinernema carpocapsae와 Xenorhabdus nematophila 14 층 사이의 연결을 조사하기 위해 개발되었습니다. 비슷한 방법은 다른 선충류 - 세균 협회 9, 15-18을 조사하는 데 사용하고 접근 방식은 따라서 일반적으로 적용되었습니다. 메트로폴리스석탄 통은 협회와 식민지 14, 16, 19 과정의 성격에 통찰력을 제공, 개발의 여러 단계에서 nematodes에서 박테리아의 존재 및 현지화의 특성 수 있습니다. 현미경 분석은 조직 14, 16, 19-21를 호스팅하는 박테리아의 인구 및 현지화에서 식민지 주파수를 모두 보여줍니다. 여기에는 sonication 22 또는 식민지의 평균 수준을 제공 할 수있는 23, 연삭 등의 선충류의 인구, 내 세균을 모니터링하는 다른 방법에 비해 장점이지만, 예를 들어,있는 인구에서 낮은 symbiont로드의 높은 빈도로 인구 차별하지 않을 수 있습니다 높은 symbiont로드의 낮은 주파수. 식민지의 phenotypes 21 세균 돌연변이를 검사하거나 특성화 때 주파수 및 colonizing 박테리아의 부하를 차별하는 것은 특히 중요 할 수 있습니다24. 사실, 형광 현미경은 식민지 17, 18의 결함에 대한 세균의 돌연변이의 높은 처리량 검사에 사용하고, sonication 22, 25-27 및 개인 선충류의 해부 28, 29 등의 다른 방법보다 덜 힘든 것입니다되었습니다.

Watch this Scientific Journal Video about Visualizing Bacteria in Nematodes using Fluorescent Microscopy at JoVE.com

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

사이의 mutualism을 공부하려면<em&gt; Xenorhabdus</em&gt; 박테리아와<em&gt; Steine​​rnema</em&gt; nematodes, 방법은 세균의 존재와 nematodes 내 위치를 모니터링하기 위해 개발되었습니다. 다른 시스템에 적용 할 수있는 실험 방법은, 엔지니어링 박테리아가 투명 선충류에서 형광 현미경 박테리아를 사용하여 녹색 형광 단백질과 시각화를 표현하는 수반.

형광 현미경을 사용하여 Nematodes에 박테리아를 머릿속으로

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

visualizing-bacteria-in-nematodes-using-fluorescent-microscopy

Research

JoVE Journal

Biology

18.6K 조회수.  University of Wisconsin-Madison. 사이의 mutualism을 공부하려면 Xenorhabdus 박테리아와 Steine​​rnema nematodes, 방법은 세균의 존재와 nematodes 내 위치를 모니터링하기 위해 개발되었습니다. 다른 시스템에 적용 할 수있는 실험 방법은, 엔지니어링 박테리아가 투명 선충류에서 형광 현미경 박테리아를 사용하여 녹색 형광 단백질과 시각화를 표현하는 수반.

비디오: Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

라이브를 떠올리 Drosophila</em형광 염료와 함께> Glial - 신경근육학 융티온

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

연구

생물학

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

단일 분자 단지를 떠올리 VIVO에서 고급 형광 현미경을 사용하여

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

형광 현미경과 함께 머릿속으로 단일 분자의 DNA 복제

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

형광 단백질을 표현 라이브 마우스의 이미징 세포 이하의 구조물의 intravital 현미경

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

고속 3D 구조 조명 현미경을 사용하여 라이브 박테리아의 Cytokinetic Z 반지의 슈퍼 해상도 이미징 (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

녹색 형광 단백질 박테리아에서 감염 하는 동안 전달 하는 이펙터를 시각화 하기 위해 시스템을 분하시오

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

전 생체 내 공초점 현미경을 사용하여 림프절 구조 및 세포 국소화 시각화

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.