Questo lavoro è stato sostenuto da un nuovo investigatore di Grant [2014R1A1A1005553] dalla Fondazione Nazionale delle Ricerche di Corea (NRF) per JIL; e Università di Yonsei Future Leader sovvenzione sfida [2015-22-0133] per JIL

1. Preparare Soluzioni per NGT-3D e NGB-3D <ol><li> Preparare le seguenti soluzioni sterili: 1 L di soluzione 0,1454 M di NaCl, 1 L di 1 M CaCl 2, 1 L di 1 M MgSO 4, lisogenia Broth (LB), 1 L di 1 M KPO 4 tampone (108,3 g di KH 2 PO 4 e 35,6 g di K 2 HPO 4, e riempire H 2 O a 1 L). Produzione di NGM utilizzando queste soluzioni può essere trovato in un precedente protocollo 10. </li><li> Le soluzioni autoclave a 121 ° C, 15 min. </li><li> Preparare 50 ml di 5 mg / ml soluzione colesterolo sterile. In una provetta conica da 50 ml, mescolare 0,25 g di colesterolo e 50 ml di etanolo 99,99% e mescolare bene. Non sterilizzare in autoclave. Sterilizzare la soluzione di colesterolo utilizzando una siringa da 50 ml con un filtro a siringa da 0,45 micron. Questo sarà anche eliminare ogni colesterolo non disciolto. </li><li> (Opzionale) Preparare 10 ml di una 150 mm di 2&#39;-desossi-5-fluorouridina (FUDR) stock. In un tubo conico da 15 ml, mescolare 0,3693 gdi FUDR e 10 ml di acqua distillata. Agitare bene. </li></ol> 2. Preparare Coltura dei batteri per NGT-3D e NGB-3D <ol><li> Inoculare 10 ml di brodo LB con i batteri. I batteri standard utilizzati per la C. elegans alimentazione è ceppo di E. coli OP50. </li><li> Cultura l&#39;inoculazione batterica a 37 ° C in un incubatore agitazione durante la notte. </li><li> In preparazione per una diluizione seriale, aliquote 9 ml di soluzione NaCl in sterili 15 ml provette coniche. Ad esempio, aliquota 9 ml in un totale di 7 tubi per fare una diluizione 10 -7 del ceppo OP50 E. coli. </li><li> Usando una pipetta sterile, 1.000 ml, pipetta 1 ml di coltura batterica o coltura batterica diluito nella nuova provetta sterile con 9 ml di soluzione di NaCl e vortice bene il composto 10 ml. Ripetere fino a raggiungere la diluizione batterica desiderato. NOTA: La diluizione batterica desiderata dipende dal tipo e dalle condizioni di batteri e le condizioni sperimentali esatti.Ad esempio, a 10 -6 - diluizione del ceppo OP50 E. coli 10 -8 era sufficiente a produrre da 1 - 200 colonie batteriche per 6,5 ml tubo NGT-3D. Worms affidabile sviluppati e riprodotti normalmente quando il numero delle colonie era finita 60, avvenuta ad una diluizione di 10 -6 - 10 -7. Per NGB-3D, usare una diluizione di 10 -8. </li></ol> 3. Fare NGT-3D e NGB-3D (200 ml) <ol><li> Dopo preparazione della coltura batterica, miscela 0,6 g di NaCl, 1 g granulato agar, e 0,5 g di peptone in un pallone da 500 ml. Inserire una ancoretta magnetica nel pallone. </li><li> Aggiungere 195 ml di acqua distillata e coprire la bocca del pallone con un foglio di alluminio. </li><li> Autoclave 121 ° C per 15 min. </li><li> Porre il pallone in autoclave a caldo su un piatto mescolare, e mescolare a velocità moderata per almeno 2 ore. Raffreddare il pallone a 40 ° C. Assicurati di raffreddare sufficientemente come si continua da qui a temperature elevate può portare a opacità del agar finito.Tuttavia, temperature più basse possono provocare il rapido indurimento di agar. <ol><li> (Facoltativo) Per accelerare il processo di raffreddamento, mettere il pallone in un 40 ° C bagnomaria 15 minuti prima di mettere su un piatto mescolare. </li></ol></li><li> Quando la temperatura del supporto agar raggiunge 40 ° C, aggiungere 200 ml 1 M CaCl 2, 200 ml di 5 mg / soluzione colesterolo ml, 200 ml 1 M MgSO 4 e 5 ml 1 M KPO 4 buffer come la soluzione continua a mescolare a concentrazioni finali di 1 mM CaCl 2, 5 ug / ml di colesterolo, 1 mM MgSO 4, 1 mM KPO 4. <ol><li> (Opzionale) per test di durata della vita NGT-3D aggiungere 80 ml di 150 mm FUDR ad una concentrazione finale di 120 micron 12. </li></ol></li><li> Aggiungere 6 ml di 10 ml diluiti coltura batterica dal punto 2.4 nel pallone direttamente. <ol><li> (Opzionale) per NGT-3D, rimuovere le 6 ml di terreni di coltura prima del punto 3.6 e tenere in caldo separatamente. Questo supporto sarà utilizzato per i batteri senzastrato superiore. </li></ol></li><li> Utilizzando le procedure sterili, erogare i media in una camera di cultura sterile. Assicurarsi che il coperchio della camera si chiude ermeticamente. <ol><li> (Opzionale) Per evitare colonie batteriche formazione sulla superficie superiore della agar, fare uno strato di priva di batteri agar dal punto 3.6.1 sulla sommità prima che il supporto dal punto 3.7 completamente indurisce. Ciò creerà una zona libera da batteri nella parte superiore della NGT-3D. </li><li> Per NGT-3D, versare 6,5 ml supporti nelle provette di plastica trasparenti 8 ml di fare uno strato di batteri agar, e dispensare accuratamente 200 ml di mezzi di comunicazione priva di batteri sulla parte superiore del supporto semi-indurito 3D per fare un sottile bacteria- strato libero sulla parte superiore. </li><li> Per NGB-3D, versare 65 ml di media nel 25 centimetri bottiglia coltura cellulare 2 plastica trasparente. Tale importo deve riempire il corpo della bottiglia 25 centimetri cultura 2 cellule. </li></ol></li><li> Lasciare camere verticalmente a temperatura ambiente per una settimana per consentire colonie batteriche di crescerenotevoli dimensioni del diametro minimo di 1 mm. <ol><li> (Opzionale) per test di durata della vita in NGT-3D, camere di coprire con un foglio di alluminio per evitare il degrado luce di FUDR. </li></ol></li></ol> 4. Misurare Fitness di Worm Popolazione al NGT-3D (Brood dimensione relativa del saggio) <ol><li> Scegliere un verme L4 fase utilizzando un filo di platino raccogliere e trasferire su un piatto NGM senza batteri. Lasciare verme di spostarsi liberamente per alcuni minuti per rimuovere batteri attaccati al suo corpo. </li><li> Ripetere il passaggio 4.1 e fare in modo che il worm è esente da batteri. In generale, due repliche di 4.1 sono sufficienti. </li><li> posizionare con cura il verme pulita sulla superficie del supporto 3D con un pick filo di platino. Il worm dovrebbe finalmente entrare l&#39;agar agar nella matrice 3D. </li><li> Chiudere il coperchio senza bloccare consentendo una certa aria nel tubo, ma impedendo l&#39;essiccazione dei media agar. </li><li> Incubare per 96 ore a 20 ° C. </li><li> Dopo 4 giorni, chiudere ermeticamente i coperchi e posizionare ilNGT-3D camera di cultura in un bagno d&#39;acqua C 88 ° per sciogliere l&#39;agar. Il calore uccide i vermi, ma i loro corpi rimangono intatti. NOTA: L&#39;utilizzo 8 ml tubi per NGT-3D, 20 - 30 minuti di incubazione è di solito sufficiente. </li><li> Usando una pipetta di vetro, trasferire i mezzi fuso su un piatto di plastica 9 centimetri di Petri. pipette di plastica non sono invitati, come i vermi possono spesso bastone a loro. </li><li> Utilizzando un microscopio stereoscopico dissezione trasmissione, contare il numero di vermi al L3, L4 e stadi adulti. Non contare i vermi che sono L2 fase e più giovani, come le generazioni F1 e F2 possono essere confusi qui. Così, questo test è un test relativo dimensione covata piuttosto che un dosaggio totale dimensioni covata. </li></ol> 5. Immagine e Record Worm Comportamento NGB-3D <ol><li> Scegli uno stadio verme L4 utilizzando un filo di platino raccogliere e rimuovere i batteri attaccati alla superficie trasferendo ad una piastra NGM senza batteri e permettendo di muoversi liberamente per alcuni minuti. </li><li> Ripetere il passaggio 5.1 per assicurarsi che il worm è esente da batteri. </li><li> posizionare accuratamente il verme pulito sul centro della superficie agar del NGB-3D vicino al collo della bottiglia con un pick filo di platino. Il worm dovrebbe finalmente entrare l&#39;agar agar nella matrice 3D. </li><li> Chiudere il coperchio senza bloccare consentendo una certa aria nel tubo, evitando l&#39;asciugatura dei media agar. </li><li> Immagine o registrare il verme sotto una dissezione stereo microscopio di trasmissione. Regolare la messa a fuoco come il verme si muove attraverso la matrice 3D. </li></ol>

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	<li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+Window+into+Biology:+A+Primer+on+Caenorhabditis+elegans.">A Transparent Window into Biology: A Primer on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>Consortium, C. E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282 (5396), 2012-2018 (1998).</li><li>Choi, J. I., Yoon, K. H., Kalichamy, S. S., Yoon, S. S., Lee, J. 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+natural+odor+attraction+between+lactic+acid+bacteria+and+the+nematode+Caenorhabditis+elegans.">A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans.</a> ISME J. 10 (3), 558-567 (2016).</li><li>Gaertner, B. E., Phillips, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+platform+for+molecular+quantitative+genetics+and+the+systems+biology+of+natural+variation.">Caenorhabditis elegans as a platform for molecular quantitative genetics and the systems biology of natural variation.</a> Genet Res (Camb). 92 (5-6), 331-348 (2010).</li><li>Cutter, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+evolution+in+the+wild.">Caenorhabditis evolution in the wild.</a> Bioessays. 37 (9), 983-995 (2015).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Felix, M. A., Braendle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+Caenorhabditis+elegans.">The natural history of Caenorhabditis elegans.</a> Curr Biol. 20 (58), R965-R969 (2010).</li><li>Barriere, A., Felix, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+local+genetic+diversity+and+low+outcrossing+rate+in+Caenorhabditis+elegans+natural+populations.">High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations.</a> Curr Biol. 15 (13), 1176-1184 (2005).</li><li>Lee, T. Y., Yoon, K. H., Lee, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGT-3D:+a+simple+nematode+cultivation+system+to+study+Caenorhabditis+elegans+biology+in+3D.">NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.</a> Biol Open. 5 (4), 529-534 (2016).</li><li>Lewis, J. A., Flemming, J. T., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Culture+Methods.">Basic Culture Methods.</a> Caenorhabditis elegans.: Modern Biological Analysis of an Organism. 48, 4-27 (1995).</li><li>Choi, S. Y., Yoon, K. H., Lee, J. I., Mitchell, 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=Violacein:+Properties+and+Production+of+a+Versatile+Bacterial+Pigment.">Violacein: Properties and Production of a Versatile Bacterial Pigment.</a> Biomed Res Int. , 465056 (2015).</li><li>Solis, G. M., Petrascheck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+in+96+well+microtiter+plates.">Measuring Caenorhabditis elegans life span in 96 well microtiter plates.</a> J Vis Exp. (48), (2011).</li><li>Kwon, N., Pyo, J., Lee, S. J., Je, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+worm+tracker+for+freely+moving+C.+elegans.">3-D worm tracker for freely moving C. elegans.</a> PLoS One. 8 (2), e57484 (2013).</li><li>Pierce-Shimomura, J. T., Chen, B. L., Mun, J. J., Ho, R., Sarkis, R., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+crawling+and+swimming+locomotory+patterns+in+C.+elegans.">Genetic analysis of crawling and swimming locomotory patterns in C. elegans.</a> Proc Natl Acad Sci U S A. 105 (52), 20982-20987 (2008).</li><li>Kwon, N., Hwang, A. B., You, Y. J., SJ, V. L., Je, J. 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The authors have nothing to disclose.

La coltivazione laboratorio di C. elegans utilizzando le piastre di terreni di crescita nematode classici è stato fondamentale per le centinaia di importanti scoperte che la ricerca in C. elegans ha fornito. Qui, vi presentiamo i nuovi metodi per coltivare C. elegans in un ambiente che rifletta più accuratamente i loro naturale habitat tridimensionali. Sebbene altri metodi sono stati usati per osservare C. elegans in 3D 13, questo è il primo protocollo che permette la co...

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table><tbody><tr><td>LB broth, Miller (Luria-Bertani)</td><td>Difco</td><td>224620</td><td></td></tr><tr><td>Sodium chloride</td><td>DAEJUNG</td><td>7548-4400</td><td>58.44 MW</td></tr><tr><td>Agar, Granulated</td><td>Difco</td><td>214530</td><td></td></tr><tr><td>Peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Calcium chloride, dihydrate</td><td>Bio Basic</td><td>CD0050</td><td>2*H&lt;sub&gt;2&lt;/sub&gt;O; 147.02 MW</td></tr><tr><td>Cholesterol</td><td>Bio Basic</td><td>CD0122</td><td>386.67 MW</td></tr><tr><td>Ethyl alcohol</td><td>B&amp;amp;J</td><td>RP090-1</td><td>99.99%; 46.07 MW</td></tr><tr><td>Magnesium sulfate, anhydrous</td><td>Bio Basic</td><td>MN1988</td><td>120.37 MW</td></tr><tr><td>Potassium phosphate, monobasic, anhydrous</td><td>Bio Basic</td><td>PB0445</td><td>136.09 MW</td></tr><tr><td>2&#039;-Deoxy-5-fluorouridine</td><td>Tokyo Chemical Industry</td><td>D2235</td><td>246.19 MW</td></tr><tr><td>Potassium phosphate, dibasic, anhydrous</td><td>Bio Basic</td><td>PB0447</td><td>174.18 MW</td></tr><tr><td>Multi-Purpose Test Tubes</td><td>Stockwell Scientific</td><td>ST.8570</td><td>8 ml</td></tr><tr><td>Test Tube Closures</td><td>Stockwell Scientific</td><td>ST.8575</td><td></td></tr><tr><td>Cell Culture Flask</td><td>SPL Lifescience</td><td>70125</td><td>25 cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Research Stereo Microscope</td><td>Nikon</td><td>SMZ18</td><td></td></tr><tr><td>High-Definition Color Camera Head</td><td>Nikon</td><td>DS-Fi2</td><td></td></tr><tr><td>PC-Based Control Unit</td><td>Nikon</td><td>DS-U3</td><td></td></tr><tr><td>NIS-Elements Basic Research, Microscope Imaging Software</td><td>Nikon</td><td>MQS32000</td><td></td></tr></tbody></table>

cultivation of caenorhabditis elegans in three dimensions in the laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

La costruzione di NGT-3D è un protocollo semplice e lineare che si traduce in una provetta agar-riempita con piccole colonie batteriche distanziati tutto il agar (Figura 1A). Worms possono muoversi liberamente attraverso la matrice agar, trovare e consumare le colonie batteriche. Per confermare se C. elegans possono riprodursi e di crescere normalmente in NGT-3D, abbiamo confrontato la fertilità e lo sviluppo larvale in 3D con piastre standard 2D NGM. Nel sagg...

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Vi presentiamo un metodo semplice per la costruzione di sistemi di coltivazione nematode 3D chiamati NGT-3D e NGB-3D. Questi possono essere utilizzati per studiare nematode fitness e comportamenti in habitat che sono più simili a naturali Caenorhabditis elegans habitat rispetto alle laboratorio C. elegans piastre di coltura 2D standard.

La coltivazione di<em&gt; Caenorhabditis elegans</em&gt; In tre dimensioni in laboratorio

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

cultivation-caenorhabditis-elegans-three-dimensions

Research

JoVE Journal

Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.7K Views.  Yonsei University.  Vi presentiamo un metodo semplice per la costruzione di sistemi di coltivazione nematode 3D chiamati NGT-3D e NGB-3D. Questi possono essere utilizzati per studiare nematode fitness e comportamenti in habitat che sono più simili a naturali Caenorhabditis elegans habitat rispetto alle laboratorio C. elegans piastre di coltura 2D standard. 

Video: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

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

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

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

Il Nematode Caenorhabditis Elegans - un modello Versatile In Vivo per studiare le interazioni ospite-microbo

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

Ricerca

Genetica

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Piastra-base di coltivazione su larga scala di Caenorhabditis elegans: preparazione del campione per la studio di alterazioni metaboliche nel diabete

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Biochimica

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Cultura e dosaggio delle popolazioni di caenorhabditis elegans a grande scala

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Di base<em&gt; Caenorhabditis elegans</em&gt; Metodi: sincronizzazione e di osservazione

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Biologia

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Un semplice chip microfluidico per la crescita a lungo termine e l'imaging di Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Bioingegneria

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Un metodo per la coltura embrionale<em&gt; C. elegans</em&gt; Celle

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Culturing<em&gt; Caenorhabditis elegans</em &gt; In axeniche Liquid Media e la creazione di transgeniche Worms da microparticelle Bombardamento

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Approcci quantitativi per studiare le strutture cellulari e la morfologia delle orgine in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Coltura a lungo termine di singoli Caenorhabditis elegans su terreni solidi per il monitoraggio longitudinale della fluorescenza e interventi avversivi

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

Coltura a lungo termine e monitoraggio di Caenorhabditis elegans isolati su terreni solidi in dispositivi multi-pozzetto

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

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