Name: rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions
Uploaded: 2016-07-30T14:00:00
Description: Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

Noen detaljer om denne protokollen ble optimalisert med hjelp av Dr. Adam Dobson, som også gitt nyttige kommentarer til manuskriptet. Dette arbeidet ble støttet av stiftelsen for National Institutes of Health (FNIH) stipend nummer R01GM095372 (JMC, A (CN) W, ajd, og AED). FNIH tilskuddet antallet 1F32GM099374-01 (PDN) og Brigham Young University oppstartsfond (JMC, MLK, MV). Publiseringskostnadene ble støttet av Brigham Young University College of Life Sciences og Institutt for plante- og dyre Sciences.

1. Kultur Bakterier (Start ~ 1 uke før Plukke egg) <ol><li> Forbered endret MRS 20 (mMRS) plater og buljong rør (tabell 1). Hell 20 ml mMRS agar i hver 100 mm Petri plate og avkjøl / tørke over natten, eller 5 ml mMRS kokes i 18 mm prøverør. </li><li> Streak Acetobacter pomorum, A. tropic, Lactobacillus brevis, og L. plantarum på mMRS agarskåler. Inkuber Acetobacter over natten ved 30 ° C. Inkuber Lactobacillus anaerobt ved å p...

<ol>
	<li>McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giving+microbes+their+due--animal+life+in+a+microbially+dominant+world.">Giving microbes their due--animal life in a microbially dominant world.</a> J Exp Biol. 218, 1968-1973 (2015).</li><li>Smith, K., McCoy, K. D., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+axenic+animals+in+studying+the+adaptation+of+mammals+to+their+commensal+intestinal+microbiota.">Use of axenic animals in studying the adaptation of mammals to their commensal intestinal microbiota.</a> Semin Immunol. 19 (2), 59-69 (2007).</li><li>Rieder, L. E., Larschan, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wisdom+from+the+fly.">Wisdom from the fly.</a> Trends Genet. 30 (11), 479-481 (2014).</li><li>Arias, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+and+the+development+of+biology+in+the+20th+century.">Drosophila melanogaster and the development of biology in the 20th century.</a> Methods Mol Biol. 420, 1-25 (2008).</li><li>Lee, W. J., Brey, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+microbiomes+influence+metazoan+development:+insights+from+history+and+Drosophila+modeling+of+gut-microbe+interactions.">How microbiomes influence metazoan development: insights from history and Drosophila modeling of gut-microbe interactions.</a> Annu Rev Cell Dev Biol. 29, 571-592 (2013).</li><li>Erkosar, B., Leulier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+adult+microbiota,+gut+homeostasis+and+longevity:+novel+insights+from+the+Drosophila+model.">Transient adult microbiota, gut homeostasis and longevity: novel insights from the Drosophila model.</a> FEBS Lett. 588 (22), 4250-4257 (2014).</li><li>Chandler, J. A., Lang, J. M., Bhatnagar, S., Eisen, J. A., Kopp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+communities+of+diverse+Drosophila+species:+ecological+context+of+a+host-microbe+model+system.">Bacterial communities of diverse Drosophila species: ecological context of a host-microbe model system.</a> PLoS Genet. 7 (9), e1002272 (2011).</li><li>Broderick, N. A., Buchon, N., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota-induced+changes+in+drosophila+melanogaster+host+gene+expression+and+gut+morphology.">Microbiota-induced changes in drosophila melanogaster host gene expression and gut morphology.</a> MBio. 5 (3), 01117 (2014).</li><li>Wong, C. N., Ng, P., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-diversity+bacterial+community+in+the+gut+of+the+fruitfly+Drosophila+melanogaster.">Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster.</a> Environ Microbiol. 13 (7), 1889-1900 (2011).</li><li>Staubach, F., Baines, J. F., Kunzel, S., Bik, E. M., Petrov, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+species+and+environmental+effects+on+bacterial+communities+associated+with+Drosophila+in+the+laboratory+and+in+the+natural+environment.">Host species and environmental effects on bacterial communities associated with Drosophila in the laboratory and in the natural environment.</a> PLoS One. 8 (8), e70749 (2013).</li><li>Brummel, T., Ching, A., Seroude, L., Simon, A. F., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+lifespan+enhancement+by+exogenous+bacteria.">Drosophila lifespan enhancement by exogenous bacteria.</a> Proc Natl Acad Sci U S A. 101 (35), 12974-12979 (2004).</li><li>Cox, C. R., Gilmore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+microbial+colonization+of+Drosophila+melanogaster+and+its+use+as+a+model+of+Enterococcus+faecalis+pathogenesis.">Native microbial colonization of Drosophila melanogaster and its use as a model of Enterococcus faecalis pathogenesis.</a> Infect Immun. 75 (4), 1565-1576 (2007).</li><li>Ridley, E. V., Wong, A. C., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe-dependent+and+nonspecific+effects+of+procedures+to+eliminate+the+resident+microbiota+from+Drosophila+melanogaster.">Microbe-dependent and nonspecific effects of procedures to eliminate the resident microbiota from Drosophila melanogaster.</a> Appl Environ Microbiol. 79 (10), 3209-3214 (2013).</li><li>Rogers, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+divergence+in+gastrointestinal+microbiota+in+physically-separated+genetically+identical+mice.">Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.</a> Sci Rep. 4, 5437 (2014).</li><li>Chaston, J. M., Newell, P. D., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenome-wide+association+of+microbial+determinants+of+host+phenotype+in+Drosophila+melanogaster.">Metagenome-wide association of microbial determinants of host phenotype in Drosophila melanogaster.</a> MBio. 5 (5), 01631-01714 (2014).</li><li>Huang, J. H., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consumption+of+dietary+sugar+by+gut+bacteria+determines+Drosophila+lipid+content.">Consumption of dietary sugar by gut bacteria determines Drosophila lipid content.</a> Biology Letters. , (2015).</li><li>Shin, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+microbiome+modulates+host+developmental+and+metabolic+homeostasis+via+insulin+signaling.">Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling.</a> Science. 334 (6056), 670-674 (2011).</li><li>Storelli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactobacillus+plantarum+promotes+Drosophila+systemic+growth+by+modulating+hormonal+signals+through+TOR-dependent+nutrient+sensing.">Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing.</a> Cell Metab. 14 (3), 403-414 (2011).</li><li>Wong, A. C., Chaston, J. M., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inconstant+gut+microbiota+of+Drosophila+species+revealed+by+16S+rRNA+gene+analysis.">The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis.</a> ISME J. 7 (10), 1922-1932 (2013).</li><li>Newell, P. D., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+interactions+determine+the+impact+of+the+gut+microbiota+on+nutrient+allocation+in+Drosophila+melanogaster.">Interspecies interactions determine the impact of the gut microbiota on nutrient allocation in Drosophila melanogaster.</a> Appl Environ Microbiol. 80 (2), 788-796 (2014).</li><li>Broderick, N. A., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut-associated+microbes+of+Drosophila+melanogaster.">Gut-associated microbes of Drosophila melanogaster.</a> Gut Microbes. 3 (4), 307-321 (2012).</li><li>Ren, C., Webster, P., Finkel, S. E., Tower, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+internal+and+external+bacterial+load+during+Drosophila+aging+without+life-span+trade-off.">Increased internal and external bacterial load during Drosophila aging without life-span trade-off.</a> Cell Metab. 6 (2), 144-152 (2007).</li><li>Wong, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Host+as+the+Driver+of+the+Microbiota+in+the+Gut+and+External+Environment+of+Drosophila+melanogaster.">The Host as the Driver of the Microbiota in the Gut and External Environment of Drosophila melanogaster.</a> Appl Environ Microbiol. 81 (18), 6232-6240 (2015).</li><li>Dobson, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+genetic+determinants+of+microbiota-dependent+nutrition+revealed+by+genome-wide+analysis+of+Drosophila+melanogaster.">Host genetic determinants of microbiota-dependent nutrition revealed by genome-wide analysis of Drosophila melanogaster.</a> Nat Commun. 6, 6312 (2015).</li><li>Bakula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+persistence+of+a+microbial+flora+during+postembryogenesis+of+Drosophila+melanogaster.">The persistence of a microbial flora during postembryogenesis of Drosophila melanogaster.</a> J Invertebr Pathol. 14 (3), 365-374 (1969).</li><li>Ryu, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+homeostasis+by+the+homeobox+gene+caudal+and+commensal-gut+mutualism+in+Drosophila.">Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila.</a> Science. 319 (5864), 777-782 (2008).</li><li>Blum, J. E., Fischer, C. N., Miles, J., Handelsman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+replenishment+sustains+the+beneficial+microbiome+of+Drosophila+melanogaster.">Frequent replenishment sustains the beneficial microbiome of Drosophila melanogaster.</a> MBio. 4 (6), 00860 (2013).</li><li>Bitner-Mathe, B. C., Klaczko, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+Drosophila+melanogaster+wing+morphology:+effects+of+sex,+temperature+and+density.">Plasticity of Drosophila melanogaster wing morphology: effects of sex, temperature and density.</a> Genetica. 105 (2), 203-210 (1999).</li><li>Edward, D. A., Chapman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+effects+of+developmental+environment+on+reproductive+trait+expression+in+Drosophila+melanogaster.">Sex-specific effects of developmental environment on reproductive trait expression in Drosophila melanogaster.</a> Ecol Evol. 2 (7), 1362-1370 (2012).</li><li>Ridley, E. V., Wong, A. C., Westmiller, S., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+resident+microbiota+on+the+nutritional+phenotype+of+Drosophila+melanogaster.">Impact of the resident microbiota on the nutritional phenotype of Drosophila melanogaster.</a> PLoS One. 7 (5), e36765 (2012).</li><li>Newell, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+function+and+comparative+genomic+analyses+of+the+Drosophila+gut+microbiota+identify+candidate+symbiosis+factors.">In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors.</a> Front Microbiol. 5, 576 (2014).</li><li>Dewhirst, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+the+defined+murine+microbiota:+altered+Schaedler+flora.">Phylogeny of the defined murine microbiota: altered Schaedler flora.</a> Appl Environ Microbiol. 65 (8), 3287-3292 (1999).</li><li>Min, K. T., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia,+normally+a+symbiont+of+Drosophila,+can+be+virulent,+causing+degeneration+and+early+death.">Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death.</a> Proc Natl Acad Sci U S A. 94 (20), 10792-10796 (1997).</li></ol>

<html> Metoden som beskrives her er en av flere tilnærminger for embryo dechorionation 8,11,18,25,26,27, sammen med alternative metoder for oppdrett av axenic fluer, inkludert seriell overføring av axenic voksne 18,27 eller antibiotikabehandling 13,18. Andre dechorionation metoder omfatter etanol vasker og redusere 11,25,26 eller forlenge 8 hypokloritt behandling. Ulike vasketrinn kan hjelpe oppdragelse forskjellige flue genotyper: I en tidligere studie fleste av ~...

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. The Protocol was updated.
Step 6.1.4.2 of the Protocol was updated from:
If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation: 
E = ((O-B) x V x D)/C 
where E = volume to resuspend pellet in (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use &#34;O&#34; in place of &#34;O-B&#34;. 
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.052), A. pomorum (0.038), L. brevis (0.056), L. plantarum (0.077).
to:
If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation: 
E = ((O-B) x V x D)/C 
where E = volume to resuspend pellet in (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use &#34;O&#34; in place of &#34;O-B&#34;. 
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.053), A. pomorum (0.038), L. brevis (0.077), L. plantarum (0.056).

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. The Protocol was updated.

Erratum: Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

De fleste dyr er nært forbundet med bakterier ( &#39;bakterieflora&#39;) fra fødsel til død en. Sammenligninger av mikroorganismer fritt ( &#39;axenic&#39;) og mikro-assosiert ( &#39;vanlig&#39;) dyr har vist mikrober påvirke ulike aspekter av dyrehelse, inkludert metabolske, ernæringsmessige, vaskulær, lever, luftveier, immunologiske, endokrine og nevrologiske funksjon 2. Bananflue Drosophila melanogaster er en nøkkel modell for forståelse av mange av disse prosesser i nærvær av mikrober 3,4 og for å studere floraen innflytelse på dyrehelse 5,6. Ingen bakteriearter er til stede i hver enkelt ( &quot;kjerne&quot;), men Acetobacter og Lactobacillus arter numerisk dominere microbiota av både laboratorie oppdrettet og villfanget D. melanogaster. Andre Acetobacteraceae (inkludert Komagataeibacter og Gluconobacter), Firmicutes (for eksempel Enterococcus og Leuconostoc), og Enterobacteriaceae enten er ofte til stede i Drosophila individer ved lav overflod, eller uregelmessig tilstede ved høy overflod 7-12. Den bakterieflora av Drosophila og pattedyr er ustadige innenfor og på tvers av generasjoner 14,19. Microbiota inconstancy kan føre til fenotypiske støy ved måling microbiota avhengige egenskaper. For eksempel Acetobacteraceae innflytelse lipid (triglyserider) lagring i Drosophila 15-18. Hvis Acetobacteraceae er mer rik på fluer i ett hetteglass enn i en annen 19, kan isogene fluer ha forskjellige fenotyper 20. En løsning på problemet med microbiota ustadighet i mus 14 har vært i praksis siden 1960-tallet, ved å innføre et definert fellesskap av 8 dominerende mikrobielle arter på museunger hver ny generasjon (endret Schaedler flora),sikre at hver valp er utsatt for de samme sentrale medlemmer av musebakterieflora. Denne praksisen kontroller for bakterieflora sammensetning, selv når den bakterieflora ikke er det primære målet for studium 32, og setter presedens for å sikre tilstedeværelse av nøkkel mikrober i en rekke eksperimentelle betingelser. For å definere påvirkning av mikrober på Drosophila ernæring, har flere protokoller for å utlede axenic snører blitt utviklet, inkludert hypokloritt dechorionation av embryoer (enten avledet de novo hver generasjon eller vedlikeholdes generationally ved overføring til sterile dietter) og antibiotikabehandling 13. Det er fordeler med å ulike tilnærminger, for eksempel letthet og hurtighet for begge antibiotika behandling og seriell overføring, kontra større kontroll over konfunderende variabler med de novo dechorionation (f.eks egg tetthet, rest forurensende mikrober, off-target antibiotika effekter). Uavhengig av fremgangsmåten ifølgeforberedelse, innføring av spesifiserte mikrobielle arter axenic embryoer tillater kulturen i Drosophila med definerte ( &#39;gnotobiotiske&#39;) samfunn. Alternativt, etterligne bruk av Schaedler flora, dette fellesskapet kan bli vaksinert med konvensjonelt lagt egg (Følgende trinn 6-7) for å sikre tilstedeværelse av egenskap-påvirke mikrober i hvert hetteglass og unngå komplikasjoner av bakterieflora ustadighet. Her beskriver vi protokollen for å heve axenic og gnotobiotiske Drosophila av de novo dechorionation embryoer, og for å bekrefte tilstedeværelse av introduserte eller forurensende mikrobiell taxa.

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Brewer&#39;s Yeast</td>
			<td style="width:291px;">MP Biomedicals, LLC.</td>
			<td style="width:108px;">903312</td>
			<td style="width:195px;">http://www.mpbio.com/product.php?pid=02903312</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma Aldrich</td>
			<td>158968-3KG</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/158968?lang=en&#38;region=US</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Fisher--Lab Scientific</td>
			<td>fly802010</td>
			<td>https://www.fishersci.com/shop/products/drosophila-agar-8-100mesh-10kg/nc9349177</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Welch&#39;s 100% Grape Juice Concentrate</td>
			<td>Walmart or other grocery store</td>
			<td>9116196</td>
			<td>http://www.walmart.com/ip/Welch-s-Frozen-100-Grape-Juice-Concentrate-11.5-oz/10804406</td>
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			<td height="20" style="height:20px;">Cage: 32 oz. Translucent Round Deli Container</td>
			<td>Webstaurant Store</td>
			<td>999L5032Y</td>
			<td>http://www.webstaurantstore.com/newspring-delitainer-sd5032y-32-oz-translucent-round-deli-container-24-pack/999L5032Y.html</td>
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			<td height="20" style="height:20px;">Translucent Round Deli Container Lid</td>
			<td>Webstaurant Store</td>
			<td>999YNL500</td>
			<td>http://www.webstaurantstore.com/newspring-delitainer-ynl500-translucent-round-deli-container-lid-60-pack/999YNL500.html</td>
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			<td height="20" style="height:20px;">Stock Bottles</td>
			<td>Genesee Scientific</td>
			<td style="width:108px;">32-130</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=32-130</td>
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			<td height="20" style="height:20px;">Droso-Plugs</td>
			<td>Genesee Scientific</td>
			<td style="width:108px;">49-101</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=49-101</td>
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			<td height="20" style="height:20px;">Nylon Mesh</td>
			<td>Genesee Scientific</td>
			<td>57-102&#160;</td>
			<td>https://geneseesci.com/shop-online/product-details/715/?product=57-102</td>
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			<td height="20" style="height:20px;">Plastic Bushing</td>
			<td>Home Depot</td>
			<td style="width:108px;">100343125</td>
			<td>http://www.homedepot.com/p/Halex-2-1-2-in-Rigid-Insulated-Plastic-Bushing-75225/100343125</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specimen Cup</td>
			<td>MedSupply Partners</td>
			<td>K01-207067</td>
			<td>http://www.medsupplypartners.com/covidien-specimen-containers.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Repeater M4</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td>https://online-shop.eppendorf.us/US-en/Manual-Liquid-Handling-44563/Dispensers--Burettes-44566/Repeater-M4-PF-44619.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml Centrifuge Tubes</td>
			<td>Laboratory Product Sales</td>
			<td>TR2003</td>
			<td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=TR2003</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Food Boxes</td>
			<td>USA Scientific</td>
			<td>2316-5001</td>
			<td>http://www.usascientific.com/search.aspx?find=2316-5001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysing Matrix D Bulk</td>
			<td>MP Biomedicals, LLC.</td>
			<td>116540434</td>
			<td>http://www.mpbio.com/search.php?q=6540-434&#38;s=Search</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter Pipette Tips, 300&#956;l</td>
			<td>USA Scientific</td>
			<td>1120-9810</td>
			<td>http://www.usascientific.com/search.aspx?find=1120-9810</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dishes</td>
			<td>Laboratory Product Sales</td>
			<td>M089303</td>
			<td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=M089303</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Decon Laboratories, INC.</td>
			<td>2701</td>
			<td>http://www.deconlabs.com/products.php?ID=88</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paintbrush</td>
			<td>Walmart</td>
			<td>5133</td>
			<td>http://www.walmart.com/ip/Chenille-Kraft-5133-Acrylic-Handled-Brush-Set-Assorted-Sizes-colors-8-Brushes-set/41446005</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Fisher</td>
			<td>08-882</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-medium-pointed-forceps-3/p-128693</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Household Bleach (6-8% Hypochlorite)</td>
			<td>Walmart</td>
			<td>550646751</td>
			<td>http://www.walmart.com/ip/Clorox-Concentrated-Regular-Bleach-121-fl-oz/21618295</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Universal Peptone</td>
			<td>Genesee Scientific</td>
			<td>20-260</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=20-260</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast Extract</td>
			<td>&#160;Fisher Scientific</td>
			<td>BP1422-500</td>
			<td>https://www.fishersci.com/shop/products/fisher-bioreagents-microbiology-media-additives-yeast-extract-3/bp1422500?matchedCatNo=BP1422500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dipotassium Phosphate</td>
			<td>Sigma Aldrich</td>
			<td>P3786-1KG</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=P3786-1KG&#38;interface=All&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Citrate</td>
			<td>Sigma Aldrich</td>
			<td>25102-500g</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=25102-500g&#38;interface=All&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Acetate</td>
			<td>VWR</td>
			<td>97061-994</td>
			<td>https://us.vwr.com/store/catalog/product.jsp?catalog_number=97061-994</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?matchedCatNo=M63500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Manganese Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>10034-96-5</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=10034-96-5&#38;interface=CAS%20No.&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MRS Powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:108px;">69966-500G</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/69966?lang=en&#38;region=US</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Plate Reader</td>
			<td>BioTek (Epoch)&#160;</td>
			<td>NA</td>
			<td>http://www.biotek.com/products/microplate_detection/epoch_microplate_spectrophotometer.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.7 ml Centrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>http://www.usascientific.com/search.aspx?find=1615-5500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter Pipette Tips, 1000&#956;l</td>
			<td>USA Scientific</td>
			<td>1122-1830</td>
			<td>http://www.usascientific.com/search.aspx?find=1122-1830</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Plates</td>
			<td>Greiner Bio-One</td>
			<td>655101</td>
			<td>https://shop.gbo.com/en/usa/articles/catalogue/article/0110_0040_0120_0010/13243/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ceramic Beads</td>
			<td>MP Biomedicals, LLC.</td>
			<td>6540-434</td>
			<td>http://www.mpbio.com/product.php?pid=116540434</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Homogenizer</td>
			<td>MP Biomedicals, LLC.</td>
			<td>116004500</td>
			<td>http://www.mpbio.com/product.php?pid=116004500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Class 1 BioSafety Cabinet</td>
			<td>Thermo Scientific&#160;</td>
			<td>Model 1395</td>
			<td>http://www.thermoscientific.com/en/product/1300-series-class-ii-type-a2-biological-safety-cabinet-packages.html</td>
		</tr>
	</tbody>
</table>

rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Vellykket oppdrett av axenic fluer bekreftes av isolering av ingen CFU fra hele kroppen homogenizations av D. melanogaster voksne (figur 1). Alternativt, hvis belagt homogenate gir kolonier, blir ampullene forurenset og bør kastes. For gnotobiotiske fluer, ble hver av de fire bakterieisolater isolert fra bassenger av 5 voksne menn, viser forskjeller i totale levedyktige CFU assosiert med voksne fluer (figur 1). Hver bakteriearter har en distink...

Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

En fremgangsmåte for oppdrett Drosophila melanogaster henhold axenic og gnotobiotiske betingelser er presentert. Fly embryoer er dechorionated i natriumhypokloritt, overføres aseptisk til sterilt kosthold, og alet opp i lukkede beholdere. Inoculating kosthold og embryoer med bakterier fører til gnotobiotiske foreninger, og bakteriell tilstedeværelse er bekreftet av plating hele kroppen Drosophila homogenates.

Oppdragelse bananflue<em&gt; Drosophila melanogaster</em&gt; Under Axenic og gnotobiotiske betingelser

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Research

JoVE Journal

Developmental Biology

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Analyse av blodcellepopulasjoner i<em&gt; Drosophila melanogaster</em&gt; Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Produksjon og Administrasjon av Therapeutic Mesenchymale Stem / stromal Cell (MSC) Spheroids Grunnet i 3-D kulturer Under Xeno-frie betingelser

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Protokoller for Visualisering Steroidogenic organer og deres Interaktiv organer med Farging i bananflue<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Disseksjon og Immunofluorescent flekker av sopp kropp og Photoreceptor Neurons i voksen Drosophila melanogaster hjernen

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Induksjon av Endothelial differensiering i Cardiac Progenitor celler Under lav Serum forhold

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Tiner dyrking og Cryopreserving Drosophila linjer

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

Methods to Test Endocrine Disruption in Drosophila melanogaster

Metoder for å teste endokrine forstyrrelser i Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Analysen for Blood-Brain Barrier integritet i Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Effektiv differensiering av postganglioniske sympatiske nevroner ved hjelp av humane pluripotente stamceller under materfrie og kjemisk definerte kulturforhold

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...