Name: rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions
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Sommige details van dit protocol werden geoptimaliseerd met de hulp van Dr. Adam Dobson, die ook voorzien van nuttige opmerkingen over het manuscript. Dit werk werd ondersteund door de Stichting voor de National Institutes of Health (FNIH) subsidie ​​nummer R01GM095372 (JMC, A (CN) W, AJD, en AED). FNIH subsidie ​​nummer 1F32GM099374-01 (PDN), en Brigham Young University opstarten fondsen (JMC, MLK, MV). Publicatiekosten werden gesteund door de Brigham Young University College van Life Sciences en het ministerie van Plant and Wildlife Sciences.

1. Cultuur Bacteriën (Start ~ 1 week voor Picking eieren) <ol><li> Bereid gemodificeerde MRS 20 (Mmrs) borden en bouillon buizen (tabel 1). Giet 20 ml Mmrs agar in elk 100 mm Petri plaat en laat het afkoelen / droog &#39;s nachts, of 5 ml Mmrs bouillon in 18 mm reageerbuizen. </li><li> Streak Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, en L. plantarum op Mmrs agarplaten. Incubeer Acetobacter overnacht bij 30 ° C. Incubate Lact...

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<html> De hier beschreven methode is een van de verschillende benaderingen voor het embryo dechorionation 8,11,18,25,26,27, samen met alternatieve houderijsystemen axenic vliegen, met inbegrip van seriële overdracht van axenic volwassenen 18,27 of een behandeling met antibiotica 13,18. Andere dechorionation methoden omvatten ethanol wast en verminderen 11,25,26 of uit te breiden 8 hypochloriet behandeling. Verschillende wasstappen kunnen helpen grootbrengen verschil...

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 meeste dieren zijn nauw verbonden met bacteriën ( &#39;microbiota&#39;) vanaf de geboorte tot de dood 1. Vergelijkingen van micro-organisme-vrij ( &#39;axenic&#39;) en micro-organisme geassocieerde ( &#39;conventionele&#39;) dieren hebben aangetoond microben invloed op diverse aspecten van de gezondheid van dieren, met inbegrip van metabole, nutritionele, bloedvaten, lever, respiratoire, immunologische, endocriene en neurologische functie 2. De fruitvlieg Drosophila melanogaster is een belangrijk model voor het begrijpen van vele van deze processen in de aanwezigheid van microben 3,4 en voor het bestuderen microbiota invloed diergezondheid 5,6. Geen bacteriesoorten aanwezig in ieder individu ( &#39;core&#39;), maar Acetobacter en Lactobacillus soorten numeriek domineren de microbiota van zowel laboratorium gekweekte en in het wild gevangen D. melanogaster. Andere Acetobacteraceae (inclusief Komagataeibacter en Gluconobacter), Firmicutes (zoals Enterococcus en Leuconostoc), en Enterobacteriaceae zijn ofwel vaak aanwezig in Drosophila personen bij lage overvloed, of onregelmatig aanwezig bij hoge overvloed 7-12. De microbiota van Drosophila en zoogdieren is wisselvallig binnen en tussen de generaties 14,19. Microbiota wisselvalligheid kan leiden tot fenotypische lawaai bij het meten van microbiota-afhankelijke eigenschappen. Bijvoorbeeld, de Acetobacteraceae invloed lipide (triglyceride) opslag in Drosophila 15-18. Als Acetobacteraceae meer overvloedig in vliegen van een flacon is dan in een andere 19, kan isogene vliegen verschillende fenotypes 20 hebben. Een oplossing voor het probleem van microbiota onbestendigheid bij muizen 14 is in de praktijk sinds de 1960&#39;s, door invoering van een bepaalde gemeenschap van 8 dominante microbiële soort muizen- pups elke nieuwe generatie (gewijzigde Schaedler flora),zodat elk pup wordt blootgesteld aan dezelfde belangrijke leden van de muis microbiota. Deze praktijk bedieningsorganen voor microbiota samenstelling zelfs wanneer de microbiota is niet het primaire doel van de studie 32 en stelt precedent de aanwezigheid van belangrijke microben in verschillende experimentele omstandigheden te waarborgen. Om de invloed van microben op Drosophila voeding te bepalen, hebben een aantal protocollen voor het afleiden van axenic fly lijnen ontwikkeld, met inbegrip van chloorbleekloog dechorionation van embryo&#39;s (hetzij afgeleid de novo elke generatie of generationally onderhouden door overschrijving op steriele diëten) en behandeling met antibiotica 13. Er zijn voordelen aan verschillende benaderingen, zoals gemak en snelheid voor beide antibiotica behandeling en seriële overdracht versus meer controle verstorende variabelen de novo dechorionation (bijvoorbeeld ei dichtheid resterende contaminerende microben, off-target antibiotische effecten). Ongeacht de wijze vanvoorbereiding, de introductie van bepaalde microbiële soorten embryo axenische maakt cultuur van Drosophila met gedefinieerde ( &#39;gnotobiotische&#39;) communities. Alternatief nabootsen het gebruik van Schaedler flora die gemeenschap kunnen worden geïnoculeerd conventioneel eitjes (volgende stappen 6-7) om de aanwezigheid van eigenschap beïnvloeden microben Elke flacon en ter voorkoming van complicaties van microbiota onbestendigheid. Hier beschrijven we het protocol voor het verhogen van axenic en gnotobiotische Drosophila door de novo dechorionation van embryo&#39;s, en voor het bevestigen van de aanwezigheid van ingevoerd of vervuilende microbiële taxa.

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			<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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			<td>Genesee Scientific</td>
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			<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.

Succesvolle opfok van axenic vliegen wordt bevestigd door isolatie van geen CFU&#39;s uit het hele lichaam homogenisaties van D. melanogaster volwassenen (figuur 1). Als alternatief, als de vergulde homogenaat levert kolonies, de flesjes zijn vervuild en moet worden weggegooid. Voor gnotobiotische vliegen, elk van de vier bacteriële isolaten werden geïsoleerd uit pools van 5 volwassen mannen, waaruit verschillen in de totale levensvatbare CFU&#39;s geassocieer...

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

Een methode voor het kweken van Drosophila melanogaster onder axenic en gnotobiotische voorwaarden wordt gepresenteerd. Fly embryo&#39;s worden dechorionated in natriumhypochloriet, aseptisch in steriele voeding en grootgebracht in gesloten containers. Inenten van voeding en embryo&#39;s met bacteriën leidt tot gnotobiotische verenigingen, en bacteriële aanwezigheid wordt bevestigd door plating whole-body Drosophila homogenaten.

Het grootbrengen de fruitvlieg<em&gt; Drosophila melanogaster</em&gt; Onder Axenische en gnotobiotische voorwaarden

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

Testen Bloedcel Populaties van de<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

Productie en toediening van therapeutische mesenchymale stamcellen / Stromal Cell (MSC) sferoïden primer in 3-D Cultures Onder Xeno-free Voorwaarden

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

Protocollen voor het visualiseren steroidogenic organen en hun Interactive Organen met Immunokleuring in de fruitvlieg<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

Dissectie en immunefluorescentie kleuring van Mushroom lichaam en fotoreceptor neuronen in de hersenen van de volwassen Drosophila melanogaster

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

Inductie van Endothelial differentiatie in cardiale voorlopercellen onder lage Serum voorwaarden

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

Ontdooien, kweken en Cryopreserving van Drosophila cellijnen

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

Methoden voor het testen van endocriene verstoring in fruitvliegje 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

Test voor de integriteit van de bloed-hersen barrière in 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

Efficiënte differentiatie van postganglionic Sympathische neuronen met behulp van menselijke pluripotente stamcellen onder Feeder-vrije en chemisch gedefinieerde cultuurvoorwaarden

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