Name: rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions
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Alguns detalhes deste protocolo foram otimizados com a assistência do Dr. Adam Dobson, que também forneceram comentários úteis sobre o manuscrito. Este trabalho foi financiado pela Fundação para o National Institutes of Health (FNIH) número de concessão R01GM095372 (JMC, A (CN) W, AJD, e AED). FNIH número de concessão 1F32GM099374-01 (PDN) e da Universidade Brigham Young fundos de inicialização (JMC, MLK, MV). Os custos de publicação foram apoiadas pela Academia Brigham Young University de Ciências da Vida e do Departamento de plantas e animais selvagens Sciences.

1. Cultura das bactérias (Start ~ 1 semana antes da colheita dos ovos) <ol><li> Prepare MRS modificado 20 (RMM) placas e tubos de caldo (Tabela 1). Introduzir 20 ml de agar RMM em cada placa de Petri de 100 mm e deixe esfriar / secar durante a noite, ou 5 ml Mmrs caldo para 18 tubos de ensaio mm. </li><li> Streak Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, e L. plantarum na RMM placas de agar. Incubar durante a noite a Acetobacter ...

<ol>
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<html> O método descrito aqui é uma das várias abordagens para embrião dechorionation 8,11,18,25,26,27, juntamente com métodos alternativos de criação de moscas axênicas, incluindo a transferência de série dos adultos axênicas 18,27 ou tratamento antibiótico 13,18. Outros métodos incluem dechorionation lavagens de etanol e reduzir ou estender 11,25,26 8 tratamento de hipoclorito. Diferentes etapas de lavagem pode auxiliar criação de diferentes genótip...

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

A maioria dos animais estão intimamente associados com bactérias ( &#39;microbiota&#39;) desde o nascimento até a morte 1. Comparações de ( &quot;convencional&quot;) animais livres de microrganismos ( &#39;axênico&#39;) e associada a microorganismos têm demonstrado micróbios influenciar diversos aspectos da saúde animal, incluindo metabólica, nutricional, vascular, hepática, respiratória, imunológicos, endócrino e função neurológica 2. A mosca da fruta Drosophila melanogaster é um modelo fundamental para a compreensão de muitos desses processos na presença de micróbios 3,4 e para o estudo da influência microbiota em 5,6 a saúde animal. Nenhuma espécie bacteriana está presente em cada indivíduo ( &#39;core&#39;), mas Acetobacter e Lactobacillus espécies numericamente dominar a microbiota de ambos D. pego wild-criados em laboratório e melanogaster. Outros Acetobacteraceae (incluindo Komagataeibacter e Gluconobacter), Firmicútis (tais como Enterococcus e Leuconostoc) e Enterobacteriaceae ou são frequentemente presentes em indivíduos de Drosophila em baixa abundância, ou irregularmente presente em grande abundância 7-12. A microbiota de Drosophila e mamíferos é inconstante dentro e entre gerações 14,19. Microbiota inconstância pode levar ao ruído fenotípica ao medir traços dependente da microbiota. Por exemplo, o armazenamento Acetobacteraceae influência lipídico (triglicéridos) em Drosophila 15-18. Se Acetobacteraceae são mais abundantes em moscas de um frasco do que nos outros 19, moscas isogênicos podem ter diferentes fenótipos 20. Uma solução para o problema da inconstância microbiota em camundongos 14 tem sido na prática desde 1960, através da introdução de uma comunidade definida de 8 espécies microbianas dominantes para filhotes de rato cada nova geração (alterada SCHAEDLER flora),garantindo que cada filhote está exposto aos mesmos membros-chave da microbiota mouse. Esta prática controla a composição da microbiota, mesmo quando a microbiota não é o alvo primário do estudo 32, e define precedente para assegurar a presença de micróbios chave numa variedade de condições experimentais. Para definir a influência de micróbios em Drosophila nutrição, vários protocolos para derivar linhas mosca axênicas têm sido desenvolvidos, incluindo dechorionation hipoclorito de embriões (derivada quer de novo cada geração ou mantida generationally por transferência para uma alimentação esterilizada) e 13 tratamento com antibióticos. Há benefícios para diferentes abordagens, tais como a facilidade e rapidez para os dois tratamentos antibióticos e transferência de série, contra um maior controle de variáveis ​​de confusão com de novo dechorionation (por exemplo, densidade de ovos, micróbios contaminantes residuais, longe do alvo efeitos antibióticos). Independentemente do método depreparação, a introdução de espécies microbianas especificados para axênicas embriões permite cultura de Drosophila com as comunidades ( &#39;gnotobióticos&#39;) definidas. Alternativamente, imitando o uso de flora SCHAEDLER, esta comunidade poderia ser inoculado aos ovos convencionalmente-estabelecidas (seguindo os passos 6-7 somente) para assegurar a presença de micróbios que influenciam a característica em cada frasco e evitar complicações da inconstância microbiota. Aqui descrevemos o protocolo para elevar e gnotobiótico axénica de Drosophila por dechorionation de novo de embriões e para a confirmação da presença dos taxa microbiana introduzido ou contaminantes.

<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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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		<tr height="20">
			<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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		<tr height="20">
			<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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		<tr height="20">
			<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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		<tr height="20">
			<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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		<tr height="20">
			<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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		<tr height="20">
			<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.

Criação bem-sucedida de moscas axênicas é confirmado pelo isolamento de não UFCs de homogeneizações todo o corpo de D. adultos melanogaster (figura 1). Alternativamente, se o homogeneizado chapeado produz colónias, os frascos são contaminada e deve ser descartado. Para gnotobióticos moscas, cada um dos quatro isolados bacterianos foram isolados a partir de misturas de 5 adultos do sexo masculino, demonstrando diferenças em CFUs totais viávei...

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

Um método para a criação de Drosophila melanogaster sob condições axénicas e gnotobióticos é apresentada. embriões voam são dechorionated em hipoclorito de sódio, transferidos assepticamente à dieta estéril, e criados em recipientes fechados. Inoculando dieta e embriões com bactérias leva a associações gnotobióticos e presença de bactérias é confirmada por plaqueamento de corpo inteiro de Drosophila homogeneizados.

Elevando a mosca da fruta<em&gt; Drosophila melanogaster</em&gt; Sob axênicas e gnotobióticos Condições

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

Ensaiando populações de células do sangue<em&gt; Drosophila melanogaster</em&gt; Larva

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

Biologia do Desenvolvimento

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

Produção e Administração de Therapeutic-tronco mesenquimais / celular estromal (MSC) Spheroids condicionadas em culturas 3-D Sob Condições Xeno-livres

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

Protocolos para a visualização de esteroidogênicas Órgãos e seus órgãos interativa com imunocoloração na mosca da fruta<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

Dissecção e imunofluorescência coloração de cogumelo corpo e fotoreceptor neurônios em cérebros adultos 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

Indução de diferenciação endotelial em células progenitoras cardíacas sob condições de baixo de soro

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

Descongelamento, cultivo e Cryopreserving linhas de células de Drosophila

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

Métodos para testar a perturbação endócrina na 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

Ensaio para a integridade da barreira hematoencefálica na 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

Diferenciação eficiente de neurônios simpáticos pós-ganglionários usando células-tronco pluripotentes humanas em condições de cultura sem alimentadores e quimicamente definidas

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