Alcuni dettagli di questo protocollo sono state ottimizzate con l&#39;aiuto del Dr. Adam Dobson, che ha anche fornito utili commenti sul manoscritto. Questo lavoro è stato sostenuto dalla Fondazione per il National Institutes of Health (FNIH) codice di autorizzazione R01GM095372 (JMC, A (CN) W, AJD, e AED). FNIH codice di autorizzazione 1F32GM099374-01 (PDN), e Brigham Young University fondi di avvio (JMC, MLK, MV). costi di pubblicazione sono stati sostenuti dalla Brigham Young University College of Life Sciences e del Dipartimento di Scienze della flora e fauna.

1. Coltura dei batteri (Inizio ~ 1 settimana prima di prendere le uova) <ol><li> Preparare MRS modificato (20) mmrs piastre e tubi di brodo (Tabella 1). Versare 20 ml mMRS agar in ogni piatto 100 millimetri di Petri e lasciare raffreddare / asciugare durante la notte, o 5 ml mMRS brodo in provette mm 18. </li><li> Streak Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, e L. plantarum su mMRS piastre di agar. Incubare Acetobacter notte a 30 ° C. Incubare anaerobicamente Lactobacillus posizionando le piastre in un contenitore ermetico e inondazioni con anidride carbonica prima della sigillatura. Incubare a 30 ° C per una notte. Nota: L. colonie brevis potrebbero non essere visibili fino 24-48. Le piastre possono essere conservati a 4 ° C e le colonie possono essere usati fino a 3 settimane. </li><li> Due o tre giorni prima di trasferire le uova dechorionated alla dieta sterile (sezione 5), scegliere una singola colonia dalla piastra mMRS in una provetta conmMRS tenenti brodo. Crescere Acetobacter con agitazione e crescere Lactobacillus statico per 24 ore o fino a quando torbido, sia a 30 ° C. </li></ol> 2. Preparare sterile dieta <ol><li> Preparare dieta sterile (Tabella 2) in un pallone da 2 L Erlenmeyer. Microonde la dieta fino a quando non ha bollito per 3 volte consecutive; mix tra ogni ebollizione. </li><li> Porre il pallone su un piatto mescolare per mantenere mescolando durante il trasferimento dieta per provette da centrifuga coniche. Trasferire 7,5 ml di dieta per 50 ml provette da centrifuga. Liberamente tappare i tubi, e mettere in un rack in polipropilene autoclavabile coperto. </li><li> Sterilizzare dieta volo usando autoclave a 121 ° C e 15 psi per 25 min. Rimuovere rack da autoclave, e scuotere immediatamente ciascun rack orizzontale per garantire la dieta non separarsi durante il raffreddamento. Fare attenzione a non scuotere i rack in verticale per impedire il trasferimento di dieta per il coperchio o bordi dei tubi. Lasciare che la dieta raffreddare su un agitatore per esattamente 45 minuti eancora una volta scuotere la dieta in senso orizzontale a mano. Nota: La dieta può essere conservato a 4-15 ° C per un massimo di una settimana. </li><li> Come alternativa all&#39;utilizzo di rack autoclavabili coperte (passaggi 2.1-2.3), o ad alzare le mosche sulle diete contenenti conservante acido, effettuare le seguenti operazioni: <ol><li> Preparare 1 L dieta liquida in un pallone 2 L con un ancoretta nel pallone. Autoclave la dieta. </li><li> Dopo la sterilizzazione in autoclave, aggiungere 10 ml di conservanti e mescolare continuamente su un piatto mescolare riscaldata. Quando l&#39;agar è raffreddato a 50-60 ° C, spostare il pallone in una piastra di mescolare riscaldata in un armadio biosicurezza e mantenere la temperatura pallone per riscaldamento a 50-60 ° C. </li><li> Nel gabinetto biosicurezza, pipetta ~ 7,5 ml individualmente in tubi conici. </li></ol></li></ol> 3. Preparare Gabbie deposizione delle uova <ol><li> Fai piastre di agar succo d&#39;uva da microonde 100 ml di acqua, lievito di birra 10 g di birra, 10 g di glucosio, e 1 g di agar. Portare a ebollizione per 3 volte nella fase 1.1 e aggiungere 10 g di uva congelato succo concentrate per aumentare la visibilità delle uova sulla piastra di agar. </li><li> Quando agar è raffreddato a 55 ° C, versare 20 ml in 100 mm Petri e lasciare solidificare. </li><li> Coprire la superficie delle piastre di agar con una pasta di lievito mescolando lievito 1 g di birra con acqua 15 g. Versare pasta di lievito sulla piastra di agar assicurandosi che la superficie è coperta, quindi versare l&#39;eccesso, lasciando dietro di sé una sottile residuo di lievito. Se si utilizzano più piastre, la pasta può essere sequenziale versato a più piastre. </li><li> Trasferire piastre di agar nel fondo di una gabbia. Nota: Un contenitore deli 32 oz è un buon sostituto per gabbie Fly acrilici. <ol><li> Rendere coperchi per contenitori deli 32 oz tagliando un foro nella parte superiore e incollando una mesh traspirante sopra il foro con colla non tossici. Se la colla è idrosolubili impediscono esposizione all&#39;acqua al coperchio. </li></ol></li><li> Trasferimento 200-300 vola nel contenitore e coprire con il coperchio. Coprire il buco rete protetta con un coperchio piatto di Petri vuota Prevent evaporazione dalla superficie agar e aggiungere una carta velina umida all&#39;interno del coperchio se desiderato. Incubare mosche a 25 ° C durante la notte per 16-20 ore. </li></ol> 4. raccogliere le uova <ol><li> Preparare un setaccio per la raccolta delle uova, ponendo rete di nylon in una boccola in plastica. </li><li> Per recuperare la piastra con le uova su di esso, rimuovere mosche dalla gabbia trasferendo in un contenitore vuoto. Se stessi mosche saranno utilizzati il ​​giorno successivo, trasferire immediatamente ad una nuova gabbia contenente una piastra di agar succo d&#39;uva appena yeasted. Le mosche possono essere utilizzati per 3-5 giorni sequenziali </li><li> Rimuovere mosche morte dal agar con un pennello pulito, facendo attenzione a non rompere l&#39;agar. </li><li> Raccogliere uova sciacquando piastra di agar con acqua distillata, delicatamente spazzolatura uova dalla superficie agar, e versando l&#39;impasto sulla maglia. Ripetere 3-4 volte fino a quando tutti o la maggior parte delle uova sono state rimosse dalla piastra di agar. </li></ol> 5. Dechorionate uova e trasferimento sterile DIET <ol><li> Preparare la armadio biosicurezza spruzzando l&#39;interno (compresi i lati) con il 70% di etanolo. Pulire il fondo con un tessuto di laboratorio, e sterilizzare il cappuccio con luce UV per ~ 15 min. Sterilizzare tutti i materiali di consumo non-biologici (campioni tazze, pennello, pinze, contenitori di rifiuti, 400 ml di acqua e 100 ml di ipoclorito di sodio allo 0,6%), a spruzzo con etanolo e subito messa in cabinet biosicurezza. Sterilizzare con luce UV per 15 min. </li><li> Avviare il primo di 2 lavaggi di ipoclorito di sodio posizionando la bussola con le uova in una tazza 120 ml campione o altro contenitore sterile. Versare lentamente ~ 90 ml di soluzione di ipoclorito di sodio 0,6% nella boccola fino a poco sotto il bordo. </li><li> Lavare le uova per 2,5 min. Periodicamente risospendere le uova utilizzando pinze per spostare la boccola su e giù nella soluzione di ipoclorito. </li><li> Trasferire la bussola direttamente in una seconda tazza esemplare, pre-riempita con 90 ml di candeggina, all&#39;interno dell&#39;armadio biosicurezza. </li><li> Ripeterepasso 5.3 all&#39;interno dell&#39;armadio biosicurezza. Alla fine del secondo trattamento candeggiante, le uova dovrebbero iniziare ad aderire ai lati della boccola. </li><li> Effettuare i passaggi 5,7-5,8 nell&#39;armadio biosicurezza. </li><li> Eliminare la candeggina e lavare la bussola con acqua sterile per 3 volte. Risospendere le uova più volte durante ogni lavaggio spostando la bussola con una pinza. Alla fine del terzo lavaggio più uova devono essere fissati al lato della boccola. </li><li> Utilizzando un pennello sterilizzati in etanolo, trasferire uova dal lato della boccola alla dieta sterile. Trasferire le uova singolarmente o in piccoli lotti. Obiettivo per 30-50 uova per fiala. Lasciare i tappi allentato per permettere l&#39;ossigeno di entrare nel tubo. Se fiale devono rimanere axeniche, trasferimento in un incubatore di insetti; altrimenti, aggiungere batteri come sotto. </li></ol> 6. Fare Gnotobiotic Flies Utilizzando 4 batteriche Specie <ol><li> preparare batteri <ol><li> Preparare un armadio biosicurezza sterilizzato con forniture necessarie (pipettes, scatole punta pipette, provette da centrifuga sterilizzati, brodo MRS, e rastrelliere provetta) come al punto 4.1. Pulire l&#39;esterno di provette con un laboratorio di etanolo imbevuto wipe prima di mettere in armadio biosicurezza. </li><li> Pellet i batteri in primo luogo il trasferimento di 500 ml di crescita durante la notte per una provetta per microcentrifuga sterile. Se la densità batterica è bassa, aggiungere fino a 1,5 ml a ciascuna provetta o in sequenza rimuovere surnatante e aggiungere la cultura in più per lo stesso tubo. Rimuovere campioni dal cabinet biosicurezza e centrifugare per 10 minuti a 10.000 x g. Utilizzare puntali con filtro per evitare la contaminazione tra i campioni. </li><li> Determinare la densità di ciascuna coltura misurando OD 600. Se si utilizza un lettore di piastre multi-bene, trasferire 200 ml di ogni cultura ad una piastra a 96 pozzetti in 1, 2 e 4- diluizioni. </li><li> Determinare la quantità di mMRS in cui diluire ciascun pellet cellulare (5.2.2) utilizzando uno spettrofotometro lettura della piastra e le seguenti equazioni. Piano di aggiungere abbastanza brodo per inoculare 50 &amp;# 956; l per ogni flacone mosca. <ol><li> Raccogliere OD 600 letture di 1: 1, 1: 2 e 1: 4 diluizione ciascuna coltura batterica su uno spettrofotometro piastriforme lettura. Selezionare la diluizione per ogni ceppo batterico che produce un valore di 600 OD sono compresi tra 0,1 e 0,2 e utilizzare questo valore e il suo fattore di diluizione corrispondente come &#39;O&#39; e &#39;D&#39; nelle formule indicate in 6.1.4.2 o 6.1.4.3. </li><li> Se si utilizza le 4 specie qui descritte, normalizzare le cellule per unità formanti colonia equivalente (CFU) / ml densità (OD 600 alla conversione CFU determinato in precedenza 20) utilizzando questa equazione: E = ((OB) x V x D) / C dove E = volume di risospendere pellet in (ml), O = OD 600 batteri, B = OD 600 supporti vergini, D = fold-diluizione, V = coltura batterica ml prima della centrifugazione, C = OD 600 di costante predeterminata. Vedere file di codice supplementare per gli esempi di calcoli utilizzando queste equazioni. per spettrofotometros che automaticamente vuoto, usare &quot;O&quot; al posto di &quot;OB&quot;. Nota: Le costanti predeterminate (unità OD 600, normalizzati a 10 7 CFU ml -1, costanti derivati ​​in 20) sono i seguenti: A. tropicalis (0.052), A. pomorum (0,038), L. brevis (0,056), L. plantarum (0,077). </li><li> Se si utilizzano altre specie batteriche (senza CFU / OD 600 costante è disponibile), normalizzare la densità di OD 600 = 0.1 utilizzando questa equazione: E = ((OB) x V x D) /0.1 OD 600 Nota: Le unità sono le stesse come nel passaggio 6.1.4.2. Vedere file di codice supplementare per gli esempi di calcoli utilizzando queste equazioni. </li></ol></li><li> Nel gabinetto biosicurezza, rimuovere il surnatante con una pipetta e risospendere il pellet in mMRS freschi o PBS come calcolato al punto 6.1.4.2. </li></ol></li><li> Seminare batteri <ol><li> Trasferimento 50 ml di batteri ai tubi conici con sterili dieta und dechorionated uova in armadio biosicurezza. Aggiungere batteri dopo il trasferimento uovo per evitare contaminazione tra fiale. </li><li> Posizionare provette inoculate in un incubatore a 25 ° C. </li></ol></li></ol> 7. Misurare CFU carico / prova di sterilità <ol><li> Per misurare il carico CFU in tutto omogenati corpo mosca, trasferimento 5 mosche (5-7 giorni dopo Eclosion) in una provetta da 1,7 ml microcentrifuga contenente 125 ml di sfere di ceramica e 125 ml di brodo mMRS. Omogeneizzare mosche usando un omogeneizzatore tessuto per 30 secondi a 4,0 m / sec. <ol><li> In alternativa, omettono perline e mano omogeneizzare in provette da microcentrifuga con pestelli in plastica per 1 min. </li><li> Se la quantificazione del microbiota intestinale, superficie sterilizzare le mosche per rimuovere i microbi esogeni 22. Trasferimento vola a una provetta contenente 100 ml etanolo al 70% per 1 min, aspirare l&#39;etanolo, e trasferimento in una nuova provetta per homogenizations. Se il contenuto di DNA dell&#39;intestino sarà misurato, sciacquare fo 1 min con ipoclorito di sodio 0,6% prima del lavaggio etanolo. </li></ol></li><li> Diluire l&#39;omogeneizzato con 875 microlitri mMRS, vortex per 5 secondi, e pipetta 120 ml di omogeneizzato nel primo pozzetto di una micropiastra. </li><li> Eseguire due sequenziali 1: 8 diluizioni con 10 microlitri omogeneizzato e 70 microlitri MRS nei prossimi due pozzi. <ol><li> Rimuovere 10 ml dal primo pozzo e aggiungerlo al secondo pozzetto contenente 70 ml MRS. Mescolare il contenuto del secondo pozzo accuratamente, trasferimento 10 microlitri dal secondo pozzo alla terza pozzetto contenente 70 microlitri MRS, e omogeneizzare. Questo porta a 3 le concentrazioni totali di originale omogeneizzato 1.000 ml: Non diluito, 1: 8, e 1:64. </li></ol></li><li> Trasferire 10 ml di ogni diluizione ad una lastra mMRS (utilizzando una pipetta multicanale se desiderato). Leggermente inclinare il piatto per diffondere la diluizione diversi millimetri verso il basso la superficie agar e consentire il liquido si asciughi prima di spostare il piatto. Il liquido si asciuga in The piastra rapidamente se le piastre sono 2 giorni di età, riducendo miscelazione di due goccioline adiacenti. </li><li> Incubare a 30 ° C per 1-2 giorni. Rimuovere le piastre dal termostato volta distinti, singole colonie sono visibili, e contare da una diluizione con 10-100 colonie isolate. </li><li> Calcola CFU per volare utilizzando l&#39;equazione E = C x D / P x V / F, dove E = CFU per volare, C = numero di colonie contato, D = diluizione, P = ml placcato, V = volume di volare omogenato, e F = numero di mosche omogeneizzati. </li></ol>

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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. 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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>

Gli autori non hanno nulla da rivelare.

<html> Il metodo qui descritto è uno dei diversi approcci per embrione dechorionation 8,11,18,25,26,27, insieme con altri metodi di allevamento mosche axeniche, tra cui il trasferimento seriale di adulti axeniche 18,27 o 13,18 trattamento antibiotico. Altri metodi includono dechorionation lavaggi etanolo e ridurre 11,25,26 o estendono 8 Trattamento ipoclorito. Diverse fasi di lavaggio può essere di aiuto l&#39;allevamento di diversi genotipi mosca: in un precedente...

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

La maggior parte degli animali sono intimamente associati con i batteri ( &#39;microbiota&#39;) dalla nascita alla morte 1. I confronti di ( &#39;convenzionale&#39;) gli animali privi di microrganismi ( &#39;axeniche&#39;) e di microrganismi-associati hanno dimostrato microbi influenzano diversi aspetti della salute degli animali, tra cui metabolica, nutrizionale, vascolari, epatiche, respiratorio, immunologico, endocrino, e la funzione neurologica 2. La mosca della frutta Drosophila melanogaster è un modello chiave per comprendere molti di questi processi in presenza di microbi 3,4 e per studiare l&#39;influenza microbiota su 5,6 salute degli animali. Nessuna specie batterica è presente in ogni individuo ( &#39;core&#39;), ma Acetobacter e Lactobacillus specie numericamente dominano il microbiota di entrambi D. laboratorio-allevati e catturati in natura melanogaster. Altro Acetobacteraceae (compresi Komagataeibacter e Gluconobacter), Firmicutes (come Enterococcus e Leuconostoc), e Enterobacteriaceae sono sia spesso presente in individui Drosophila a bassa abbondanza, o irregolarmente presenti in grande abbondanza 7-12. Il microbiota di Drosophila e mammiferi è incostanti all&#39;interno e attraverso le generazioni 14,19. Microbiota incostanza può portare a rumore fenotipica quando si misurano i tratti microbiota-dipendente. Ad esempio, la memorizzazione Acetobacteraceae influenza lipidi (trigliceridi) in Drosophila 15-18. Se Acetobacteraceae sono più abbondanti nella mosche di una fiala che in un altro 19, mosche isogenici possono avere diversi fenotipi 20. Una soluzione per il problema del microbiota incostanza nei topi 14 è stato in pratica dal 1960, con l&#39;introduzione di una comunità definita di 8 specie microbiche dominanti per cuccioli di topo ogni nuova generazione (alterata Schaedler flora),assicurando che ogni cucciolo è esposto agli stessi membri chiave del microbiota del mouse. Questa pratica controlla per la composizione microbica anche quando il microbiota non è l&#39;obiettivo primario di studio 32, e imposta precedente per assicurare la presenza di microbi chiave in una varietà di condizioni sperimentali. Per definire l&#39;influenza dei microbi sulla Drosophila nutrizione, diversi protocolli per derivare linee di volo axenici sono state sviluppate, compresa ipoclorito dechorionation di embrioni (sia derivato de novo ogni generazione o mantenuta generationally mediante trasferimento diete sterili) e trattamento antibiotico 13. Ci sono benefici per diversi approcci, come la facilità e rapidità sia per il trattamento di antibiotici e di trasferimento seriale, contro un maggior controllo delle variabili confondenti con de novo dechorionation (ad esempio, la densità d&#39;uovo, i microbi contaminanti residui, fuori bersaglio effetti antibiotici). Indipendentemente dal metodo dipreparazione, introduzione di specie microbiche specificati axeniche embrioni permette cultura della Drosophila con definite le comunità ( &#39;gnotobiotic&#39;). In alternativa, mimando l&#39;uso di flora Schaedler, questa comunità potrebbe essere inoculato alle uova convenzionalmente stabilite (seguenti passaggi 6-7 solo) per assicurare la presenza di microbi trait-influenzano in ogni flacone ed evitare complicazioni del microbiota incostanza. Qui si descrive il protocollo per l&#39;allevamento axeniche e gnotobiotic Drosophila da de novo dechorionation di embrioni, e per confermare la presenza di introdotto o contaminazione microbica taxa.

<table><tbody><tr><td>Brewer&#039;s Yeast</td><td>MP Biomedicals, LLC.</td><td>903312</td><td>http://www.mpbio.com/product.php?pid=02903312</td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>158968-3KG</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/158968?lang=en&amp;amp;region=US</td></tr><tr><td>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><td>Welch&#039;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></tr><tr><td>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></tr><tr><td>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></tr><tr><td>Stock Bottles</td><td>Genesee Scientific</td><td>32-130</td><td>https://geneseesci.com/shop-online/product-details/?product=32-130</td></tr><tr><td>Droso-Plugs</td><td>Genesee Scientific</td><td>49-101</td><td>https://geneseesci.com/shop-online/product-details/?product=49-101</td></tr><tr><td>Nylon Mesh</td><td>Genesee Scientific</td><td>57-102&amp;nbsp;</td><td>https://geneseesci.com/shop-online/product-details/715/?product=57-102</td></tr><tr><td>Plastic Bushing</td><td>Home Depot</td><td>100404002</td><td>http://www.homedepot.com/p/Carlon-1-in-Non-Metallic-Terminal-Adapter-E943FR-CTN/100404002</td></tr><tr><td>Plastic Bushing Cap</td><td>Home Depot</td><td>100153897</td><td>http://www.homedepot.com/p/1-1-4- in-Rigid-Plastic-Insulated-Bushing-2-Pack-27529/100153897</td></tr><tr><td>Specimen Cup</td><td>MedSupply Partners</td><td>K01-207067</td><td>http://www.medsupplypartners.com/covidien-specimen-containers.html</td></tr><tr><td>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><td>50 ml Centrifuge Tubes</td><td>TrueLine Centrifuge Tubes</td><td>TR2003</td><td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=TR2003</td></tr><tr><td>Food Boxes</td><td>USA Scientific</td><td>2316-5001</td><td>http://www.usascientific.com/search.aspx?find=2316-5001</td></tr><tr><td>Lysing Matrix D Bulk</td><td>MP Biomedicals, LLC.</td><td>116540434</td><td>http://www.mpbio.com/search.php?q=6540-434&amp;amp;s=Search</td></tr><tr><td>Filter Pipette Tips, 300 &amp;mu;l</td><td>USA Scientific</td><td>1120-9810</td><td>http://www.usascientific.com/search.aspx?find=1120-9810</td></tr><tr><td>Petri Dishes</td><td>Laboratory Product Sales</td><td>M089303</td><td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=M089303</td></tr><tr><td>Ethanol</td><td>Decon Laboratories, INC.</td><td>2701</td><td>http://www.deconlabs.com/products.php?ID=88</td></tr><tr><td>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><td>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><td>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><td>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><td>Yeast Extract</td><td>&amp;nbsp;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><td>Dipotassium Phosphate</td><td>Sigma Aldrich</td><td>P3786-1KG</td><td>http://www.sigmaaldrich.com/catalog/search?term=P3786-1KG&amp;amp;interface=All&amp;amp;&lt;br/&gt;
N=0&amp;amp;mode=match%20partialmax&amp;amp;lang=&lt;br/&gt;
en&amp;amp;region=US&amp;amp;focus=product</td></tr><tr><td>Ammonium Citrate</td><td>Sigma Aldrich</td><td>25102-500g</td><td>http://www.sigmaaldrich.com/catalog/search?term=25102-500g&amp;amp;interface=All&amp;amp;N&lt;br/&gt;
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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.

Allevamento di successo di mosche axeniche è confermata da isolamento di non CFU da homogenizations tutto il corpo di D. adulti melanogaster (figura 1). In alternativa, se l&#39;omogeneizzato placcato produce colonie, i flaconi sono contaminati e devono essere eliminati. Per mosche gnotobiotic, ciascuna delle quattro isolati batterici sono stati isolati da pool di 5 maschi adulti, dimostrando differenze CFU vitali totali associati con mosche adulte <st...

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

Un metodo per l&#39;allevamento di Drosophila melanogaster in condizioni axeniche e gnotobiotic è presentato. Fly embrioni vengono dechorionated in ipoclorito di sodio, trasferiti asetticamente alla dieta sterili, e allevati in contenitori chiusi. Inoculando dieta ed embrioni con i batteri porta ad associazioni gnotobiotic, e la presenza di batteri è confermata dalla placcatura di tutto il corpo omogenati Drosophila.

Allevamento la mosca della frutta<em&gt; Drosophila melanogaster</em&gt; Sotto axeniche e Gnotobiotic Condizioni

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

rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic

Research

JoVE Journal

Developmental Biology

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

21.0K Views.  Brigham Young University.  Un metodo per l'allevamento di Drosophila melanogaster in condizioni axeniche e gnotobiotic è presentato. Fly embrioni vengono dechorionated in ipoclorito di sodio, trasferiti asetticamente alla dieta sterili, e allevati in contenitori chiusi. Inoculando dieta ed embrioni con i batteri porta ad associazioni gnotobiotic, e la presenza di batteri è confermata dalla placcatura di tutto il corpo omogenati Drosophila. 

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

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Un modello di ferita Drosophila In Vivo per lo studio della neurorigenerazione la periferica e del sistema nervoso centrale

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Ricerca

Medicina

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

Drosophila melanogaster Protocollo di iniezione della larva

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

Immunologia e infezioni

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. We found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, we aim to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

A method to reactivate quiescent neural stem cells in cultured Drosophila brain explants has been established. Using this method, the role of systemic signals can be uncoupled from tissue-intrinsic signals in the regulation of neural stem cell quiescence, entry and exit.

Riattivazione delle cellule staminali neurali negli espianti cerebrali di Drosophila in coltura

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes ...

Neuroscienze

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Mantenimento di un<em&gt; Drosophila melanogaster</em&gt; Cage Popolazione

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Biologia

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Protocollo sperimentale per l'utilizzo di Drosophila come sistema modello invertebrati marini per test di tossicità in laboratorio

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Infezione batterica orale e spargimento in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

Semplici strumenti fatti in casa per gestire le mosche della frutta -Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

An Introduction to Drosophila melanogaster

Drosophila melanogaster, also known as the fruit fly, is a powerful model organism widely used in biological research that has made significant contributions to the greater scientific community over the last century. First, this video introduces the fruit fly as an organism, including its physical characteristics, life cycle, environment, and diet. Next, the reasons why fruit flies make an excellent model organism are discussed. For example, fruit flies are inexpensive to maintain in the laboratory, have simplified genetics, and short generation times allow for quick experiments with high sample numbers. Then, key discoveries and important Drosophila researchers, such as Thomas Hunt Morgan are profiled. Finally, applications of Drosophila research, ranging from genetics to cardiac and neurological development and disease, are provided. This video serves as an overview of the highly-important and influential model organism that is Drosophila melanogaster.

Drosophila melanogaster Fruit Fly as a Model Organism

Un'introduzione a Drosophila melanogaster

Drosophila melanogaster, also known as the fruit fly, is a powerful model organism widely used in biological research that has made significant ...

Didattica

JoVE Science Education

Biologia di base

Biologia I: lieviti, drosophila e c. elegans

Drosophila Maintenance

Drosophila melanogaster, commonly known as fruit flies, are a frequently used model organism for life science research. Although starting a collection of these critters may seem as easy as leaving a banana on your kitchen counter for too long, a productive fly colony in the lab requires careful husbandry and maintenance.
This video demonstrates the necessary steps for maintaining a healthy fly stock. The overview begins with the preparation and storage of the yeast and sugar-containing media on which flies feed. Next, the vessels most commonly used for housing Drosophila are shown, as well as how and when to move flies between these containers. Finally, the presentation also includes examples of the ways in which housing and feeding conditions are manipulated for biological experiments.

Maintenance and Husbandry of Drosophila melanogaster

Drosophila Manutenzione

Drosophila melanogaster, commonly known as fruit flies, are a frequently used model organism for life science research. Although starting a collection of ...

Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer

Source: Koyle, M. L., et al. <a href="https://www.jove.com/video/54219/" target="_blank">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. J. Vis. Exp. (2016).
The Drosophila embryo is surrounded by protective outer membranes. To gain access to the embryo, the chorion&#8211;the outermost of the membranes&#8211;is often removed. This video describes a commonly used method of dechorionation using sodium hypochlorite. The example protocol demonstrates the procedure while employing sterile techniques necessary for axenic or gnotobiotic animal rearing.

drosofila Raccolta e decorionazione degli ovuli: un metodo per rimuovere lo strato di uova più esterno

Source: Koyle, M. L., et al. Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions. J. Vis. Exp. (2016).
The Drosophila ...

Allevamento la mosca della frutta

Riepilogo

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