Vorremmo ringraziare Anthony Weinhold e i numerosi studenti che hanno lavorato con noi nel corso degli anni per il supporto tecnico, nonché suggerimenti per migliorare il nostro insetto procedure di allevamento.

1. uovo insieme (Figura 1)
<ol><li>Preparare tazza incita, inserire un'estremità del panno in Becher ~ 500 mL riempita con acqua calda (~ 40 ° C). Si sovrappongono i lati della tazza e fissare con un elastico. Piegare l'estremità libera del panno indietro sopra la parte superiore della Coppa.</li>
	<li>Posto Coppa incita in gabbia di mosche di 8 – 10 giorni vecchio per femmina Gravid h. ~ 2 stabile vola sarà oviposit sul panno.</li>
	<li>Allontana da uova in gabbia e risciacquare fuori il panno egging egging calice in un pentolino con bottiglia di lavaggio a pressione.</li>
</ol>2. preparare Pan allevamento larvale
<ol><li>
Preparare il supporto (quantità per 1 vaschetta di allevamento)
	<ol>
<li>Unire la crusca di frumento (500 g), trucioli di legno (200 g) e farina di pesce (115g) in vaschetta di plastica piatto di 10 L e mescolare bene.</li>
		<li>Aggiungere bicarbonato di ammonio (50 g) di acqua (~ 25 ° C, 1600 mL) e mescolare fino a completa dissoluzione.</li>
		<li>Aggiungere acqua soluzione gli ingredienti secchi e mescolare affinché nessun materiale unwetted. Livello medio in padella, ma non comprimere.</li>
	</ol>
</li>
	<li>
Aggiungere le uova volare stabile
	<ol>
<li>Fare un solco poco profondo in mezzo la lunghezza della padella.</li>
		<li>Depositare 1 mL di stabile volare uova (~ 8.000 uova) in solco con pipetta di uovo (Figura 2). Nota: Pipetta uovo fatta tagliando una pipetta graduata in plastica al punto desiderato, inserendo 100 mesh schermo e incollaggio pipetta tornare insieme.</li>
		<li>Coprire le uova con un sottile strato di mezzo per prevenire il disseccamento.</li>
		<li>Coprire la pentola con federa, chiudere con un elastico e l'etichetta. Mettere pentole in camera larvale (23 ± 2 ° C, 30 – 50% RH e 12:12 [L:D] h fotoperiodo). Nota: Lo sviluppo larvale dura 10 – 14 giorni dal momento della deposizione delle uova.</li>
	</ol>
</li>
</ol>3. pupal elaborazione, larve spostarsi sul margine del mezzo 7 – 9 giorni dopo la deposizione delle uova e Pupariate di giorni 13 o 14
<ol><li>Paletta puparia, principalmente situato intorno ai bordi della vaschetta sotto la crosta che si forma sulla superficie del mezzo, fuori e messo in una pentola pulita.</li>
	<li>Riempire la pentola, con puparia, ½ a ¾ piena d'acqua. Rompere con ciuffi di puparia e medie. Componenti di crusca e proteine di frumento del mezzo lavandino mentre puparia > 1 giorno vecchio galleggiante19.</li>
	<li>Rimuovere materiale galleggiante, puparia e alcuni componenti medi con un colino e lavare attraverso una serie di setacci (n. 5, #7, #12, #20) per rimuovere il mezzo rimanente. Il setaccio #12 raccoglie il puparia e il #20 mantiene rifiuti solidi di entrare lo scarico. Lavare puparia tra setacci con uno spruzzatore di lavandino.</li>
	<li>Risciacquare il setaccio #12, puparia, in una pentola pulita. Riempire la pentola a ½ pieno di acqua. Versare puparia galleggiante in un colino e lasciare drenare l'acqua in eccesso.</li>
	<li>Trasferimento drenati puparia a schermo di secchezza con ventilatore e lasciare fino a secco (Figura 3).</li>
</ol>4. pupal scaffale, un metodo alternativo per la raccolta Puparia è con un ripiano Pupal18
Nota: La mensola fatta da un pezzo di plastica tagliato dalla fine di una padella piatto tipico di 10 L (10,2 cm di altezza x 10,2 cm di larghezza x 31,9 cm di lunghezza).
<ol><li>Preparare il supporto come descritto al punto 2.1. Cono medio in padella da ~2.5 cm profondo su un'estremità a ~7.5 cm profondo su altro.</li>
	<li>
Inserire ripiano su supporto nella parte bassa di allevamento padella e nastro adesivo per evitare le larve dallo strisciare lungo la mensola (Figura 4).
	<ol>
<li>Saturare la spugna (~14.5 x 9.0 x 4,5 cm3, cellulosa rigenerata) e avvolgerlo in un panno imbevuto di acqua (30,5 x 42 cm2, cotone) e posto sullo scaffale circa mezzo pollice dal mezzo.</li>
	</ol>
</li>
	<li>
Aggiungere le uova, come descritto al punto 2.2 e procedere come prima.
	<ol>
<li>Verifica padelle ogni giorno per assicurare che la spugna rimanga umida.</li>
	</ol>
</li>
	<li>Risciacquo puparia dallo scaffale, spugna e panno in un piatto pulito padella e versare nel colino 14 giorni dopo la deposizione delle uova.</li>
</ol>5. controllo di qualità
<ol><li>Pesare tutti i puparia prodotta in padella (Totale wt.).</li>
	<li>Isolare 100 puparia, pesare e inseriti in una capsula di Petri di 9 cm.</li>
	<li>Conteggio e sesso adulti ~ 5 – 8 giorni più tardi per determinare il tasso di emersione e rapporto del sesso. Conservare capsule di Petri con adulti è emersi in un congelatore per il conteggio in un secondo momento, se necessario. Mosche sono sessuati dalla forma dei loro occhi e larghezza della placca fronto-orbitale20 o genitali sotto ingrandimento basso.</li>
	<li>Peso totale record, peso di 100 puparia e numero di emersi adulti maschi e femmine.</li>
</ol>6. preparazione di sangue
<ol><li>Raccogliere sangue fresco di bovini da un macello locale a secchi 19 L contenenti 70 g di sodio citrato tribasico diidrato in 500 mL di acqua. Mescolare vigorosamente per 5 – 10 min di sangue e restituito al laboratorio.</li>
	<li>Sangue di ceppo attraverso un colino per rimuovere la formazione di coaguli e partizionato in contenitori 2L, etichetta con la data di raccolta e conservare in congelatore (-20 ° C).</li>
	<li>Rimuovere il contenitore dal freezer e posto in frigorifero 2 giorni prima del necessario. Sangue può essere utilizzato per ~ 2 settimane una volta scongelati e conservati nel congelatore per fino a 1 anno.</li>
</ol>7. adult Maintenance
<ol><li>Preparare gabbie, fondo gabbia linea con carta da macellaio per facilitare la pulizia.</li>
	<li>Mettere 50 g di secco pupe volare stabile (~ 3.500) in ogni gabbia. Adulti eclose poche ore a un paio di giorni dopo l'elaborazione pupal.</li>
	<li>Posto un sangue fresco imbevuto femminile tovagliolo sulla cima di ogni gabbia per l'alimentazione. Avviare l'alimentazione entro 24 h dalla eclosion adulto e ripetere ogni giorno fino al giorno prima della deposizione delle uova (9 – 10 giorni dopo la nascita).</li>
	<li>Dopo la deposizione delle uova (sezione 1), posizionare la gabbia in freezer per 4 – 8 h e poi pulire rimuovendo la carta, risciacquo frass dai lati e piano con acqua calda e strofinare tutte le superfici con detersivo e candeggina. Sciacquare le gabbie con acqua calda e lasciare per asciugare.</li>
</ol>

<ol>
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Vet. 24 (4), 387-395 (2015).</li><li>Sol&#243;rzano, J. -. A., Guilles, J., Bravo, O., Vargas, C., Gomez-Bonilla, Y., Bingham, G., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+trapping+of+stable+flies+(Diptera:+Muscidae)+developing+in+pineapple+residues+(Ananas+comosus)+in+Costa+Rica.">Biology and trapping of stable flies (Diptera: Muscidae) developing in pineapple residues (Ananas comosus) in Costa Rica.</a> J. Insect Sci. 15 (1), 145 (2015).</li><li>Berry, I. L., Stage, D. A., Campbell, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+and+economic+impacts+of+stable+flies+on+cattle.">Populations and economic impacts of stable flies on cattle.</a> Trans. Am. Soc. Agric. Eng. 26, 873-877 (1983).</li><li>Taylor, D. B., Vreysen, J., Hendrichs, R., Cardoso Pereira, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Area-wide+management+of+stable+flies.">Area-wide management of stable flies.</a> Area-wide management of insect pests. , (2017).</li><li>Bishopp, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stable+fly+(Stomoxys+calcitrans+L.),+an+important+live+stock+pest.">The stable fly (Stomoxys calcitrans L.), an important live stock pest.</a> J.Econ. Entomol. 6 (1), 112-126 (1913).</li><li>Hogsette, J. A., Ruff, J. P., Jones, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+fly+biology+and+control+in+northwest+Florida.">Stable fly biology and control in northwest Florida.</a> J. Agric. Entomol. 4 (1), 1-11 (1987).</li><li>Lysyk, T., Kalischuk-Tymensen, L., Selinger, L., Lancaster, R., Wever, L., Cheng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+stable+fly+larvae+(Diptera:+Muscidae)+on+an+egg+yolk+medium.">Rearing stable fly larvae (Diptera: Muscidae) on an egg yolk medium.</a> J. Med. Entomol. 38, 382-388 (1999).</li><li>Romero, A., Broce, A., Zurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+bacteria+in+the+oviposition+behaviour+and+larval+development+of+stable+flies.">Role of bacteria in the oviposition behaviour and larval development of stable flies.</a> Med. Vet. Entomol. 20, 115-121 (2006).</li><li>Glaser, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+collecting+clean+stable+fly+(Diptera:Muscidae)+pupae+of+known+age.">A new method for collecting clean stable fly (Diptera:Muscidae) pupae of known age.</a> Southwest. Entomol. 34 (4), 469-476 (2009).</li><li>Champlain, R. A., Fisk, F. W., Dowdy, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+improvements+in+rearing+stable+flies.">Some improvements in rearing stable flies.</a> J. Econ. Entomol. 47 (5), 940-941 (1954).</li><li>Zumpt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The stomoxyine biting flies of the world. , (1973).</li><li>Wienhold, B. J., Taylor, D. 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Mosche stabili possono trovare lo sviluppo in un'ampia varietà di substrati in natura e possono essere allevati in molti tipi di media in laboratorio. Abbiamo usato dei trucioli di legno e vermiculite come agenti volumizzanti. La vermiculite ha funzionato bene, ma fatto puparia separazione da allevamento medio difficile ed era costoso (~$0.60/pan). Forse a causa il rigore aggiunto di separare puparia dal mezzo, eclosion era inoltre più basso con vermiculite, 57% vs 75% per trucioli di legno. Trucioli di Cottonwood eran...

Mosche stabili, Stomoxys calcitrans (L.), sono ematofagi mosche cui punture dolorose può interferire il comportamento al pascolo del bestiame, causano dolore e sofferenza per animali da compagnia e interferire con attività ricreative umane in tutto il mondo. Mosche stabili immaturi sviluppano nella fermentazione materia vegetativa, spesso contaminato da rifiuti di origine animale. Colture e mutevoli pratiche agronomiche hanno prodotto gravi episodi di mosche stabili in presenza di residui colturali, verdure in Australia1, canna da zucchero in Brasile2e l'ananas in Costa Rica3. Anche se solo 14 mosche stabili per animale sono considerati la soglia economica4, sono state fatte osservazioni di più di 2.000 mosche per animale durante recenti focolai5. Tali livelli di infestazione riducono la produttività di host per vicino allo zero e possono causare mortalità6. A seguito di focolai agronomico associati, mosche stabili stanno ricevendo rinnovato interesse e domanda per colonie di laboratorio è aumentato drammaticamente.
Per quanto riguarda tutti gli insetti holometamorphic, mosche stabili ottenere tutti i nutrienti necessari per la crescita durante la fase larvale o immatura. Pertanto, una componente importante di un sistema di allevamento è la dieta larvale o substrato. Le larve del Mosca stabile sono state osservate lo sviluppo in un'ampia gamma di substrati in campo7 e sono dipendente della comunità microbica del substrato8,9. Substrati naturali larvali sono principalmente composte di decomposizione o vegetativi materiali spesso contaminati con rifiuti azotati di fermentazione.
Per l'allevamento di laboratorio, substrati larvale volare stabile sono di solito composte da un materiale vegetativo e una fonte di azoto aggiunto. Numerosi materiali sono stati usati per le diete larvale volare stabile. Le diete larvale prime ha imitato substrati naturali e incluso di fermentazione paglia di avena e letame di cavallo o mucca10,11. Fonti di carboidrati includono frumento crusca12,13,14, erba medica pasto12,13,14 e una formulazione commerciale sviluppato da specialità chimiche Produttori Association (CSMA, crusca di grano 33%, 27% di farina di erba medica, lievito di birra 40% granuli)13,14,15,16. Fonti di azoto includono lievito sospensione12, farina di pesce e di bicarbonato di ammonio17. Materiali inerti volumizzanti sono spesso inclusi nelle diete tra cui avena scafi12, bagassa13, vermiculite16, trucioli di legno13,18 e gusci di arachidi pellettato14.
Un obiettivo primario di laboratorio allevamento è quello di produrre un prodotto che è come fisiologicamente simile a "wild type" possibile in modo che gli esperimenti del laboratorio produrrà risultati riflettono quelle delle popolazioni di campo. Ciò richiede che essere minimizzato in allevamento e selezione per mantenere genetica diversità e risorse nutritive essere paragonabili a quelle nel campo. Gli obiettivi secondari sono per ridurre al minimo il lavoro e le spese. Una componente importante di minimizzare le spese è l'utilizzo di componenti di alimentazione disponibile localmente. Al volo stabile del sistema presentato di allevamento è stato sviluppato per raggiungere questi obiettivi.

<table>
	<tbody>
		<tr>
			<td>Diamalt</td>
			<td>Premier Malt Products, Inc., Saddle Brook, NJ</td>
			<td>2540</td>
			<td></td>
		</tr>
		<tr>
			<td>CSMA Fly media</td>
			<td>Purina Animal Nutrition, Arden Hills, MN</td>
			<td>5S6Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Maxi Pad</td>
			<td>The Tranzonic Co., Cleveland, OH, USA</td>
			<td>5001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf Manna</td>
			<td>MannaPro, Chesterfield, MO, USA</td>
			<td>Manna Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Spectrum Chemical Manufacturing Corp, Gardena, CA</td>
			<td>A1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat bran, Coarse</td>
			<td>Siemer Milling Company, Teutopolis, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wood shavings</td>
			<td>Tractor Supply Company, Brentwood, TN</td>
			<td>502770699</td>
			<td></td>
		</tr>
		<tr>
			<td>Fishmeal</td>
			<td>Consumer Supply Distributing, North Sioux City, SD</td>
			<td>F1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult cages</td>
			<td>All Aluminum Window Company, Lincoln, Ne</td>
			<td>Custom</td>
			<td>45 &#215; 45 &#215; 45 cm, 18 &#215; 16 mesh aluminum screen, stockinette access</td>
		</tr>
		<tr>
			<td>9 &#215; 28 cm black cotton cloth</td>
			<td>Robert Kaufman Fabrics, Los Angeles, CA</td>
			<td>K040-114</td>
			<td>Egging cloth</td>
		</tr>
		<tr>
			<td>10 liter plastic dish pans</td>
			<td>Rubbermaid, Saratoga Springs, NY</td>
			<td>FG2951ARWHT</td>
			<td>Larval pans</td>
		</tr>
		<tr>
			<td>Stockinette, Cotton, 12&#34; X 25 yd roll</td>
			<td>Tex-Care Medical Company, Burlington, NC</td>
			<td>91311-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory rearing of stable flies and other muscoid diptera

Mosche stabili, Stomoxys calcitrans, sono parassiti gravi di bestiame, gli esseri umani, animali da compagnia e animali selvatici in tutto il mondo. Durante gli ultimi 20 + anni, cambiamenti nelle pratiche agronomiche ha provocato gravi epidemie di mosche stabili in diversi paesi. Questi focolai perturbato produzione zootecnica e ricreazione umana conseguente richieste pubbliche per aumentare gli sforzi di ricerca e gestione per questo parassita. Una procedura semplice e poco costoso per l'allevamento di mosche stabili per le ricerche di laboratorio è presentata. La procedura utilizza componenti localmente disponibili dieta, attrezzature e forniture. La procedura può essere adattata per l'allevamento di altre mosche muscoidi inclusi viso volare (Musca autumnalis), corno fly (da schistosoma haematobium irritans) e Mosca domestica (Musca domestica). La procedura produce puparia Vola stabile con una media di 12,5 mg e ~ 35% uovo alla sopravvivenza adulto. Circa 3000 mosche sono prodotte in ogni vaschetta.

Pupariate 10 – 14 giorni di larve e adulti emergono 14 – 16 giorni dopo la deposizione delle uova. Tempo di generazione, uovo, è ~ 24 giorni. Allevamento dei dati per maggio 2013 al gennaio 2017 con tre diversi agenti volumizzanti e due colonie sono presentati nella Figura 5. Cottonwood ha dato il miglior rendimento, 3867 ±1442 (<img alt="Equation" src="/files/ftp_upload/57341/57341eq1.jpg"> </img> ± deviazione standard) pupe peso 12,5 ± 1,6 mg con 74...

Watch this Scientific Journal Video about Laboratory Rearing of Stable Flies and Other Muscoid Diptera at JoVE.com

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Una procedura per l'allevamento di mosche stabili (Stomoxys calcitrans) è presentata. La procedura utilizza materiali localmente disponibili per dieta componenti, attrezzature e forniture.

Laboratorio di allevamento di mosche stabili e altri ditteri muscoidi

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

laboratory-rearing-of-stable-flies-and-other-muscoid-diptera

Research

JoVE Journal

Biology

8.5K Views.  ARS-USDA. Una procedura per l'allevamento di mosche stabili (Stomoxys calcitrans) è presentata. La procedura utilizza materiali localmente disponibili per dieta componenti, attrezzature e forniture.

Video: Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Microbial Communities in Nature and Laboratory - Interview

Comunità microbica in natura e laboratorio - Intervista

Ricerca

Biologia

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protocollo per la cura dei Mosquito (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Raccolta e Preparazione Drosophila Gli embrioni per la registrazione elettrofisiologici e altre procedure

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA Stabile-isotopica Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Un protocollo ottimizzato per la cura dei Fopius arisanus, Un parassitoide di frutta Tephritid mosche

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

Tecniche di laboratorio asettico: Metodi Placcatura

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

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 ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>Dissezione del sacco endolinfatico da topi

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

Dissezione del sacco endolinfatico da topi

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Isolamento scalabile e purificazione di vescicole extracellulari da Escherichia coli e altri batteri

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Laboratorio di allevamento di mosche stabili e altri ditteri muscoidi

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Esplora Altri Video

Capitoli in questo video

Comunità microbica in natura e laboratorio - Intervista

Protocollo per la cura dei Mosquito (A. gambiae)

Raccolta e Preparazione Drosophila Gli embrioni per la registrazione elettrofisiologici e altre procedure

DNA Stabile-isotopica Probing (DNA-SIP)

Un protocollo ottimizzato per la cura dei Fopius arisanus, Un parassitoide di frutta Tephritid mosche

Tecniche di laboratorio asettico: Metodi Placcatura

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

Dissezione del sacco endolinfatico da topi

Dissezione del sacco endolinfatico da topi

Isolamento scalabile e purificazione di vescicole extracellulari da Escherichia coli e altri batteri