I finanziamenti per lo sviluppo di questo protocollo è stato fornito dal Consorzio Pennsylvania sud-est per l&#39;istruzione superiore e il Dipartimento di Scienze Cabrini College. Il finanziamento e il sostegno aggiuntivo provenivano da Arcadia University e Immacolata University. Abbiamo particolarmente Ringraziamo il Dr. Karen Snetselaar al St. Joseph University per prendere gentilmente le immagini al microscopio elettronico dei nostri fagi isolati. Ulteriore supporto è stato fornito dai Cacciatori Howard Hughes Medical Institute Science Education Alliance fagi Advancing Genomica e Evolutionary Science programma (SEA-fagi).

1. Preparazione di cellule Arthobacter per Phage Isolation <ul><li> Cultura Arthrobacter sp. KY3901 colonie striati su Luria Bertaini (LB) piastra agar incubata a 30 ° C per 2-3 giorni. Scegli una colonia e utilizzare un&#39;ansa sterile per aggiungerlo a 250 ml di brodo LB in un pallone di coltura sconcertato e incubare in un incubatore agitazione a 225 rpm a 30 ° C. </li><li> Lasciare circa 24 ore di crescita per ottenere cellule in fase esponenziale fine / inizio stazionarie per esperimenti di infezione fagi. Monitorare lo stato di crescita batterica strettamente per evitare che le cellule di entrare nel centro di stato di crescita stazionaria tardiva. NOTA: la crescita delle cellule deve essere composto da un OD densità ottica di 600 tra 0,5 .0.7 per le procedure di isolamento fago e purificazione / passaggi di seguito descritti. </li></ul> 2. Raccolta dei fagi da campioni di suolo <ol><li> Raccogliere 200-400 g di suolo da posizione desiderata. Nota: Poiché i batteri nel Arthrobagenere CTER sono batteri del suolo comuni che e fagi che li infettano possono trovare in e sono presenti in quasi tutti i tipi di terreno. </li><li> Aggiungere almeno 400 ml di Phage Buffer (PB) [68 mM NaCl, 10 mM MgSO 4, 10 mM Tris-Cl (pH 7,5)] al terreno e miscelare in un matraccio abbastanza grande in modo che almeno 200 ml di PB è nel surnatante ed è in grado di essere estratto. </li><li> Mescolare agitando o agitando delicatamente fino a quando il terreno è sospeso e lasciare il sedimento terreno di stabilirsi, di solito 30 min. </li><li> Passare il &quot;estratto terreno&quot; su carta da filtro per filtrazione a gravità per rimuovere i detriti detriti sedimenti. </li><li> Aggiungere la polvere LB e miscelare per ottenere una soluzione al 2% (4 g LB polvere in 200 ml di estratto suolo). </li><li> Far passare la soluzione attraverso un filtro di 0,22 micron per filtrazione sotto vuoto per ottenere un filtrato sterile. Se possibile, passare alla fase 2.7 immediatamente. Se del caso, conservare i campioni filtrati a 4 ° C per una settimana prima di procedere, tuttavia, il numero di particelle fagiche vitalisarà ridotto. </li><li> Aliquota più 50 ml di miscela di estratto LB / terreno filtro sterilizzato in singole da 250 ml sterile agitando cultura palloni sconcertato utilizzando tecniche asettiche. Aggiungere sterile M CaCl soluzione 2.0 2 alle concentrazioni finali desiderati (0-50 mm) in quanto diversi fagi Arthrobacter crescono in modo ottimale in diverse CaCl 2 condizioni. NOTA: Troviamo che la maggioranza dei fagi Arthrobacter individuati da noi sono all&#39;interno della gamma di 1 mM-2.5 mM CaCl 2. Tuttavia, molti dei fagi isolati cresciuto da noi esclusivamente a 0 mM CaCl 2 .o ad altissima CaCl 2 concentrazioni. Procedere immediatamente al punto 3.1. </li></ol> 3. Phage isolamento <ol><li> Aggiungere 1 e 2,5 ml di ritardo esponenziale / inizio stazionaria coltura batterica di fase a ciascuno dei flaconi. Per un OD 600 0,5-0,7, utilizzare 1 ml di cellule. Per una OD 600 superiore un OD di 0,7 utilizzare 2,5 ml dicellule. NOTA: Le cellule in fase di crescita esponenziale in genere producono più fagi e titoli superiori rispetto alle cellule in crescita fase stazionaria. </li><li> Agitare beute a 250 rpm a 30 ° C per circa 24 ore in un incubatore agitazione. </li><li> Dopo un periodo di incubazione 24 ore rimuovere fiaschi di arricchimento dal termostato scuotimento e diluire i campioni di arricchimento di dieci volte in tampone fago. </li><li> Istituito tubi di coltura con 0,5 ml di ritardo esponenziale / inizio stazionario coltura batterica di fase e aggiungere diverse quantità di coltura di arricchimento (5 ml a 500 ml di una diluizione 10 -1 in PB) ai tubi di coltura. NOTA: Si consiglia di testare una vasta gamma di concentrazioni dal momento che ogni campione di suolo genera diversi titoli fagi dopo arricchimento. </li><li> Aggiungere CaCl 2 per provette di coltura per effettuare una concentrazione finale pari alla concentrazione presente nel pallone arricchimento originale. Utilizzare una gamma di CaCl 2 concentrazioni di selezionare per molti fagi wesima variabile CaCl 2 dipendenze. NOTA: La maggior parte dei fagi saranno isolati all&#39;interno della gamma CaCl 2 di 1-2,5 mm, ma ci sono fagi isolati più in modo ottimale ovunque 0-50 mM CaCl 2 concentrazioni. Per questo motivo è meglio controllare una gamma di CaCl 2 concentrazioni per massimizzare il numero di unici isolati fagi Arthrobacter. </li><li> Mescolare 4,5 ml di LB top agar nel tubo cultura e versare il composto su una piastra di agar LB. Agitare delicatamente per distribuire attraverso la piastra in modo uniforme e consentire top agar a solidificare per circa 15 min. NOTA: Top agar può essere preparato in lotti più grandi (250 ml) e top agar extra può essere conservato a temperatura ambiente e riutilizzato per successivi esperimenti per almeno due settimane. Anche se ci sono diverse formule utilizzate per fare top agar, facciamo top agar utilizzando 7g / L di agar mescolato con LB. Inserire superiore agar in un bagno di acqua 60 ° C per mantenere allo stato liquido durante il corso di un esperimento. </li><li> Inverti tegli piastra e incubare a temperatura desiderata (temperatura di 30 ° C) O / N a 48 hr. Vary queste condizioni per ottimizzare i fagi attuali. Fagi più isolati provengono da piastre incubate a 30 ° C a 1-2.5 CaCl 2 mM concentrazione dopo una incubazione di 24 ore. </li></ol> 4. Phage Purificazione <ol><li> Dopo un controllo di incubazione 24 ore per la formazione della placca sui piatti. Continuare l&#39;incubazione per un massimo di 72 ore se non placche sono evidenti o per determinare se appariranno targhe supplementari. Se necessario, le piastre negozio con targhe putativi per un massimo di una settimana a 4 ° C prima di procedere. NOTA: Non è raro trovare una varietà di differenti morfologie placca su una piastra. Trovare placche dopo 3 giorni di incubazione è possibile ma rara. </li><li> Purificare fagi desiderati da placche chiaramente isolati con il metodo striscia targa. Toccare una targa con un bastone di legno sterile. Streak la targa eseguendo un bastone di legno con una fiamma, allowing bastone raffreddare brevemente, e poi strofinando il bastone sulla piastra di agar nel moto dolce come mostrato in Figura 1. Ripetere questo utilizzando uno scovolo per ogni passaggio. </li><li> In alternativa, utilizzare il tradizionale saggio di placca titolo di isolare i singoli placche fagi. Il saggio di placca titolo richiede l&#39;uso di più reattivi come LB agar LB e superiore agar e materiali come piastre Petri per l&#39;isolamento fago placca. Abbiamo avuto un grande successo con il metodo striscia targa ma è tecnicamente più facile isolare singole placche utilizzando il saggio di placca titolo. </li><li> Una volta che la piastra di agar LB è striata almeno tre volte, segnare l&#39;area con la più bassa concentrazione di particelle fagiche putativi e delicatamente applicano 0,5 ml di Arthrobacter e 4,5 ml di LB agar fuso superiore (contenente la stessa concentrazione di cloruro di calcio come la piastra madre ) e permettono di diffondere in modo uniforme in tutto il piatto. </li><li> Capovolgere e incubare piastra O / N. Ripetere la targa strisciatecnica per almeno tre iterazioni per assicurare una specie fagi puri è isolato. Conservare le piastre a 4 ° C per un massimo di una settimana, se necessario tra strisce senza una significativa perdita di redditività fago. </li><li> Placche volta desiderati sono stati coltivati ​​e isolato sul piatto striscia finale, toccare uno ben isolato targa con una punta micropipetta e risospendere in 100 microlitri PB. Serialmente diluire questa soluzione 10 0 fuori della diluizione 10 -8 passando 20 microlitri del campione in 180 ml ​​di PB fresco. </li><li> Aggiungere 10 ml di ciascuna diluizione di 0,5 ml di Arthrobacter cresciuto in una provetta per 5 a 10 min a temperatura ambiente e la piastra mescolando i batteri infettati con 4,5 ml di LB agar fuso superiore contenente la stessa concentrazione di CaCl 2 utilizzati per isolare il fago dal campione originale suolo. Salvare tutte le diluizioni effettuate a 4 ° C fino al giorno successivo. </li><li> Determinare la diluizione in serie fago necessario per fare un modello web utilizzando il tradizionale titolo placcadire. Lo scopo del saggio di placca titolo è determinare il titolo fago necessario per ottenere una placca modello web. Identificare un modello piastra tela come in gran parte privi di batteri, ma contenente resti di batteri che non sono ancora stati lisati per fagi. Aggiungere 5 ml di cultura Arthrobacter stazionario esponenziale / inizio tardi per un 50 ml pallone di coltura sterile. <ol><li> Aggiungere 100 ml di diluizione (che ha dato un modello web alla cultura Arthrobacter) nel pallone di coltura e permettono fagi di infettare le cellule batteriche per 5 a 10 minuti a RT. Mescolare con fusa superiore agar LB contenente la stessa concentrazione di CaCl 2 come utilizzato per identificare inizialmente fago e piastra 5 ml di questa miscela su 10 piastre LB fresco. Lasciar solidificare ed incubare le piastre capovolte a 30 ° C. </li></ol></li><li> Il giorno successivo, inondare le placche modello web con 5 ml di PB e negozio O / N a 4 ° C o 4 ore a temperatura ambiente. </li><li> Filtrare il lisato fago attraverso un filtro siringa da 0,22 microne conservare a 4 ° C. Utilizzare questo fago magazzino finale per ulteriori esperimenti come le immagini di microscopia elettronica di particelle dei fagi, fago isolamento DNA genomico per l&#39;analisi di restrizione del DNA enzima e il sequenziamento genomico utilizzando le procedure standard. Per la conservazione a lungo termine del fago magazzino, aggiungere una quantità uguale di lisato fago di glicerolo sterile in piccole fiale per azione archiviazione a -80 ° C. NOTA: Per chi non sperimentato con queste tecniche fagi, una grande risorsa è il sito online www.phagesdb.org accessibili. </li></ol>

<ol>
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AD26 and Use of this Strain in Bioremediation of Contaminated Soil.</a> J Environ Sci. 20, 1226-1230 (2008).</li><li>Wang, J., Zhu, L., Liu, A., Ma, T., Wang, Q., Xie, H., Wang, J., Jiang, T., Zhao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+an+Arthrobacter+sp.+Strain+HB-5+that+Transforms+Atrazine.">Isolation and characterization of an Arthrobacter sp. Strain HB-5 that Transforms Atrazine.</a> Environ Geochem Hlth. 33, 259-266 (2011).</li><li>Mages, I. S., Frodl, R., Bernard, K. A., Funke, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identities+of+Arthrobacter+spp.+And+Arthrobacter-Like+Bacteria+Encountered+in+Human+Clinical+Specimens.">Identities of Arthrobacter spp. And Arthrobacter-Like Bacteria Encountered in Human Clinical Specimens.</a> J Clin Microbiol. 46 (9), 2980-2986 (2008).</li><li>Brown, D. R., Holt, J. G., Pattee, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Arthrobacter+Bacteriophages+and+Their+Application+to+Phage+Typing+of+Soil+Arthrobacters.">Isolation and Characterization of Arthrobacter Bacteriophages and Their Application to Phage Typing of Soil Arthrobacters.</a> Appl Environ Microbiol. 35 (1), 185-191 (1978).</li><li>Petrovski, S., Seviour, R. J., Tillett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Gordonia+and+Nocardia+Stabilized+Foam+Formation+by+Using+Bacteriophage+GTE7.">Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7.</a> Appl Environ Microbiol. 77 (21), 7864-7867 (2011).</li><li>Le Marrec, C., Moreau, S., Loury, S., Blanco, C., Trautwetter, a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Characterization+of+Site-specific+Integration+Functions+of+phi+AAU2+infecting+&#8220;Arthrobacter+aureus&#8221;+C70.">Genetic Characterization of Site-specific Integration Functions of phi AAU2 infecting &#8220;Arthrobacter aureus&#8221; C70.</a> J Bacteriology. 178 (7), 1996-2004 (1996).</li><li>Casida, L. E., Liu, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthrobacter+globiformis+and+Its+Bacteriophage+in+Soil.">Arthrobacter globiformis and Its Bacteriophage in Soil.</a> J Appl Microbiol. 28 (6), 951-959 (1974).</li><li>Einck, K. H., Pattee, P. A., Holt, J. G., Hagedorn, C., Miller, J. A., Berryhill, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+a+Bacteriophage+of+Arthrobacter+globiformis.">Isolation and Characterization of a Bacteriophage of Arthrobacter globiformis.</a> J Virol. 12 (5), 1031-1033 (1973).</li><li>Twest, R., Kropinski, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+Enrichment+from+Soil+and+Water.">Bacteriophage Enrichment from Soil and Water.</a> Methods Mol Bio. 501, 15-21 (2009).</li><li>Fullner, K. J., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacteriophage+L5+Infection+of+Mycobacterium+bovis+BCG:+Implications+for+Phage+Genetics+in+the+Slow-growing+Mycobacteria.">Mycobacteriophage L5 Infection of Mycobacterium bovis BCG: Implications for Phage Genetics in the Slow-growing Mycobacteria.</a> Mol Microbiol. 26 (4), 755-766 (1997).</li><li>Robb, S. M., Woods, D. R., Robb, F. 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Gli autori dichiarano interessi finanziari concorrenti.

Nonostante i molti precedenti tentativi di isolare i fagi in grado di infettare gli host Arthrobacter, abbiamo avuto poco successo utilizzando le procedure di arricchimento standard. Il metodo generalizzato di arricchimento batterica sviluppato e adattato da van Twest e Kropinski 10 per arricchire fagi da campioni ambientali resta la base per la maggior parte delle procedure di arricchimento. Prove da studi precedenti suggeriscono che i metodi di placcatura diretta hanno prodotto placche rilevabili s...

L&#39;ubiquità di specie Arthrobacter in ambienti di terreno offre un vasto numero e la diversità dei fagi che possono essere isolati da questa specie di batteri ospiti. Membri batteriche della famiglia Acintobacteriaceae sono più notevole per i loro percorsi catabolici di degradare composti recalcitranti come l&#39;atrazina e vari altri pesticidi e diserbanti 1,2,3. Sebbene la maggior parte della ricerca è stata effettuata utilizzando ceppi ambientali di Arthrobacter, isolati clinici di questo genere si trova nel sangue, urine, gli occhi, e molte altre fonti umane tutte espongono eterogeneità filogenetico 4. Mentre vi è un corpo piuttosto ampio di ricerche sui batteri Arthrobacter, solo pochi studi riportano sui fagi capaci di infettare i membri di questo genere vario. Interessante notare, però, il lavoro fatto in precedenza su Arthrobacter fagi tocca diversi argomenti distinti chiave come la tipizzazione del suolo &lt;em&gt; specie Arthrobacter 5, usi industriali con lo scopo di ridurre la schiuma deleterio in attivati ​​impianti di trattamento dei fanghi 6, e il lavoro evidenziando ricombinazione sito specifico e geni integrasi 7. Vari protocolli tecnica di arricchimento sono stati impiegati per generare pura fagi isolati nelle specie Arthrobacter. Procedure I primi comprendono incubazione del terreno con l&#39;aggiunta di agenti tossici, come i sali di nicotina per periodi di oltre un anno 8 dando luogo a fagi capaci di infettare solo A. globiformis. Studi condotti utilizzando terreno percolati con sostanze organiche labili sembravano produrre fagi rilevabili attraverso tecniche di analisi della placca, omettendo i periodi di incubazione lunghi 8. Interessante notare, però, una tecnica simile a semina diretta è stata utilizzata in passato ha dato origine a diversi fagi, pur avendo un tasso di successo notevolmente basso dagli investigatori 5, citando studi precedenti con basse percentuali di successo <sup&gt; 8. In generale, le tecniche di isolamento utilizzate in passato sono stati notevoli per avere poca efficacia nella pratica, nonostante le genere Arthrobacter rappresentano il terreno aerobico più comune isolare in natura 4,9,, Van Twest e Kropinski 10 attuali metodi di arricchimento per isolare i fagi da acqua e suolo adattato dalle tecniche precedenti utilizzate per arricchire isolati batterici ambientali, ma queste tecniche di arricchimento dimostrato inefficiente a isolare fagi Arthrobacter. Lo scopo del metodo qui descritto è quello di mostrare &quot;proof of concept&quot;, che i metodi di arricchimento primi possono essere adattati per isolare in modo coerente ed efficace fagi Arthrobacter, superando precedenti sfide tecniche associate a isolare fagi da questo genere di batteri.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>LB Broth powder</td>
			<td>Fisher</td>
			<td>BP9722-2</td>
			<td>It&#39;s best to order these in bulk.</td>
		</tr>
		<tr>
			<td>Granulated Agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>It&#39;s best to order these in bulk.</td>
		</tr>
		<tr>
			<td>0.22 um syringe filters</td>
			<td>Fisher</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>.22 um buchner filters</td>
			<td>Fisher</td>
			<td align="right">430320</td>
			<td>More than 50 mL of liquid can be obtained by carefully swapping the receiving tube.</td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher</td>
			<td align="right">76002844</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher</td>
			<td align="right">76002845</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-676-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman qualitative filter paper</td>
			<td>Fisher</td>
			<td>1001-824</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized enrichment technique for the isolation of arthrobacter bacteriophage species from soil sample isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

Per dimostrare la riproducibilità della tecnica di arricchimento migliorata per fagi Arthrobacter, 30 diversi campioni di terreno sono stati utilizzati in tempi e luoghi diversi durante la primavera e l&#39;estate del 2014. Di questi campioni di suolo 30 fagi Arthrobacter unici sono stati ottenuti da 22 campioni di terreno raccolti utilizzando questo arricchimento procedura. La procedura standard di arricchimento prodotto fagi unici da 3 degli stessi campioni di suolo. I campioni di arricchimento poss...

Watch this Scientific Journal Video about An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates at JoVE.com

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Una tecnica di arricchimento Ottimizzato per l&#39;isolamento di<em&gt; Arthrobacter</em&gt; Batteriofago Specie campione di terreno Isolati

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

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An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

19.4K Views.  University of the Sciences. We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Video: An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Progettazione e funzionamento di un continuo<sup&gt; 13</sup&gt; C e<sup&gt; 15</sup&gt; N Etichettatura Camera di Uniform o differenziale, metaboliche e strutturali, Flora Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

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Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

Progettazione e Costruzione di un Research Facility deflusso urbano

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Campionamento e Isolamento di Nematodi entomopatogeni Suolo (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

A CO<sub&gt; 2</sub&gt; Gradiente di concentrazione strumento per il test di CO<sub&gt; 2</sub&gt; Arricchimento e del suolo Effetti sulla Prateria Ecosistema Funzione

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

Spotting Cheetahs: Identificazione degli individui per le loro impronte

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Protocollo per microplastiche campionamento sulla superficie del mare e Analisi dei campioni

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

Protocolli per la quantificazione residui di antiparassitari Valori in Turfgrass Sistemi

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Isolamento ed analisi di comunità microbiche nel suolo, rizosfera e radici negli esperimenti di erba perenne

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Un protocollo di Rhizobox ottimizzato per visualizzare la crescita delle radici e tempi di risposta alle sostanze nutrienti localizzate

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Utilizzo di Caenorhabditis elegans per studiare gli effetti trans e multigenerazionali dei tossici

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...