We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

1. Preparazione di cristalleria e Filter Contenitori <ol style="margin-left: 40px;"><li> Se non diversamente specificato, sterilizzare tutti i bicchieri, contenitori e soluzioni di filtro a 121 ° C per 15 minuti. Per garantire la sterilità, coprire tutte le aperture o le superfici esposte sia con fogli di alluminio o di carta nastro fissato prima della sterilizzazione. </li><li> Montare apparato di filtrazione allegando alloggiamento del filtro (diametro di 47 mm) a 1 L braccio laterale beuta. Raccogliere filtri necessari: 47 mm di diametro filtri a disco elettropositivi / electronegative. </li><li> Preparare 1 L di estratto di carne bovina 1,5% (flocculanti; produce particelle Floc quando pH della soluzione si abbassa a 3,5, o non flocculante, che non fa aggregato a pH 3,5), con 0,05 M glicina, sciogliendo 15 g di estratto di carne bovina in 1 L di acqua deionizzata e l&#39;aggiunta di 3,75 g di glicina. NOTA: estratti di carne non floccaggio richiedono l&#39;aggiunta di celite prima concentrazione terziaria. </li><li> Aggiungi eluizione polyph additivo sodioosphate di estratto di carne bovina ad una concentrazione di 0,1%. </li><li> Aggiungere 1 soluzione di acido cloridrico M, gocciolamento, di miscelazione estratto di carne, pH della soluzione a pH desiderato. Estratto di carne bovina Autoclave per 15-30 minuti a 121 ° C. </li><li> Misura 0,1 g di fine, media, o grande celite particelle (per l&#39;utilizzo con estratto di carne non-flocculanti solo). </li><li> Preparare vuoto / aspirazione collegando tubazioni compatibile beuta braccio laterale e alla presa di vuoto. </li></ol> 2. preparazione di soluzioni e virus Stock <ol style="margin-left: 40px;"><li> Preparare 1 L di 1 M HCl. </li><li> Preparare 1 L di 1 M NaOH. </li><li> Preparare la soluzione PBS 1x (137 mM NaCl, 2.7 mM KCl, 1.47 mm K 2 PO 4 e 4,3 mm NaH 2 PO 4). <ol><li> Regolare il pH di 1x PBS a 9,0 con 1 M NaOH. </li></ol></li><li> Preparare e diluire magazzino virus per 10 4 -10 5 numero più probabile (MPN) ml -1 mescolando 1 ml di virus con 9 ml di PBS (pH7), come precedentemente descritto 8,13. Conservare a -80 ° C in aliquote di 1 ml. <ol><li> Sterilizzare virus magazzino filtrando siringa (0,22 micron dimensione dei pori) per rimuovere potenziali contaminanti. </li></ol></li><li> Declorata Tap Water <ol><li> Misura 1 L di acqua di rubinetto sterile utilizzando una sterile cilindro graduato e versare in un bicchiere contenente 2 L ancoretta magnetica. </li><li> Aggiungere 0,7 g di tiosolfato di sodio e mescolare fino a granuli si dissolvono. Regolare il pH dell&#39;acqua di rubinetto a 7-7,5 se necessario con 1 M HCl o soluzione 1 M NaOH. </li><li> Spike virus Thaw (1 ml) e aggiungere intero volume di picco per 1 L di acqua di rubinetto sterili senza cloro. Mescolare per 10 min. </li><li> Preparare un metodo vuoto (acqua di rubinetto declorurata senza adenovirus) con ogni esperimento e processo nello stesso modo come il campione spillo. </li></ol></li></ol> 3. Preparazione di Millipore Filtro Apparatus <ol style="margin-left: 40px;"><li> Rimuovere copertura sterile su filtro. Garantirela misura adeguata tra il corpo del filtro ciotola superiore e schermo del filtro inferiore per evitare perdite campione durante la filtrazione. </li><li> Rimuovere copertura sterile dal 1 boccetta L Erlenmeyer. Assicurarsi che il tappo del gruppo filtrante adatta comodamente in cima apertura beuta, creando una buona tenuta. </li><li> Rimuovere ciotola superiore dell&#39;unità filtrante dallo schermo del filtro inferiore e portare 47 millimetri disco filtro elettropositivo esattamente sopra lo schermo con una pinza sterile. Attaccare il filtro ciotola alloggi a schermo inferiore e bloccare in posizione ruotando in senso antiorario. </li></ol> 4. Toccare filtrazione dell&#39;acqua (concentrazione primaria) <ol style="margin-left: 40px;"><li> Misurare 100 ml di virus a spillo l&#39;acqua del rubinetto con sterile cilindro graduato. </li><li> Versare 100 ml di virus spillo l&#39;acqua del rubinetto in scatola del filtro contenente sia un / filtri di cellulosa 47 millimetri di vetro pieghettata o un filtro rigido in fibra di nano-allumina / vetro. </li><li> Consentire all&#39;acqua di passare lentamente attraverso il filtro. Applicare delicato vuoto per rimuovere residui residui oacqua di rubinetto f dalla superficie del filtro. </li><li> Se si utilizza il trattamento tripsina, aggiungere 5 ml di 0,25% tripsina per filtrare la superficie e incubare per 5 min. </li><li> Lavare tripsina dalla superficie del filtro con 50 ml di acqua deionizzata sterile. </li></ol> 5. Virus eluizione da filtro <ol style="margin-left: 40px;"><li> Smontare la scatola del filtro da 2 fiasco L Erlenmeyer, e posizionarlo sul pallone da 250 ml braccio laterale. </li><li> Misurare 100 ml di estratto di carne utilizzando cilindro graduato. Versare lentamente 100 ml di estratto di carne in scatola del filtro. </li><li> Lasciare estratto di carne bovina per versare attraverso il filtro, catturando estratto di carne in un piccolo (250 ml) beuta. Applicare il vuoto per tirare attraverso estratto di carne residuo dal filtro. </li></ol> 6. Celite Concentrazione secondaria <ol style="margin-left: 40px;"><li> Flaconi di trasferimento contengono i virus eluiti ad una piastra scalpore. Aggiungere sterile ancoretta magnetica e posto sul piatto mescolare, mescolare per creare una leggera vortice. </li><li> Aggiungere 0,1 g di celitepolvere 100 ml di estratto di carne e consentire celite per disperdere. Posizionare la sonda pH sterile in estratto di carne. </li><li> Aggiungere goccia a goccia 1 M HCl per estratto di carne per raggiungere pH 4,0. Lasciare miscelare lentamente per 10 min. </li><li> Mettere 47 millimetri di pre-filtro sul corpo del filtro (come descritto in precedenza) con un pallone 2 L Erlenmeyer. </li><li> Versare 100 ml di estratto di manzo / miscela celite over pre-filtro. Lasciare estratto di carne bovina per versare attraverso. Applicare leggero vuoto per tirare attraverso estratto di carne residuo dal filtro. </li><li> Posizionare la clip tubo metallico sulla fine del becco filtro. Attaccare sterile 15 ml tubo di raccolta in polipropilene a clip di tubo di metallo. Posizionare la scatola del filtro di nuovo in beuta. </li><li> Aggiungere 5 ml di tampone 1x PBS, pH 9.0 su celite raccolte sul pre-filtro. </li><li> Lasciare PBS goccioli attraverso pre-filtro. Applicare leggero vuoto per tirare attraverso residuo PBS in 15 ml tubo di raccolta. </li></ol> 7. Flocculazione Organic Concentrazione secondaria <ol style="margin-left: 40px;"><li>Trasferire i bicchieri contenenti il ​​virus eluito a un piatto mescolare. Aggiungere sterile ancoretta magnetica a 100 ml di estratto di carne e mescolare per creare una leggera vortice. </li><li> Posizionare la sonda pH sterile in estratto di carne. </li><li> Aggiungere 1 M HCl goccia saggio estratto di carne e aggiustare il pH a 3,5. Mescolare per 30 min. </li><li> Versare l&#39;eluato in 250 ml provette coniche e centrifugare a 2500 xg per 15 minuti a 4 ° C. </li><li> Eliminare il surnatante facendo attenzione a non a non disturbare il pellet. Risospendere il pellet in 5 ml di PBS 1x, pH 9. </li><li> Centrifugare la sospensione a 4.000 xg per 10 minuti a 4 ° C. Eliminare il surnatante e scartare il pellet. </li><li> Regolare il pH a 7-7.5, filtrare sterilizzare attraverso 0,22 micron filtro siringa e congelamento a -80 ° C. </li></ol> 8. Viral Nucleic Acid Extraction <ol style="margin-left: 40px;"><li> Estrarre gli acidi nucleici virali utilizzando un kit commerciale adeguata secondo le istruzioni del produttore. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 9. AdV 40/41 qPCR <ol style="margin-left: 40px;"><li> Utilizzare primer e sonde, nonché miscela di reazione e profili cicli termici come pubblicato 8,13. NOTA: limite stimato di rilevamento per il dosaggio è di circa 5 unità qPCR per reazione. </li><li> Aggiungere componenti di miscele di amplificazione PCR a concentrazioni come segue: Tris 10 mM (pH 8,3), 50 mM KCl, 4,5 mM MgCl 2, 10 mM dNTPs, 10 mM primer e della sonda 1 mM e 0,5 ml di polimerasi in modo che i volumi finali di 45 microlitri può essere aggiunto al numero appropriato di pozzetti in una piastra di reazione PCR da 96 pozzetti. </li><li> Carico 45 ml di miscela di amplificazione PCR per ciascun pozzetto appropriata di un pozzo 96, reazione veloce PCR piastra. </li><li> Aggiungere 5 ml di DNA estratto campione adeguati pozzetti della piastra PCR. </li><li> Coprire piastra con calore guarnizione del coperchio resistente, girare la piastra per spostare eventuali gocce di fondo dei pozzetti della piastra PCR. </li><li> Impostare PCR cicli di amplificazione come follOWS: 95 ° C per 10 minuti, seguiti da 40 cicli di 95 ° C per 15 sec e 60 ° C per 1 min. </li></ol> 10. Analisi dei dati <ol style="margin-left: 40px;"><li> Analizzare misurazioni in triplicato delle diluizioni seriali 5-fold dei campioni abbracciano 1: 5 a 1: 625 gamma di diluizione per determinare il numero più probabile (MPN) delle particelle virali, come precedentemente descritto 8,13. </li><li> Analizzare diluizioni seriali 5-fold di picchi sperimentali adenovirus abbracciano 1: 5 a 1: 15.625 serie di diluizione per determinare il numero più probabile (MPN) delle particelle virali. </li><li> Utilizzando la calcolatrice EPA MPN (http://www.epa.gov/nerlcwww/online.html), regolare la calcolatrice per spiegare il tipo di diluizione (1: 5 o 1:10), il numero di diluizioni effettuate, e il numero di replicare campioni in serie di diluizioni. </li><li> Eseguire log 10 trasformazione dei dati, seguiti normalizzando ad una unità di volume (ad esempio, 1 ml, 100 ml, etc. </em&gt;). Calcola recuperi per dato esperimento dividendo il numero di particelle virali presenti nel campione concentrati con il numero di particelle virali presenti in picchi sperimentali. </li><li> Valutare l&#39;effetto delle diverse variabili sperimentali (ad esempio, diversi tipi Celite, intervalli di pH, diversi tipi di filtro e diverse tecniche di concentrazione secondari) sui recuperi Adv eseguendo test statistici parametrici e non parametrici (es paired t-test, uno o due -way ANOVA) a seconda della distribuzione dei dati e il disegno sperimentale. </li></ol>

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No competing financial interests. 
The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Filtri electropositive sono utili nella concentrazione virus dall&#39;acqua; Tuttavia questi filtri possono differire nella loro struttura e composizione che potrebbe a sua volta alterare la loro efficacia. Ad aggravare questo problema, le strutture e le spese del capside variano tra i ceppi virali che richiedono tecniche di concentrazione essere adattati per garantire il recupero ottimale 15. Attraverso semplici modifiche delle tecniche di concentrazione esistenti (ad esempio, filtri elettropositivi...

Virus enterici umani sono importanti agenti eziologici di malattie trasmesse dall&#39;acqua 1-3, ma sono generalmente presenti in numero ridotto nelle acque ambientali contaminate, rendendo la loro individuazione difficile senza concentrazione. Le procedure utilizzate per concentrare virus includono tipicamente una fase di filtrazione, seguita da filtro eluizione, e concentrazione secondaria dell&#39;eluato filtro. Una procedura di filtrazione comune si basa sull&#39;uso di membrane cariche quali filtri elettropositivi (recentemente recensito in 4,5). Questi filtri si basano sulla cattura virus sospese in acqua utilizzando interazioni elettrostatiche tra la superficie del filtro (carica positiva) e particelle di virus mirati (carica negativa). Due filtri elettropositivi che sono commercialmente disponibili si basano su questa tecnologia, i filtri di vetro / cellulosa e nano-allumina / fibra di vetro. I costi filtro di vetro / cellulosa sono fino a 10 volte quella del nano-allumina / fibra di vetro, che limitano l&#39;uso del vetro / ceFiltri llulose per il monitoraggio virus di routine. Recenti studi hanno concluso differenze sono nominali tra questi due filtri in recupero di enterovirus da 6,7 all&#39;acqua ambiente, giustificare l&#39;uso di una più economica alternativa filtro. Altre opzioni di filtro come filtri elettronegativi e lana di vetro sono stati studiati, tuttavia, richiedono sia il pretrattamento dell&#39;acqua di fonte (filtri elettronegativi) o non sono disponibili in commercio (filtri di lana di vetro). Lo sviluppo di procedure di concentrazione del virus si è prevalentemente concentrata sull&#39;ottimizzazione tecniche di concentrazione primari (filtri) al fine di migliorare i recuperi virus dall&#39;acqua. Tuttavia, le procedure di concentrazione secondarie, che riducono il volume di eluente tipicamente da 1 L a millilitro volumi, possono anche avere un impatto significativo sui recuperi virus 8. Concentrazione media di virus enterici basa tipicamente su un agente flocculante come alcuni tipi di estratto di carne (floccu organicamento) o il latte scremato flocculazione 9-12 per rimuovere particelle di virus da superfici filtranti. Recentemente, un&#39;altra procedura concentrazione secondaria con estratto di carne insieme con l&#39;aggiunta di celite (particelle fini) ha mostrato risultati promettenti per il recupero adenovirus, enterovirus e norovirus 8,13,14. Opere concentrazione Celite sotto principi simili a quella del metodo di flocculazione organica che particelle virali connettono e vengono rilasciate da particelle (floc o celite) alterando il pH della soluzione di sospensione. Il confronto tra queste due tecniche di concentrazione secondari sono stati valutati nel recupero di adenovirus spillo (ADV) i tipi 40 e 41 8. Questo studio ha concluso che le due tecniche di concentrazione secondari erano statisticamente simili nel recupero di adenovirus. Tuttavia, il metodo richiede una flocculazione organica 30 min. incubazione a pH 3,5, mentre la tecnica celite richiede una breve incubazione (10 min) a pH 4.0. Il flocculat organicaion richiede anche l&#39;uso di costose attrezzature di laboratorio (centrifughe) per raccogliere particelle Floc momento della concentrazione terziaria, la tecnica celite in contrasto utilizza solo attrezzature di laboratorio di base (filtrazione sotto vuoto) per separare particelle Celite da sospensione. Alcune combinazioni di filtri e tecniche di eluizione secondaria può colpire anche i recuperi di virus. Uno studio ha concluso che alcune combinazioni di primari (filtri elettropositivi) e tecniche di concentrazione secondari (Celite o flocculazione organica) hanno avuto un impatto significativo recupero di adenovirus 13. Questi risultati suggeriscono che l&#39;ottimizzazione è necessaria al fine di recuperare in modo ottimale virus bersaglio da una data matrice acqua quando utilizzando queste tecniche. L&#39;ottimizzazione è un dispendio di tempo, di processo arduo molti ricercatori evitare attivamente poiché numerose variabili saranno valutati (tipo di filtro / marca, soluzione di eluizione pH, celite / flocculazione organica). Per questo study, una procedura è stata sviluppata per individuare le condizioni ottimali per la concentrazione del virus da acqua utilizzando spillo adenovirus umano ceppi 40 e 41. Presumibilmente, dal momento che ogni tipo di virus visualizza una morfologia capside unica e carica capside specifico, possono avere bisogno di essere ottimizzato per ogni virus protocolli di concentrazione obiettivo per raggiungere il recupero virale ottimale. Questo studio fornisce un approccio per AdV 40 e 41 concentrazione mediante: 1) valutare recuperi virus in acqua di rubinetto utilizzando dischi filtranti elettropositivi seguiti da 2) valutazione di un metodo di flocculazione organica stabilita rispetto alla tecnica celite come concentrazione secondaria, e 3) valutazione buffer di eluizione per la concentrazione terziaria.

<table border="0" cellpadding="0" cellspacing="0" width="786">
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			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:295px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 40 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 41 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Thiosulfate</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">72051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Celites #577</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">22142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">NanoCeram 47mm</td>
			<td style="width:143px;">Argonide</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1MDS 47mm</td>
			<td style="width:143px;">3M</td>
			<td style="width:119px;">6408502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AP-20 Prefilter 47mm</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td style="width:119px;">AP2004700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glycine&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">50046-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Polyphosphate</td>
			<td style="width:143px;">Acros Organics</td>
			<td style="width:119px;">390930010</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:231px;">Trypsin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">PBS</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hydrochloric Acid</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">A481-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">BBL Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">212303</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Difco Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">211520</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:231px;">ABI 7900 Real-time PCR system</td>
			<td style="width:143px;">ABI</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stainless Steel Filter Housing</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td>XX2004720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Blood DNA Extraction Kit</td>
			<td style="width:143px;">Qiagen&#160;</td>
			<td style="width:119px;">51104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EPA MPN Calculator</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.epa.gov/nerlcwww/online.html</td>
		</tr>
	</tbody>
</table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

<html> Selezione Celite Tre diversi tipi di celite stati testati prima della selezione della variante migliore performante. Celites con ammenda di particelle di medie dimensioni realizzati i più alti recuperi adenovirus. L&#39;uso di grandi celites comportato recuperi inferiori sia AdV40 e 41 (range 32% -100%) (Figura 1). Recuperi medi di adenovirus 40 erano 144% ± 52% (fine), 115% ± 28% (medio) e il 82% ± 53% celites (grande) di particelle e per AdV41, 132% ± 39% (fine)...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

A Procedura piccolo volume per concentrazione virale da Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

a-small-volume-procedure-for-viral-concentration-from-water

Research

JoVE Journal

Environment

10.0K Views.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

Video: A Small Volume Procedure for Viral Concentration from Water

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Laboratorio-determinato Fosforo Flux dal lago di sedimenti come misura di fosforo interno Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Ricerca

Ambiente

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Un piccolo volume Bioassay per valutare batterica / fitoplancton Co-cultura Utilizzando WATER-Pulse-modulata in ampiezza (WATER-PAM) fluorometria

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

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 ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Un metodo semplice per Automated fase solida estrazione di campioni di acqua per l&#39;analisi immunologiche di piccole inquinanti

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

Estrazione metriche per sistemi di radici tridimensionali: volume e superficie di analisi da In-terreno a raggi X Tomografia Computerizzata dati

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Terreno lisimetro scavo per accoppiate idrologici, geochimiche e microbiologiche Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Estrazione di pesticidi di organochlorato da pellicole di plastica e analisi di tipo plastico

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

Acquisizione ponderati per il flusso di acqua e particelle in sospensione dai canali agricoli durante eventi di drenaggio

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Una semplice tecnica di impianto per ristabilire gli alberi dove si verifica frequente Inundation

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

Procedura ottimizzata per determinare l'adsorbimento di fosfonati su idrossido ferrico granulare utilizzando un metodo di determinazione del fosforo miniaturizzato

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...