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

1. Herstellung der Glasgeschirr und Filtergehäuse <ol style="margin-left: 40px;"><li> Wenn nicht anders angegeben, sterilisieren alle Gläser, Filtergehäuse und Lösungen bei 121 ° C für 15 min. Um die Sterilität zu gewährleisten, decken alle Öffnungen oder freiliegenden Flächen entweder mit Aluminiumfolie oder Klebeband gesicherten Papier vor der Sterilisation. </li><li> Montieren Sie Filtervorrichtung durch Anbringen von Filter-Gehäuse (47 mm Durchmesser) auf 1 L Seitenarm Erlenmeyerkolben. Filter erforderlich sammeln: 47 mm Durchmesser elektro / elektroScheibenFiltern. </li><li> Herstellung von 1 l 1,5% Rinderextrakt (Ausflocken, produziert Flockenpartikel beim pH der Lösung wird auf 3,5 oder nichtflock abgesenkt, das aber nicht bei pH 3,5 hat Aggregat) durch Auflösen von 15 g Rinderextrakt in, mit 0,05 M Glycin, 1 l entionisiertem Wasser und Zugabe von 3,75 g Glycin. HINWEIS: Nicht-Flockungsmitteln Rindfleischextrakten wird die Zugabe von Celite vor tertiären Konzentration erfordern. </li><li> Fügen Elution Additiv Natrium polyphosphate um Rindfleischextrakt in einer Konzentration von 0,1%. </li><li> 1 M Salzsäurelösung tropfenweise von dem Mischungsrindfleischextrakt, pH-Lösung um den gewünschten pH. Autoklav Rindfleischextrakt für 15-30 min bei 121 ° C. </li><li> Messen Sie 0,1 g feine, mittlere oder große Partikel Celite (zur Verwendung mit nicht-Flockungsmittel Rindfleischextrakt nur). </li><li> Bereiten Vakuum / Sog durch das Anbringen kompatibler Schlauch an Erlen Seitenarm Kolben und an die Vakuumsteckdose. </li></ol> 2. Herstellung von Lösungen und Virenvektor <ol style="margin-left: 40px;"><li> Herstellung von 1 l 1 M HCl. </li><li> Herstellung von 1 l 1 M NaOH. </li><li> Bereiten 1x PBS-Lösung (137 mM NaCl, 2,7 mM KCl, 1,47 mM K 2 PO 4 und 4,3 mM NaH 2 PO 4). <ol><li> Der pH-Wert von 1 × PBS auf 9,0 unter Verwendung von 1 M NaOH. </li></ol></li><li> Vorbereiten und verdünnen Lager Virus bis 10 4 -10 5 wahrscheinlichste Zahl (MPN) ml-1 durch Mischen von 1 ml Stammvirus mit 9 ml PBS (pH7), wie zuvor beschrieben, 8,13. Bei -80 ° C in 1 ml-Teilmengen. <ol><li> Sterilisieren Virusstamm mit Hilfe einer Spritze Filterung (0,22 um Porengröße), um mögliche Verunreinigungen zu entfernen. </li></ol></li><li> Entchlortem Leitungswasser <ol><li> Maßnahme 1 l sterilem Leitungswasser mit einer sterilen Messzylinder abgemessen und in einen 2 l-Becherglas mit Magnetrührer. </li><li> Hinzufügen von 0,7 g Natriumthiosulfat und mischen, bis Granulate aufzulösen. Der pH-Wert des Leitungswassers auf 7-7,5 bei Bedarf mit 1 M HCl oder 1 M NaOH-Lösung. </li><li> Thaw Virus Spike (1 ml) und fügen gesamte Volumen der Spitze zu 1 l dechloriert sterilem Leitungswasser. Mischen Sie für 10 Minuten. </li><li> Bereiten Sie eine leere Methode (dechloriert Leitungswasser ohne Adenovirus) mit jedem Experiment und Verfahren in der gleichen Weise wie der angereicherten Probe. </li></ol></li></ol> 3. Herstellung von Millipore Filtergerät <ol style="margin-left: 40px;"><li> Entfernen steriler Verband Filtergehäuse. Dafür Sorgengute Passform zwischen den Filtergehäuseoberschale und unteren Filter Bildschirm Probe Leckage während der Filtration zu vermeiden. </li><li> Entfernen sterilen Abdeckung von 1 l Erlenmeyerkolben. Sicherzustellen, dass der Korken von der Filtereinheit paßt genau in die obere Öffnung des Erlenmeyerkolben, der eine gute Abdichtung. </li><li> Entfernen Sie die obere Schale mit Filtereinheit von der unteren Filterscheibe, und legen Sie 47 mm elektroFilterScheibe direkt über Bildschirm mit einer sterilen Pinzette. Befestigen Sie Filtergehäuse Schüssel zum unteren Bildschirm und einrasten durch Verdrehen gegen den Uhrzeigersinn. </li></ol> 4. Tippen Sie auf Wasserfiltration (Primary Konzentration) <ol style="margin-left: 40px;"><li> Messen Sie 100 ml Virus versetzt Leitungswasser mit sterilen Messzylinder. </li><li> Gießen Sie 100 ml Virus versetzt Leitungswasser in das Filtergehäuse, die entweder ein 47 mm Falten Glas / Cellulose-Filter oder eine Nano-Aluminiumoxid / Glasfaser-Scheibenfilter. </li><li> Damit das Wasser langsam durch Filter. Bewerben leichtem Vakuum, um die verbleibenden Reste zu entfernen of Leitungswasser aus Filterfläche. </li><li> Bei der Verwendung von Trypsin-Behandlung, 5 ml 0,25% Trypsin, um Oberflächen filtern und Inkubation für 5 min. </li><li> Von Filterfläche mit 50 ml sterilem deionisiertem Wasser waschen Trypsin. </li></ol> 5. Virus Elution von Filter <ol style="margin-left: 40px;"><li> Entfernen Sie Filtergehäuse aus zwei L-Erlenmeyerkolben, und setzen Sie auf 250 ml Seitenarm Kolben. </li><li> Messen Sie 100 ml Fleischextrakt unter Verwendung von Messzylinder. Gießen Sie langsam 100 ml Fleischextrakt in das Filtergehäuse. </li><li> Lassen Rindfleischextrakt durch Filter gießen, der Erfassung Rindfleischextrakt in einem kleinen (250 ml) Erlenmeyerkolben. Gib Vakuum durch Restfleischextrakt aus dem Filter zu ziehen. </li></ol> 6. Celite Sekundär Konzentration <ol style="margin-left: 40px;"><li> Transfer-Kolben, die die eluierten Viren auf einer Rührplatte. In sterilen Magnetrührer und auf Rührplatte, Mischen zu leichten Wirbel zu erzeugen. </li><li> Mit 0,1 g CelitePulver zu 100 ml Fleischextrakt und lassen Celite zu zerstreuen. Zeigen sterile pH-Sonde in Fleischextrakt. </li><li> Fügen tropfenweise 1 M HCl auf Rindfleischextrakt auf pH 4,0 zu erreichen. Lassen Sie langsam mischen für 10 min. </li><li> Platz 47 mm-Vorfilter auf Filtergehäuse (wie zuvor beschrieben) mit einem 2 l-Erlenmeyerkolben. </li><li> Gießen Sie 100 ml Fleischextrakt / Celite-Mischung über Vorfilter. Lassen Rindfleischextrakt durch gießen. Mit leichtem Unterdruck, durch Restfleischextrakt aus dem Filter zu ziehen. </li><li> Zeigen Metallrohr Clip auf Ende des Filtergehäuses Auslauf. Bringen sterile 15 ml-Polypropylen-Sammelrohr, um Metallrohr Clip. Zeigen Filtergehäuse zurück in Erlenmeyerkolben. </li><li> 5 ml 1x PBS-Puffer, pH 9,0 über Celite am Vorfilter gesammelt. </li><li> Lassen PBS durch Vorfilter tropfen. Mit leichtem Unterdruck, durch Rest PBS in 15 ml Sammelröhrchen zu ziehen. </li></ol> 7. Organische Flockung Sekundär Konzentration <ol style="margin-left: 40px;"><li>Übertragen Sie die Becher, die das eluierte Virus auf einer Rührplatte. In sterilen Magnetrührer zu 100 ml Fleischextrakt und mischen, um leichten Wirbel zu erzeugen. </li><li> Zeigen sterile pH-Sonde in Fleischextrakt. </li><li> 1 M HCl tropfenweise zu Rindfleischextrakt und pH-Wert auf 3,5. Mischen für 30 min. </li><li> Gießen des Eluats in 250 ml Zentrifugen konische Röhrchen und Zentrifugieren bei 2.500 xg für 15 min bei 4 ºC. </li><li> Überstand abgießen dabei nicht, das Sediment nicht zu stören. Das Pellet in 5 ml 1x PBS, pH 9. </li><li> Zentrifugieren Sie die Suspension bei 4.000 × g für 10 min bei 4 ° C. Überstand abgießen und verwerfen das Pellet. </li><li> PH-Wert auf 7-7,5 filtersterilisieren durch 0,22 &amp; mgr; m-Spritzenfilter übertragen und bei -80 ° C. </li></ol> 8. viralen Nukleinsäureextraktion <ol style="margin-left: 40px;"><li> Extrahieren viralen Nukleinsäuren unter Verwendung eines geeigneten handelsüblichen Kits nach den Anweisungen des Herstellers. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 9. AdV 40/41 qPCR <ol style="margin-left: 40px;"><li> Verwenden Primer und Sonden sowie den Reaktionsmischung und Temperaturwechselprofile veröffentlicht als 8,13. Hinweis: Das Nachweisgrenze für den Test ist ca. 5 qPCR Einheiten pro Reaktion. </li><li> Hinzufügen von Komponenten einer PCR-Amplifikation Mischungen in Konzentrationen wie folgt: 10 mM Tris (pH 8,3), 50 mM KCl, 4,5 mM MgCl 2, 10 mM dNTPs, 10 &amp; mgr; M Primer und 1 &amp; mgr; M Sonde und 0,5 &amp; mgr; l Polymerase sodass Endvolumen von 45 ul können auf geeignete Anzahl von Wells einer 96-Well-PCR-Reaktionsplatte gegeben werden. </li><li> Last 45 ul der PCR-Amplifikation Mischung zu jedem entsprechenden Vertiefung einer 96-well, schnelle Reaktion PCR-Platte. </li><li> In 5 ul DNA extrahiert Probe Vertiefungen der PCR-Platte. </li><li> Abdeckplatte mit hitzebeständigem Deckeldichtung, drehen Sie die Platte um alle Tröpfchen nach unten von PCR-Plattenvertiefungen zu bewegen. </li><li> Stellen Sie die PCR-Amplifikation Zyklen wie folgt einteilenOWS: 95 ° C für 10 min, gefolgt von 40 Zyklen bei 95 ° C für 15 sec und 60 ° C für 1 min. </li></ol> 10. Datenanalysen <ol style="margin-left: 40px;"><li> Analysieren Dreifachmessungen der serielle 5-fache Verdünnungen der Proben Spanning 1: 5 bis 1: 625 Verdünnung Bereich um die wahrscheinlichste Zahl (MPN) der Viruspartikel zu bestimmen, wie zuvor beschrieben, 8,13. </li><li> Analysieren serielle 5-fache Verdünnungen von Adenovirus experimentellen Spitzen Spanning 1: 5 bis 1: 15.625 Verdünnung Bereich um die wahrscheinlichste Zahl (MPN) der Viruspartikel zu bestimmen. </li><li> Mit dem EPA MPN Rechner (http://www.epa.gov/nerlcwww/online.html), stellen Sie den Rechner, um für Art der Verdünnung zu berücksichtigen (1: 5 oder 1:10), die Anzahl der Verdünnungen durchgeführt, und die Zahl der Parallelproben in Verdünnungsreihen. </li><li> Führen log 10-Transformation der Daten, gefolgt von der Normalisierung auf eine Volumeneinheit (beispielsweise 1 ml, 100 ml, usw. </em&gt;). Berechnen Wiedereinziehungen für gegebenen Experiment durch Division Anzahl der in Probe vorhandenen Viruspartikel konzentriert zur Zahl der in der experimentellen Spannungsspitzen vorhanden Viruspartikel. </li><li> Bewerten der Wirkung der verschiedenen experimentellen Parametern (zB unterschiedliche Celite Typen pH-Bereichen, unterschiedliche Filtertypen und verschiedene sekundäre Konzentrationstechniken) auf AdV Einziehungen durch Ausführen parametrischer oder nicht-parametrischen statistischen Tests (zB gepaarter t-Test, ein oder zwei -Wege ANOVA) in Abhängigkeit von der Verteilung der Daten und die Konzeption des Experiments. </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.

Elektro Filter sind nützlich bei der Konzentration von Viren aus Wasser; aber diese Filter können in ihrer Struktur und Zusammensetzung, die wiederum ihre Wirksamkeit verändern könnten abweichen. Compoundierung dieses Problem, Kapsidstrukturen und Gebühren schwanken zwischen Virusstämme erfordern Konzentrationstechniken zugeschnitten auf optimale Erholung 15 zu gewährleisten. Durch einfache Änderungen an den vorhandenen Konzentrierungstechniken (zB elektro Filter, Rindfleischextrakt Elution),...

Menschendarmviren sind wichtige Erreger von Wasser übertragenen Krankheiten 1-3, aber sind in der Regel in geringer Anzahl in kontaminierten Umwelt Gewässern, so dass ihr Nachweis schwierig, ohne Konzentration. Verfahren verwendet werden, um Viren zu konzentrieren umfassen typischerweise ein Filtrationsschritt, gefolgt von Filter Elution und Sekundär Konzentration des Filter Eluat. Eine gemeinsame Filtrationsverfahren stützt sich auf den Einsatz von geladenen Membranen wie elektro Filter (vor kurzem in 4,5 überprüft). Diese Filter verlassen sich auf die Erfassung von Viren in Wasser suspendiert, elektrostatische Wechselwirkungen zwischen der Filteroberfläche (positiv geladen) und gezielte Viruspartikel (negativ geladen). Zwei elektro Filter, die kommerziell verfügbar sind, beruhen auf dieser Technologie, die Glas / Cellulose und Nano-Aluminiumoxid / Glasfaser-Filtern. Die Glas / Cellulose-Filterkosten um bis zu 10-fache des Nano-Aluminiumoxid / Glasfaser, das die Verwendung des Glases / ce begrenzenllulose Filter für die routinemäßige Virenüberwachung. Jüngste Studien haben festgestellt, Unterschiede nominal zwischen diesen beiden Filter in der Wiederaufnahme von Enteroviren von Umgebungswasser 6,7 sind, rechtfertigt den Einsatz eines preiswerter Filter Alternative. Weitere Filteroptionen wie elektro und Glaswolle-Filter untersucht worden, aber sie erfordern entweder die Vorbehandlung der Quelle Wasser (elektro Filter) oder nicht im Handel erhältlich (Glaswolle-Filter). Die Entwicklung der Viruskonzentration Verfahren hat sich vor allem auf die Optimierung der primären Konzentrationstechniken (Filter), um Virenrückforderungen aus dem Wasser zu verbessern konzentriert. Allerdings Sekundär Konzentration Verfahren, die das Volumen des Elutionsmittels typischerweise 1 L senken, Volumes Milliliter, kann auch einen erheblichen Einfluss auf die Virus Erholungen 8. Sekundäre Konzentration von Darmviren beruht in der Regel auf einem Flockungsmittel, wie einige Arten von Rinderextrakt (organische floccunung) oder Magermilch Flockung 9-12 zu Viruspartikeln aus Filterflächen zu entfernen. Kürzlich wurde eine weitere sekundäre Konzentrationsverfahren unter Verwendung von Rinderextrakt zusammen mit der Zugabe von Celite (feine Teilchen) Versprechen zur Rückgewinnung Adenovirus, Enteroviren und Norovirus 8,13,14 gezeigt. Celite Konzentration Arbeiten unter ähnlichen Prinzipien wie die des organischen Ausflockungsverfahren dass Viruspartikel befestigen und von Teilchen (Flocken oder Celite) durch Änderung des pH der Suspension die Lösung freigesetzt. Vergleiche zwischen diesen beiden Sekundär Konzentration Techniken bei der Beitreibung von Stachel Adenovirus (AdV) Typen 40 und 41 8 bewertet worden. Diese Studie ergab, dass die beiden Sekundärkonzentrationstechniken waren in der Wiederaufnahme von Adenoviren statistisch ähnlich. Jedoch erfordert das organische Ausflockungsverfahren 30 min. Inkubation bei pH 3,5, während das Celite Technik erfordert eine kürzere Inkubation (10 min) bei pH 4,0. Die organische flocculatIonen erfordert auch den Einsatz von teuren Laboreinrichtungen (Zentrifugen) Flockenpartikel während tertiäre Konzentration sammeln, das Celite Technik im Gegensatz nur die wichtigsten Laborausrüstung (Vakuumfiltration), um Celite Teilchen aus der Suspension zu trennen. Bestimmte Kombinationen von Filtern und Sekundär Elutionstechniken kann auch Auswirkungen auf Virus-Rückforderungen. Eine Studie ergab, dass bestimmte Kombinationen von primären (elektro Filter) und sekundäre Konzentrationstechniken (Celite oder organischen Flokkulation) einen signifikanten Einfluss der Erholung von Adenovirus-13 hatte. Diese Ergebnisse legen nahe, dass die Optimierung ist, um optimal zu erholen Zielvirus aus einer gegebenen Wassermatrix bei der Verwendung dieser Techniken erforderlich. Optimierung ist ein zeitaufwendiger, aktiv zu vermeiden mühsamer Prozess viele Forscher seit vielen Variablen werden ausgewertet (Filtertyp / Marke pH Elutionslösung, Celite / organischen Flokkulation). Dafür study, ein Verfahren entwickelt, um optimale Bedingungen für die Viruskonzentration von Wasser zu identifizieren mit Stachel humane Adenovirus-Stämme 40 und 41 Vermutlich da jeder Virustyp zeigt eine einzigartige Kapsid Morphologie und spezifische Kapsid Ladung, müssen Konzentrationsprotokolle für jeden Virus optimiert werden zielen, um eine optimale Virusrückgewinnung zu erreichen. Diese Studie liefert einen Ansatz für die AdV 40 und 41 Konzentration: 1) Auswertung Virus Erholungen in Leitungswasser mittels elektroFilterScheiben, gefolgt von 2) Auswertung eines etablierten organischen Flockungsmethoden gegenüber dem Celite-Technik als Sekundär Konzentration, und 3) Bewertung der Elutionspuffer für tertiäre Konzentration.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		</tr>
		<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> Celite Auswahl Drei verschiedene Arten von Celite wurden vor der Auswahl des besten Variante getestet. Celite mit feinen bis mittelgroße Partikel produziert die höchsten Adenovirus Erholungen. Einsatz größerer Celite führte zu niedrigeren Wiederfindungsraten für sowohl AdV40 und 41 (Bereich 32% -100%) (Abbildung 1). Durchschnittliche Rückforderungen von Adenovirus-40 waren 144% ± 52% (fein), 115% ± 28% (mittel) und 82% ± 53% (große) Partikel Celite und AdV41...

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.

Ein kleines Volumen Verfahren für Viral Konzentration von Wasser

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 Aufrufe.  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. 

Serie: 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.

Labor-Phosphor bestimmt Flux von Seesedimenten als Maß der internen Phosphorbelastung

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

Forschung

Umwelt

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.

A Small Volume Bioassay um bakterielle / Phytoplankton Co-Kultur Bewerten Sie mit Wasser-Pulse-amplitudenmodulierten (WATER-PAM) Fluorometrie

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.

Ein CO<sub&gt; 2</sub&gt; Konzentration Gradient Facility for Testing CO<sub&gt; 2</sub&gt; Enrichment und Boden Auswirkungen auf Grünland-Ökosystemen Funktion

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.

Eine einfache Methode für die automatisierte Festphasenextraktion von Wasserproben für die immunologische Analyse von kleinen Schadstoffe

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.

Extrahieren von Metriken für die dreidimensionale Wurzelsysteme: Volumen und Oberflächenanalyse von In-Boden Röntgen Computertomographie Daten

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.

Boden Lysimeter Ausgrabung Gekoppelte hydrologische, geochemische und mikrobiologische Untersuchungen

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.

Extraktion von Organochlor-Pestiziden aus Kunststoff-Pellets und Kunststoff-Typ-Analyse

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.

Erfassung von Wasser fließen gewichtet und Schwebestaub aus landwirtschaftlichen Kanäle bei Drainage-Veranstaltungen

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.

Eine einfache Bepflanzung Technik für Wiederherstellung des Bäume wo tritt häufig Überschwemmung

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.

Optimierte Verfahren zur Bestimmung der Adsorption von Phosphonaten auf Granular Ferric Hydroxid mit einer miniaturisierten Phosphor Entschlossenheit Methode

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