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

Züccaciye ve Filtre gövdeleri 1. Hazırlık <ol style="margin-left: 40px;"><li> Aksi belirtilmediği sürece, 15 dakika için 121 ° C&#39;de tüm cam, filtre gövdesi ve çözümleri sterilize. Sterilite sağlamak için, tüm açıklıkları veya alüminyum folyo ya da sterilizasyon öncesinde bant güvenli kağıt ile ya maruz yüzeyleri kapsar. </li><li> 1 L&#39;lik bir yan kol Erlenmeyer şişesine filtre yuvası (47 mm çapında) takılarak filtreleme aparatı birleştirin. Gerekli filtreleri toplayın: 47 mm çap elektronegatif / elektropozitif disk filtreler. </li><li> 1 L&#39;lik 1.5% sığır eti ekstresi hazırlanması (topaklanması; topaklanma parçacıklarını üreten çözeltinin pH değeri 3,5, ya da olmayan flokülasyon altına düştüğünde, pH 3.5 agrega değildir) eti ekstresi, 15 g içinde çözülerek 0.05 M glisin ile, 1 L deiyonize su ve 3.75 g glisin ilave edilmesi. Not: Non-pıhtılaştırıcı sığır ekstraktlar önce yüksek konsantrasyona kadar selit kullanılmasını gerektirmektedir. </li><li> Elüsyon katkı sodyum polyph ekle% 0.1 konsantrasyonda sığır ekstraktı osphate. </li><li> 1 M hidroklorik asit solüsyonu ekleyin sığır eti ekstresi, istenilen pH pH solüsyonu karıştırma, damla damla. 121 ° C&#39;de, 15-30 dakika süreyle otoklavlanmaktadır sığır özü. </li><li> Ince, orta, ya da (non-flokülan sığır özü yalnızca kullanım için) büyük parçacık celite 0.1 g ölçün. </li><li> Erlenmeyer yan kol şişeye ve vakum prizine uyumlu boru takarak vakum / emme hazırlayın. </li></ol> Çözümler ve Virüs Stokta 2. Hazırlık <ol style="margin-left: 40px;"><li> 1 M HCl 1 L hazırlayın. </li><li> 1 M NaOH ile 1 L hazırlayın. </li><li> 1x PBS çözeltisi hazırlayın (137 mM NaCI, 2.7 mM KCI, 1.47 mM K 2 PO 4 ve 4.3 mM NaH 2 PO 4). <ol><li> 1 M NaOH ile 9.0&#39;a 1x PBS pH ayarlayın. </li></ol></li><li> Hazırlayın ve 9 ml PBS (pH stok virüsü 1 ml karıştırılarak 10 4 -10 5 en muhtemel sayı (EMS) ml -1 stok virüs sulandırmak7), daha önce tarif edildiği 8,13. 1 ml alikotlar içinde -80 ° C&#39;de saklayın. <ol><li> Potansiyel kirleri çıkarmak için şırınga filtreleme (0.22 mikron gözenek boyutu) virüs stok sterilize edin. </li></ol></li><li> Klor Musluk Suyu <ol><li> Ölçü steril kullanılarak steril musluk suyu içinde 1 litre dereceli silindir ve manyetik bir karıştırma çubuğu ihtiva eden bir 2 L&#39;lik bir kap içine dökün. </li><li> Sodyum tiyosülfat 0.7 g eklenir ve granüller eriyene kadar karıştırılır. 1 M HCI veya 1 M NaOH çözeltisi kullanılarak gerekirse 7-7.5&#39;e musluk suyu pH ayarlayın. </li><li> Çözülme virüs başak (1 ml) ve dechlorinated steril musluk suyu 1 L başak tüm hacim ekleyin. 10 dakika boyunca karıştırın. </li><li> Katkılı bir örnekle aynı şekilde, her bir deney ve yöntem ile ilgili bir yöntem boş (adenovirüs olmadan kloru giderilmiş musluk suyu) hazırlayın. </li></ol></li></ol> Millipore Filtre Aparatı 3. Hazırlık <ol style="margin-left: 40px;"><li> Filtre konut üzerinde steril örtü çıkarın. SağlamakFiltre gövdesi üst kase ve alt filtre ekranı arasında uygun uyum filtrasyon sırasında örnek sızıntıyı önlemek için. </li><li> 1 L&#39;lik bir Erlenmeyer şişesi, steril kılıf çıkarılır. İyi bir yalıtım oluşturmak, filtre ünitesinin mantar Erlenmeyer şişesi üst açıklığı içinden rahatça sığar emin olun. </li><li> Alt filtre ekranından filtre ünitesinin üst kase çıkarın ve steril forseps ile ekran üzerinde kare 47 mm elektropozitif filtre diski yerleştirin. Alt ekran filtresi konut kase takın ve saat yönünün tersine çevirerek yerine kilitleyin. </li></ol> 4. dokunun Su Filtrasyon (Birincil Konsantrasyon) <ol style="margin-left: 40px;"><li> Virüsün 100 ml steril dereceli silindir kullanarak musluk suyu çivili ölçün. </li><li> Virüs, 100 mi 47 mm&#39;lik kıvrımlı cam / selüloz filtresi veya bir nano alüminyum oksit / cam elyaf filtre diski ya da ihtiva eden filtre gövdesine musluk suyu çivili dökün. </li><li> Su yavaş yavaş filtre geçmesine izin verin. O kalan artıklar kaldırmak için nazik vakum uygulayınFiltre yüzeyinden f musluk suyu. </li><li> Tripsin tedavisi kullanıldığında, yüzey filtre edilir ve 5 dakika boyunca inkübasyona% 0.25 tripsin 5 ml ilave ediniz. </li><li> Steril deiyonize su, 50 ml kullanılarak filtre yüzeyinden tripsin yıkayın. </li></ol> Filtre 5. Virüs Elüsyon <ol style="margin-left: 40px;"><li> 2 L&#39;lik bir Erlenmeyer şişesi filtre gövdesini kaldırmak ve 250 mi yan kol şişede üzerine yerleştirin. </li><li> Silindir mezun kullanarak sığır ekstraktı 100 ml ölçün. Yavaş yavaş filtre gövdesine sığır ekstraktı 100 ml dökün. </li><li> Sığır özü küçük bir (250 mi) bir Erlenmeyer şişesi içinde sığır eti ekstresi yakalama, üzerine dökülen tipteki filtrenin izin verin. Filtreden kalan sığır özü ile çekmek için vakum uygulayın. </li></ol> 6. Celite İkincil Konsantrasyon <ol style="margin-left: 40px;"><li> Bir heyecan plaka yıkanarak virüs içeren transfer şişeler. Hafif girdap oluşturmak için karıştırma, karıştırma plaka üzerinde steril manyetik karıştırma çubuğu ve yer ekle. </li><li> Selit 0.1 g eklemesığır özü, 100 ml toz ve selit dağılmasını sağlar. Sığır özü içine steril pH probu yerleştirin. </li><li> PH değeri 4.0 ulaşmak için sığır özü akıllıca 1 M HCl damla ekleyin. Yavaş yavaş 10 dakika boyunca karıştırın izin verin. </li><li> 2 L&#39;lik bir Erlenmeyer şişesi kullanarak (daha önce tarif edildiği gibi) filtre yuvası üzerine 47 mm ön filtresi yerleştirin. </li><li> Ön filtre üzerinde 100 ml sığır özü / selit karışımı dökün. Sığır ekstraktı yoluyla dökmek için izin verin. Filtreden kalan sığır özü üzerinden çekin hafif vakum uygulayın. </li><li> Filtre konut ağzının ucunda metal boru klibi yerleştirin. Metal boru klibe steril 15 ml polipropilen toplama tüpü takın. Geri Erlenmeyer şişesi içine filtre muhafazasını yerleştirin. </li><li> Ön filtre üzerinde toplanan selit üzerinde 1x PBS tamponu 5 mi, pH 9.0 ekleyin. </li><li> PBS ön filtre ile damla izin verin. 15 ml toplama tüpüne artık PBS üzerinden çekin hafif vakum uygulayın. </li></ol> 7. Organik Flokülasyonu İkincil Konsantrasyon <ol style="margin-left: 40px;"><li>Bir heyecan plaka yıkanarak virüs içeren beher aktarın. Sığır ekstraktı 100 ml steril manyetik karıştırma çubuğu ekleyin ve hafif vorteks oluşturmak için karıştırın. </li><li> Sığır özü içine steril pH probu yerleştirin. </li><li> Sığır özü akıllıca 1 M HCI damla ekleyin ve 3.5 pH ayarlayın. 30 dakika boyunca karıştırın. </li><li> 4 ° C&#39;de 15 dakika boyunca 2500 x g&#39;de 250 ml&#39;lik santrifüj konik tüplere eluat ve santrifüj dökün. </li><li> Pelet bozmaya değil değil süpernatant alarak bakım kapalı dökün. 1x PBS, pH 9 5 ml pelletini. </li><li> 4 ° C&#39;de 10 dakika boyunca 4.000 xg&#39;de süspansiyon santrifüjleyin. Süpernatantı dökün ve pelet atın. </li><li> 7-7,5 pH ayarlayın, filtre -80 ° C&#39;de 0.22 mikron şırınga filtresi ve dondurma ile sterilize. </li></ol> 8. Viral Nükleik Asit Ekstraksiyon <ol style="margin-left: 40px;"><li> Üreticinin talimatlarına uygun olarak, uygun bir ticari kit kullanılarak viral nükleik asitlerin ekstre edilmesi. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 9. AdV 40/41 QPCR <ol style="margin-left: 40px;"><li> 8,13 yayınlanan primerler ve problar, aynı zamanda reaksiyon karışımı ve termal döngü profili kullanın. NOT: tahlil için algılama Tahmini sınırı reaksiyonu başına yaklaşık 5 qPCR birimdir. </li><li> Aşağıdaki gibi konsantrasyonlarda PCR büyütme katkı maddeleri ekleyin: 10 mM Tris (pH 8.3), 50 mM KCI, 4.5 mM MgCI2, 10 mM dNTP, 10 uM primerleri ve 1 uM probu ve polimeraz 0.5 ul olacak şekilde 45 son hacim ul, 96 oyuklu bir PCR reaksiyonu, plaka içindeki deliklerin uygun sayıda ilave edilebilir. </li><li> Bir 96-yuvalı, hızlı reaksiyon PCR plaka her biri uygun oyuğuna PCR amplifikasyon karışımı yüklenebilir 45 ul. </li><li> DNA 5 ul PCR plaka ihtiva eden uygun oyuklara örnek ekstre ekleyin. </li><li> , Isıya dayanıklı kapak contası ile plaka Kapak PCR plaka kuyuların dibine herhangi damlacıkları taşımak için plaka döndürün. </li><li> Foll olarak ayarla PCR amplifikasyon döngüleriakımları: 15 saniye için 95 ° C 40 döngü, 1 dakika süreyle 60 ° C&#39;de, ardından 10 dakika boyunca 95 ° C olarak ölçülmüştür. </li></ol> 10. Veri Analizleri <ol style="margin-left: 40px;"><li> 5-1: 1 kapsayan örneklerinin seri 5 kat seyreltmeleri üçlü ölçümleri analiz 625 seyreltme aralığı viral parçacıkların en olası sayı (EMS) belirlemek amacıyla, daha önce tarif edildiği gibi 8,13. </li><li> 5-1: 1 kapsayan adenovirüs deney sivri seri 5 kat seyreltmeleri analiz 15.625 seyreltme aralığı viral parçacıkların en olası sayı (EMS) tespit etmek için. </li><li> EPA EMS hesap (http://www.epa.gov/nerlcwww/online.html) kullanılarak, seyreltme tipi için hesap hesap ayarlayın (1: 5 ya da 1:10) dilüsyonlarda adedine ve sayısı seyrelme serisi içindeki örnekleri çoğaltırlar. </li><li> Yapın bir birim hacim vb (örneğin, 1 mi, 100 ml, normalize ardından verileri 10 log transformasyonu </em&gt;). Numunede mevcut viral parçacıkların sayısına bölünmesiyle verilen deney için kurtarma hesaplayın deneysel ani mevcut viral parçacıkların sayısı ile yoğunlaşmaktadır. </li><li> Farklı deneysel değişkenlerin etkisini değerlendirmek (örneğin, farklı celite tipleri, pH aralıkları, farklı filtre türleri ve farklı ikincil konsantrasyon teknikleri) (örn, eşleştirilmiş t-testi, bir ya da iki parametrik ya da parametrik olmayan istatistiksel testler yapılarak AdV verimleri üzerindeki veri dağıtım ve deneysel tasarım bağlı) ANOVA yollu. </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.

Elektropozitif filtreler su virüsleri konsantre yararlıdır; Ancak bu filtreler sırayla onların etkinliğini değiştirebilir kendi yapısı ve kompozisyon farklı olabilir. Bu sorunun Bileşikler, kapsid yapıları ve ücretleri konsantrasyon teknikleri optimum kurtarma 15 sağlamak için uyarlanabilir gerektiren virüs suşları arasında değişmektedir. (Örneğin, elektropozitif filtreler, dana özü elüsyon), hedef virüs daha etkili konsantrasyon 16,17 elde edilebilir, mevcut k...

İnsan enterik virüsler su kaynaklı hastalıkların 1-3 önemli etken vardır, ama konsantrasyon olmadan kendi algılama zorlaştırır, genellikle kontamine çevre sularında düşük sayılarda bulunurlar. Virüsleri konsantre için kullanılan prosedürler, genellikle filtre yıkama sıvısının bir hava filtresi, yıkama, ardından adım, ve ikinci bir konsantrasyon yer alır. Yaygın bir filtrasyon işlemi (son 4,5 &#39;de gözden geçirilmiştir), elektropozitif filtreler gibi yüklü membran kullanılması ile ilgilidir. Bu filtreler, filtre alanı (pozitif yüklü) ve hedeflenen virüs parçacıkları arasındaki elektrostatik etkileşimleri kullanılarak su içinde süspansiyon haline getirilmiş virüs yakalama dayanır (negatif yüklü). Ticari olarak mevcuttur iki elektropozitif filtreler bu teknoloji güveniyor, cam / selüloz ve nano-alümina / cam elyaf filtreler. Cam / selüloz filtre maliyetleri cam / ce kullanımını sınırlamak kadar 10 kez nano-alümina / cam elyaf ki, vardırRutin virüs kontrolü için llulose filtreler. Son çalışmalar ucuz filtre alternatif kullanımını haklı, farklılıklar ortam su 6,7 den enterovirüslerin kurtarma bu iki filtre arasındaki nominal olarak sonucuna varmışlardır. Örneğin elektronegatif ve cam yün filtre gibi diğer filtre ayarları, ancak, her iki kaynak, su (elektronegatif filtreleri) tabi tutulmasını gerektirir ya da (cam yünü filtreleri), ticari olarak mevcut olmadığında, incelenmiştir. virüs konsantrasyonu prosedürlerinin geliştirilmesi çok sudan virüs verimini artırmak amacıyla, birinci konsantrasyon teknikleri (filtre) optimize odaklanmıştır. Ancak, hacimleri mililitrelik için tipik olarak 1 L yıkama sıvısı hacmini azaltır ikincil konsantrasyon işlemleri, ayrıca virüs geri kazanımlar 8 üzerinde önemli bir etkiye sahip olabilir. Bağırsak virüslerinin ikincil konsantrasyonu tipik olarak sığır özü bazı türleri gibi bir pıhtılaştırıcı madde dayanır (organik floccuyon) veya yağsız süt flokülasyon 9-12 filtre yüzeyleri virüs parçacıkları kaldırmak için. Son zamanlarda, selit (ince parçacık) eklenmesi ile bağlanmış sığır eti ekstresi ile bir ikinci yoğunlaştırma işlemi adenovirüs, enterovirüs ve Norovirus 8,13,14 geri kazanılması için umut verici olduğu gösterilmiştir. Bu virüs parçacıklarının organik flokülasyon yöntem ile aynı prensiplere göre, Celite konsantrasyonu işleri tutunur ve süspansiyon çözeltisinin pH&#39;ını değiştirilmesiyle partiküller (topak veya selit) salınır. Bu iki ikincil konsantrasyon teknikleri arasındaki karşılaştırmalar çivili adenovirüs (AdV) türleri 40 ve 41 8 kurtarma değerlendirilmiştir. Bu çalışma iki ikincil konsantrasyon teknikleri adenovirüslerin kurtarma istatistiksel olarak benzer olduğu sonucuna varıldı. Bununla birlikte, organik çökeltme yöntemi, bir 30 dakika gerektirir. pH 3.5 inkübasyon, selit tekniği pH 4.0 kısa bir inkübasyon (10 dakika) gerektirir ise. Organik flocculatiyonu, tertier konsantrasyon sırasında topaklanma maddeleri toplamak pahalı laboratuar ekipmanları (santrifüj) kullanımını gerektirir, farklı olarak selit tekniği süspansiyondan selit parçacıkları ayırmak için sadece temel laboratuar ekipmanı (vakum filtrasyon) kullanılır. Filtreler ve ikincil yıkama teknikleri belirli kombinasyonları da virüs kurtarma etkileyebilir. Bir çalışma birincil (elektropozitif filtrelerin) belirli kombinasyonları ve ikincil konsantrasyon teknikleri (Celite veya organik flokülasyon) adenovirüs 13 kurtarma önemli bir etkiye sahip olduğu sonucuna varmıştır. Bu bulgular, bu teknikler kullanılarak zaman bu optimizasyon optimum verilen bir su matris hedef virüsü geri kazanmak için gerekli olduğunu düşündürmektedir. Optimizasyon sayısız değişkenler (filtre tipi / marka, pH elüsyon çözeltisi, selit / organik flokülasyon) değerlendirilecektir çünkü, zorlu bir süreç birçok araştırmacı aktif önlemek tüketen bir zaman. Bu s içinHer virüs tipi eşsiz bir kapsid morfolojisi ve özel kapsid şarj görüntüler beri tudy, bir prosedür, Muhtemelen çivili insan adenovirüs 40 suşlar ve 41. kullanarak sudan virüs konsantrasyonu için en uygun koşulları belirlemek için geliştirilen, konsantrasyon protokolleri her virüs için optimize edilmesi gerekebilir Optimal viral kurtarma ulaşmak için hedef. 1) elektropozitif filtre ardından 2 diskler) ikincil konsantrasyon olarak celıte tekniği karşı kurulmuş bir organik flokülasyon yöntemi değerlendirmesini kullanarak musluk suyunda virüs kurtarma değerlendirilmesi, ve 3) değerlendirme: Bu çalışma AdV 40 ve 41 ile konsantrasyon için bir yaklaşım sağlar tertier konsantrasyonu için elüsyon tamponları.

<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>
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			<td height="21" style="height:21px;width:231px;">Adenovirus 40 stock</td>
			<td style="width:143px;">ATCC</td>
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			<td></td>
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			<td height="21" style="height:21px;width:231px;">Adenovirus 41 stock</td>
			<td style="width:143px;">ATCC</td>
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			<td height="21" style="height:21px;width:231px;">NanoCeram 47mm</td>
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			<td height="21" style="height:21px;width:231px;">1MDS 47mm</td>
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			<td height="21" style="height:21px;width:231px;">PBS</td>
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			<td style="width:119px;">P5368</td>
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			<td height="21" style="height:21px;width:231px;">Hydrochloric Acid</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">A481-212</td>
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			<td height="21" style="height:21px;width:231px;">BBL Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
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			<td>http://www.epa.gov/nerlcwww/online.html</td>
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</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 seçimi Celite Üç farklı öncesinde iyi performans varyant seçimi test edildi. Orta ölçekli parçacıklara cezası ile selitler yüksek adenovirüs geri kazanımlarını üretti. Daha büyük selitler kullanımı AdV40 ve 41 (aralık% 32% 100) (Şekil 1) her ikisi için de daha düşük kurtarma ile sonuçlanmıştır. Adenovirüs 40 Ortalama kazanımları 144% ± 52% (ince), 115% ± 28% (orta) ve% 82 ± 53% (büyük) partikül selitler ve AdV41, 132% ± 3...

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.

Sudan Viral Konsantrasyon için Küçük Bir Ses Prosedürü

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 Görüntüleme.  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.

Göl tortulları Laboratuvar belirlenen Fosfor Flux İç Fosfor Yükleniyor bir tedbir olarak

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

Çevre

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.

Küçük Hacim Biyodeneyler SU-Pulse-Genlik Modülasyonlu (SU-PAM) fluorometri Kullanma Bakteriyel / Fitoplankton Co-kültür değerlendirilmesi için

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</subTest CO&gt; Konsantrasyon Gradient Tesisi<sub&gt; 2</subÇayır Ekosistem Fonksiyon&gt; Zenginleştirme ve Toprak Etkileri

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.

Küçük Kirleticilerin İmmünolojik Analizi Su Örneklerinin Otomatik Katı Faz Ekstraksiyon Basit Bir Yöntem

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

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.

Coupled Hidrolojik, jeokimyasal ve Mikrobiyolojik Araştırmalar Toprak Lizimetre Kazı

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

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

Su Kullanımı Endokrin Aktivitesi Tarama Piyasada satılan<em&gt; İn Vitro</em&gt; Transaktivasyon biyolojik tayinler

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

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.

Plastik Peletlerden Organochlorine Pestisitlerin Çıkarılması ve Plastik Tipi Analizi

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

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Ölçme ve toprak erozyon ve toprak karbonat konsantrasyonları tarım Yönetimi'nin altında ilgili ifade eşleme

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

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.

Su akışı ağırlıklı ve tarım kanallar üzerinden askıya alınan parçacıklar drenaj olaylar sırasında yakalama

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.

Bir basit tekniği sık su baskını/taşkın oluştuğu için yeniden kurulması ağaç dikme

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 ...

Sudan Viral Konsantrasyon için Küçük Bir Ses Prosedürü

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