Yazarlar Bağış No. 31003A-132358/1, 31003A_152972 ve sayılı 151948 İsviçre Ulusal Bilim Vakfı kabul ve Fundation Pierre Mercier la Science dökün.

Kimyasalların ve Ekipman 1. Hazırlık <ol><li> % 1 sodyum heksametafosfat (SHMP) (napo 3) n çözeltisi 500 ml yapmak ve otoklavda sterilize edilir. </li><li> Kapalı cam Petri kapları içinde otoklavlanarak sterilize nitroselüloz (NC) filtreleri ile (gözenek büyüklüğü 0.22 um, çap 47 mm). </li><li> Kapalı cam Petri kapları içinde otoklav ile Kuzey Carolina filtre ile (gözenek büyüklüğü 0.22 um, çap 25 mm) sterilize edin. </li><li> Tartılır ve (kapağı ile) steril 50 ml tüpler boş ağırlığı (numune başına bir tüp) not edin. </li><li> Tris-EDTA-tamponu (TE-tamponu) 1x hazırlayın: 10 mM Tris (tris (hidroksimetil) aminometan) ve 1 mM EDTA tampon solüsyonu yapmak. 8 pH ayarlayın ve otoklav ile sterilize. </li><li> 1 ml TE-tamponu içinde lizozim 0.02 g çözülerek lizozim çözeltisi (20 mg / ml) hazırlayın. İdeal olarak, her zaman taze olun. En fazla 1 hafta boyunca 4 ° C&#39;de saklayın. </li><li> 8 g / L sodyum klorid çözündürülmesiyle fizyolojik çözeltiyi hazırlayınDamıtılmış su (NaCI). Otoklav ile sterilize. </li></ol> Sedimentten Biyokütle 2. ayrılması <ol><li> Tortu numunesi 3 g etanol Alevli metal kepçe kullanarak 50 ml tüpler steril tartılmış önceden ekle. Kirlenmesini önlemek için temiz, UV steril biyogüvenlik kabini bu gerçekleştirin. </li><li> Steril bir büret kullanılarak dereceli numuneye% 1 steril (otoklav) SHMP çözeltisi 15 ml ekleyin. SHMP çözüm de kirlenmeyi önlemek için filtre edilebilir. </li><li> Bir sıvı dağıtıcı / homojenizör (örneğin, Ultra-Turrax homojenleştirici) ile tortu ve SHMP çözelti homojenize edilir. % 70 etanol-sterilize veya kullanmadan önce dağılım rotor otoklav. 17.500 rpm&#39;de 1 dakika için homojenizasyon çalıştırın. 2 dakika boyunca numune kalan verin ve rotor hızında 1 dakika için homojenizasyon tekrarlayın. </li><li> Numune 10 dakika bekletin. Bu aşamada, ağır parçacıklar (mineraller) razı olacak. Hücreleri ve bununla birlikte W numunenin herhangi bir organik bileşenlerHasta çözelti içinde kalır. Sediment pelet rahatsız etmemek için dikkat çekerken Daha sonra, temiz bir 50 ml tüp içine süpernatant solüsyonu (içeren hücre biyokütle) aktarın. </li><li> Sediman pelet steril bir büret mezun kullanarak% 1 steril (otoklav) SHMP solüsyonu tekrar 15 ml ekleyin. Sonra tekrar 2.3 ve 2.4 adımları tekrarlayın. Bu tekrarlama tortu mineral bileşeni hücreleri ve organik partiküllerin maksimum miktarını ayrılmasını sağlar. Bu ikinci ayrılık süpernatant ilk ayrılık süpernatanıyla birleştirilebilir. Not: Aşağıdaki adımlar tüm (hücre biyokütle içeren) süpernatant yapılır. Mineral bileşen (tortu topak) iptal edilebilir. </li><li> 1 dakika için 20 x g&#39;de santrifüjleyin. Biyolojik hücre malzeme hala çözelti içinde kalır Bu adım, küçük mineral parçacıkları çözmek için yeterli g-gücünü daha da arttırır. Santrifüj işleminden sonra, bir yüzer madde halinde çözeltisi içeren (Hücre biyokütle) aktarmaktemiz 50 ml tüp. Mineral pelet atın. </li><li> Numuneyı tartmak biyokütle içeren çözeltinin son hacmi belirlenir. Ağırlığı belirlemesi, dereceli bir silindire örnek aktarmak zorunda ve kirlenme riskini azaltır önler. </li></ol> Filtre Zarında Biyokütle 3. Koleksiyonu <ol><li> Filtrasyon ünitesi (47 mm çaplı membranlara) ve vakum pompası hazırlayın. Otoklav yoluyla veya% 70 etanol ile püskürtme ve Bunsen beki ile yanan tarafından (Pyrex cam varsa) ya filtrasyon ünitesi sterilize edin. Bu protokol ile devam etmeden önce soğumasını bekleyin. </li><li> Etanol-yakılmış sterilize forseps kullanarak filtrasyon ünitesi steril NC membranı ekleyin. </li><li> Vakum pompası kullanılarak membran üzerinde hücrelerin membran filtrasyon ünitesi üzerine (adım 2.6 dan itibaren) süpernatant numunenin yarısını ekleyin ve toplamak. </li><li> Sıvı tamamen filtre üzerinden geçtikten sonra, vakum pompası durdurmak ve dikkatle Ethan kullanarak membran kaldırmakol-alev forseps sterilize. Steril bir Petri kabı içine zarın yerleştirin. <ol><li> Etanol-yakılmış sterilize makas kullanarak yarısında zarı kesin. Ayrı bir 2 ml&#39;lik bir tüpe zar her yarım ekleyin. Membranın bir yarısı DNA ekstraksiyonu ve tüm bakteriyel eden analizi için kullanılır. Filtrenin diğer yarısı -80 ° C&#39;de saklanır ve yedeği olarak görev yapar edilecektir. </li></ol></li><li> Filtrasyon ünitesi üzerine yeni NC membranı yerleştirin ve vakum pompası kullanılarak (adım 2.6 dan itibaren) örnek hacmi ikinci yarısından itibaren biyokütle toplamak. </li><li> Sıvı tamamen filtre üzerinden geçtikten sonra, vakum pompası durdurmak ve dikkatle etanol-Alevli sterilize forseps kullanarak membran kaldırmak. Ayrı 2 ml tüp içine tüm membran yerleştirin. Bu örnek, bitki hücrelerinde endosporlar ayırmak için tedavi için kullanılır. Örnek kullanılana kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol> Bitkisel Hücre 4. Liziz <ol><li> GerçekleştirmekTedavi önceden NC zarın (adım 3.6) toplanan biyokütle üzerinde vejetatif hücrelerden endosporlar ayırmak için. <ol><li> Membran donmuş ise, çözülme 10 dakika boyunca oda sıcaklığında bekletin. Daha sonra steril bir Petri kabı membran yerleştirmek ve etanol-Alevli steril makaslar kullanılarak küçük parçalar halinde (yaklaşık 4 kat) kesti. Sonra steril bir 2 ml tüp içine tüm membran filtre parçaları yerleştirin. </li></ol></li><li> Örnek zarı içeren tüp 1x TE (Tris-EDTA) tamponu 900 ul (1.5 bakınız) ekleyin ve vorteks ile iyice karıştırın. Bu adımda, biyokütle TE-tamponu çözeltisi içine zardan çıkarılır. </li><li> 10 dakika karıştırıldı ve 80 rpm&#39;de 65 ° C&#39;de bir kuluçka tüp yerleştirin. Daha sonra inkübatör tüp kaldırmak ve 15 dakika soğumasını bekleyin. </li><li> 2 mg / ml&#39;lik bir nihai konsantrasyona ulaşmak için taze hazırlanmış lisozim (1.5 bakınız) 100 ul ekle. Numune 37 ° C soğuduktan önce bu deg olabilir gibi, lizozim ilave etmeyinizenzim rade. </li><li> Lizozim için en uygun koşulları vejetatif hücreleri lize etmek için, 60 dakika karıştırıldı ve 80 rpm&#39;de 37 ° C&#39;de örnek inkübe edin. </li><li> Lisis tamamlandıktan sonra, 3 N sodyum hidroksit (NaOH) ve örnek 6% sodyum dodesil sülfat (SDS) çözeltisi 250 ul 250 ul ilave edin. Bu ekleyerek, numune hacmi 1,5 ml ulaşır ve 0,5 N NaOH ile nihai konsantrasyon% 1 SDS nihai konsantrasyon vardır. </li><li> 60 dakika karıştırıldı ve 80 rpm&#39;de, oda sıcaklığında bu karışımı inkübe edin. Taban ve deterjan ekleme nihai hücre parçalama yardımcı olacaktır. Vejetatif hücreler lize ise, bu deterjan konsantrasyonu, endosporlar zarar şekilde optimize edilmiştir. </li><li> (Pyrex cam ise)% 70 etanol ile püskürtme ve Bunsen beki ile yanan otoklav ile 25 mm çaplı zarlarını tutan, ya da steril filtrasyon ünitesi hazırlayın. Soğumaya bırakın. </li><li> Etanol-alev sterilize forseps kullanarak filtreleme ünitesi üzerindeki 0.2 mikron NC membranı (25 mm çapında) yerleştirin. &lt;/ li&gt; <li> Membran üzerine adım 4.7 numuneyi ekleyin ve vakum pompası kullanılarak sıvı filtre. Sıvı içinden geçtikten sonra, vakum pompası devre dışı bırakın. Bu zar üzerinde muhafaza edilmez Bu adımda, parçalanmış bitki hücre malzemesi çıkarılır. Sadece endosporlar membran üzerinde kalacaktır. </li><li> Kalıntı deterjanlar yıkayın ve vakum pompası kullanılarak sıvıyı filtrelemek üzere steril fizyolojik solüsyon 2 ml ilave edilir. </li><li> Sıvı tamamen süzülür olduğunda, vakum pompası kapatın. Filtrasyon ünitesi ile ilgili membran bırakın. </li></ol> 5. DNase Tedavi Not: Filtre membran üzerinde doğrudan DNaz tedavi uygulayın. Bu filtreleme ünitesi akmaması önemlidir ve vakum pompası kapatılır. <ol><li> Filtre membran üzerine steril su, 450 ul doğrudan DNase reaksiyon tampon maddesi (1x) ve 0.5 ul DNaz enzimi 50 ul ilave edin ve 15 dakika boyunca bekletildi. Mümkünse, biraz savaş bir odada bu sindirim yapmakOrtalama RT daha mer, enzimin, yukarıdaki 25 ° C ve sıcaklıklarda daha iyi olduğunda işe yaramaktadır. Not: Bunsen beki tutulması kenara filtrasyon ünitesi de sıcaklığını arttırır ve steril bir ortamda tutmak yararı vardır, bu nedenle örneklerin bulaşma riskini azaltmıştır. </li><li> DNaz sindirim tamamlandığında, numuneden enzimi kaldırmak için vakum pompası açın. </li><li> Ekleme ve fizyolojik çözelti 1 ml filtre edilerek artık enzimin yıkayın. </li><li> Sıvı tamamen geçtiyse, vakum pompasını kapatın ve steril forseps kullanarak ve steril Petri kabı içine yerleştirin endosporlar içeren filtre membran kaldırmak. Not: ayrılmış endosporlar örnek şimdi aşağı analiz için hazırdır. Çimlenme ve kültivasyonu için kullanıldığında -20 ° C&#39;de saklayın DNA ekstraksiyonu için ya da alternatif olarak 4 ° C &#39;de kullanıldığında. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Dış agresif fizikokimyasal faktörlerden (örneğin, sıcaklık ya da deterjanlar) ile endosporlar direnci çevre örneklerinde bitkisel hücrelerden bakteri endosporlar ayırmak için bir yöntem bulmak için kullanıldı. Bu bir tahribatsız şekilde çevresel örneklerden izole endosporlar ilk kapsamlı yöntemdir. Ölçmek algılamak veya numunelerde endosporlar analiz etmek için önceki metotlar, dipikolinik asit veya spesifik bir işaretleyici genler gibi endosporlar özel vekiller ölçümüne dayanma...

Bu çalışmanın amacı, çevre numuneleri içinde vejetatif bakteriyel hücrelerden bakteri sporları ayrılması için bir protokol sağlamaktır. Bakteri sporları oluşumu filum Firmicutes 1 ait olan bakteri grupları bir dizi bulunan bir hayatta kalma stratejisi genellikle açlık tetiklediği vardır. Endospore oluşturan bakteriler de suşları bir dizi patojenleri ve dolayısıyla tıbbi önemi (örneğin, Bacillus anthracis veya Clostridium difficile) vardır başlıca nedeni, incelenmiştir. Endospore oluşturan bakterilerin Çevre suşları hemen hemen her ortamda (toprak, su, sediment, hava, buz, insan bağırsak, hayvanlar gut, ve daha fazla) 1-3 izole edilmiştir. Bu nedenle, Firmicutes kültür koleksiyonları 4 ikinci en bol filum vardır. Çünkü onların cesur dış korteks ve koruyucu çekirdek proteinlerinin, endosporlar fr değişen aşırı çevre koşulları yaşayabiliryüksek radyasyon, aşırı sıcaklıklara ve zararlı kimyasallar 5 om kuruma. Bu olağanüstü direnci endosporlar 6-8 DNA ayıklamak için bir meydan okuma yapar. Çevresel sıralama çalışmalarında 9,10 gözardı edilmiş bu yüzden muhtemelen açıklıyor. Floresan antikorlar 11 ile çevre numuneleri içinde endosporlar hedeflenmesi gibi diğer yöntemler, toprak 12 ve tortu 13 dipikolinik asit ölçümü (DPA), 14 akış sitometrisi veya pastörizasyon ve daha sonra kültür 15,16 endosporlar almak veya ölçmek için kullanılmıştır Çevresel örnekleri. Son yıllarda, optimize edilmiş DNA ekstraksiyon yöntemleri yanı sıra, spesifik moleküler primerler endospor-özgü gen sekansları, 10,17-20 geliştirilmiştir hedef. Bu bakteriler 21 bu grup arasında daha biyolojik çeşitlilik ortaya çıkarmak için yardımcı olan ve aynı zamanda endosporlar tespiti için endüstride ve tıpta uygulamalara yol açmıştırSüt tozu 19, örneğin. Burada sunulan protokol hücreleri vejetatif göre bakteri sporları arasında (örneğin, ısı ve deterjanlar gibi), zararlı fizikokimyasal koşullara direnç farka dayanır. Bir numunede vejetatif hücrelerini yok etmek için, biz arka arkaya ısı, lizozim ve deterjan düşük konsantrasyonlarda geçerlidir. Sporlar yok etmek ama tüm vejetatif hücreleri lize etmek için değil, böylece bu tedavilerin zaman ve güç optimize edilmiştir. Bir çevre hücre havuzu bazı hücreler diğerlerinden daha dayanıklıdır, böylece tüm vejetatif hücrelerin tahrip olasılığını artırmak amacıyla, üç farklı tedaviler uygulanır. Bu yöntemin avantajı, ve yenilik tedaviden sonra endosporlar hala sağlam ve daha fazla alt analizler için kullanılabilir olmasıdır. Bunlar d azaltılması ve böylece DNA ekstraksiyon, kantitatif PCR (qPCR) ve amplikon veya özel endosporlar grubu hedef alan metagenomic dizileme (ve dahilÇeflitlilik) kapsama artırırken. Endosporlar de mansap yetiştirme veya floresan mikroskobu ile miktar tayini için kullanılabilir, akış sitometrisi veya DPA tespiti. Bu yöntemin önemli bir özelliği, tedavi edilen bir örnek ile işlem görmemiş bir numune ile karşılaştırılması, tek bir hücre vejetatif karşılık gelen bileşene ek olarak bir çevre numunede endosporlar miktar ve çeşitliliği anlamak olabilir.

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			<td height="63" style="height:63px;width:216px;">Whatman nitrocellulose membrane filters, 0,2 um pore size, 47 mm diameter</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:285px;">WHA7182004 Aldrich&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td>252859 Sigma-Aldrich</td>
			<td>CAS 77-86-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884 Sigma-Aldrich</td>
			<td>CAS&#160; 60-00-4&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysozyme from chicken egg white</td>
			<td>Sigma-Aldrich</td>
			<td>62971 Fluka</td>
			<td>powder, CAS&#160; 12650-88-3&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-Turrax homogenizer T18 basic</td>
			<td style="width:95px;">IKA</td>
			<td>3720000</td>
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			<td height="42" style="height:42px;width:216px;">Glass filter holders for 47 mm membranes, pyrex glass</td>
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			<td>XX10 047 00</td>
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			<td height="21" style="height:21px;width:216px;">Chemical duty vacuum pump</td>
			<td style="width:95px;">Millipore</td>
			<td style="width:119px;">WP6122050</td>
			<td>220 V/50 Hz</td>
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			<td height="42" style="height:42px;width:216px;">Manifold sampling filtration for 25 mm membranes</td>
			<td style="width:95px;">Millipore</td>
			<td>1225 Sampling Manifold</td>
			<td>polypropylene</td>
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			<td height="42" style="height:42px;width:216px;">Dnase</td>
			<td style="width:95px;">New England Biolabs</td>
			<td>M0303S</td>
			<td>Rnase free</td>
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			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S5881 Sigma-Aldrich</td>
			<td>CAS&#160; 1310-73-2&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium dodecyl sulfphate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771 Sigma&#160;</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Whatman nitrocellulose membrane filters, 0,2 um pore size, 25 mm diameter</td>
			<td>Sigma-Aldrich</td>
			<td colspan="2">WHA7182002 Aldrich</td>
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physical isolation of endospores from environmental samples by targeted lysis of vegetative cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

Burada sunulan sonuçlar, daha önce 10,21 yayınlanmıştır. Verilerin çevre yorumlanması ve tartışılması için bu makaleleri bakın. Genel prosedür Şekil 1 &#39;de özetlenmiş ve üç ana adımlar karşılık gelir: ilk olarak, birikinti ya da herhangi bir diğer çevresel matris biyokütle ayrılması; İkinci, vejetatif hücrelerin tahribi; ve üçüncü olarak, ayrılmış endosporlar alt baş analizi. Alt analizi çeşitliliği belirlemek için, DN...

Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

Bitkisel Hücreler Hedefli lizis ile Çevre Örneklerden endosporlar Fiziksel İzolasyonu

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

physical-isolation-endospores-from-environmental-samples-targeted

Research

JoVE Journal

Environment

14.1K Görüntüleme.  Macquarie University. A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

Video: Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

Enterovirus Kültür ve RT-qPCR Su Norovirüs Oluşum EPA Metot 1615 Ölçüm. Virüs Örneklerinin I. Koleksiyonu

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Çevre

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

Fiziksel, Kimyasal ve için üretilen Altı Biochars Biyolojik Karakterizasyonu Kontamine Siteleri İyileştirilmesi

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

Enterovirüs Kültür ve RT-qPCR Su Norovirus Oluşum EPA Method 1615 Ölçümü. Bölüm III. RT-qPCR Virüs Algılama

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Otomatik Jel Boyut Seçimi Çevre Su Örneklerinden hazırlanmıştır nesil Sıralama Kütüphaneler Kalitesi Geliştirmek için

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

Uranyum dan Bakır Oksit Nanopartiküller tarafından İz Elementlerin çıkarılması<em&gt; In Situ</em&gt; Kurtarma Bleed Su ve Hücre Canlılığı Üzerine Etkisi

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

Hazırlık ve Test zehirlilik için hızlı değerlendirme İçme Suyu Örnekleri için RTgill-W1 Hücreleri ile akışkan bioçipler Empedans tabanlı

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

Topraklarda Atterberg Plastik Limiti Belirleme A Bükme Testi

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Filtrasyon Su Örnekleri ve Çevre DNA Ekstraksiyon için bir Filtre Kartuşu Kullanımı

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

Nanomalzemeler sulu ortamda dağılımı: doğru iletişim kuralı en iyi duruma getirme

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal &#39;explosions&#39; by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' Formicidae for Volatilome Analysis by GC-MS and MetaboliteDetector

Minor workers of the ant species Colobopsis explodens are dissected to isolate the wax-like content stored in their hypertrophied mandibular gland reservoirs for subsequent solvent-extraction and analysis by gas chromatography-mass spectrometry. The&#160;annotation and identification of volatile constituents using the open-source software MetaboliteDetector is also described.

Yalıtım Borneo dan çene kemiğinde bezi rezervuar İçindekiler ' GC-MS ve MetaboliteDetector göre Volatilome analiz için karıncalar (karınca) patlayan

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis ...