Bu protokolün geliştirilmesi için Fon Yükseköğretim için Güneydoğu Pennsylvania Konsorsiyumu ve Cabrini Koleji Fen Bölümü tarafından sağlanmıştır. Ek finansman ve destek Arcadia Üniversitesi ve Immaculata Üniversitesi&#39;nden geldi. Biz özellikle nazik bizim izole fajların elektron mikroskobik görüntüleri almak için Joseph Üniversitesi&#39;nden Dr. Karen Snetselaar teşekkür ederim. Ek destek Genomik ve Evrimsel Bilimi (SEA-fajlar) programı ilerleyen Howard Hughes Tıp Enstitüsü Fen Bilgisi Öğretmenliği İttifak Phage Hunters tarafından sağlandı.

Faj İzolasyon için Arthobacter Hücreler 1. Hazırlık <ul><li> Kültür Arthrobacter sp. KY3901 koloni 2-3 gün boyunca 30 ° C&#39;de inkübe edilen Luria Bertaini (LB) agar plaka üzerinde çizgi şeklinde. Bir koloni seçin ve 30 ° C&#39;de 225 rpm&#39;de bir çalkalama inkübatöründe saptırma plakalı bir kültür şişesi içinde LB suyu 250 ml eklemek ve inkübe etmek için steril bir döngü kullanılır. </li><li> Büyüme yaklaşık 24 saat faj enfeksiyonu deneyler için geç / erken üstel sabit faz hücreleri elde etmek izin verin. Geç sabit büyüme durumuna orta girmesini engellemek hücreleri yakından bakteriyel büyüme durumunu izleyin. Not: Hücre büyümesi, aşağıda tarif edilen faj izolasyon ve saflaştırma prosedürleri / aşamada 0.5-.0.7 arasında bir optik yoğunluk OD 600 arasında olmalıdır. </li></ul> Toprak Örneklerinden Faj 2. Koleksiyonu <ol><li> İstediğiniz yerden toprak 200-400 gr toplayın. Not: Arthroba bakteri yanacter cins onları enfekte fajlar bulunan ve toprak birçoğuna yaygın olan olabilir bunlar ve ortak toprak bakterilere vardır. </li><li> PB değildir, böylece, en azından 200 ml toprağa faj Tamponu (PB): [68 mM NaCI, 10 mM MgSO 4 ile, 10 mM Tris-CI (pH 7.5)], en az 400 ml ilave edilir ve yeterince büyük bir şişe içinde karıştırın Üstte kalan sıvı kısımdaki ve ekstre edilmesi mümkündür. </li><li> Toprak askıya kadar yavaşça karıştırarak veya dönen karıştırın ve toprak sediment, tipik olarak 30 dakika yerleşmek için izin verir. </li><li> Toprak sediment enkaz kaldırmak için yerçekimi filtrasyon ile filtre kağıdı ile &quot;toprak özü&quot; geçirin. </li><li> LB tozu eklenir ve bir% 2 solüsyon (200 mi toprak özü 4 gr LB toz) yapmak için karıştırın. </li><li> Steril bir süzüntü elde etmek için vakum filtrasyonu ile 0.22 um filtre içinden solüsyonu geçirir. Mümkünse, hemen 2.7 adıma devam. Uygun bir faj partiküllerinin, mağaza devam etmeden önce bir hafta kadar 4 ° C &#39;de numuneler, süzüldü olması gereken, ancak, sayıazalacaktır. </li><li> Kısım birden fazla aseptik teknikler kullanılarak bölmeli kültür şişeleri çalkalama sterilize ayrı 250 ml&#39;lik filtre ile sterilize edilmiş LB / kir özü karışımı, 50 ml bölümleri. Farklı Arthrobacter fajlar farklı CaCl2 koşullar altında en iyi şekilde büyür beri istenen nihai konsantrasyonlarda (0-50 mM), steril 2.0 M CaCl2 çözeltisi ekleyin. NOT: Biz, bize tarafından tanımlanan Arthrobacter fajların çoğunluğu 1 mM-2.5 mM CaCl2 aralığında olduğunu bulmak. Ancak, izole fajların birkaç çok yüksek CaCl2 konsantrasyonlarda 0 mM CaCl2 .ya münhasıran bizim büyüdü. 3.1 adıma hemen geçin. </li></ol> 3. Faj İzolasyonu <ol><li> Şişesinden her geç / erken üslü durağan faz bakteri kültürü 1-2,5 ml ilave edilir. 0,5-0,7 bir OD 600 için, hücreler 1 ml kullanılır. 0.7 OD &#39;den daha yüksek bir OD 600 için 2.5 ml kullanmakHücreler. Not: üslü büyüme fazında hücreler, tipik olarak daha fazla bağlanma fajlarından durağan fazı büyümesinde hücrelerden daha yüksek titrelerini üretmek. </li><li> Bir çalkalama inkübatör içinde yaklaşık 24 saat süreyle 30 ° C&#39;de 250 rpm&#39;de çalkalama. </li><li> 24 saatlik bir kuluçka süresinden sonra çalkalama inkübatör zenginleştirme şişeler çıkarın ve zenginleştirme numuneleri faj tamponu içinde on kat seyreltilir. </li><li> Geç / erken üslü durağan faz bakteri kültürü 0.5 ml kültür tüpleri kurmak ve çeşitli zenginleştirme kültür miktarda kültür tüplerine (PB 10 -1 konsantrasyondan 500 ul 5 ul) ilave edin. NOT: Her toprak örneği zenginleştirme sonra farklı faj titreleri üretir beri konsantrasyonlarının geniş bir yelpazede test etmek önerilir. </li><li> Orijinal zenginleştirme şişede konsantrasyonu mevcut bir nihai konsantrasyon eşit hale getirmek için, kültür tüplerine CaCl2 ekleyin. W çok faj seçmek için farklı CaCl2 bir konsantrasyonu kullanaraki CaCl2 bağımlılıkları değişen. NOT: Çoğu fajlar 1-2.5 mM CaCl2 aralığında izole edilecektir, ancak 0-50 mM CaCl2 konsantrasyonları her yerde en iyi şekilde izole fajlar vardır. Bu nedenle bu eşsiz izole Arthrobacter fajların sayısını arttırmak için CaCl2 konsantrasyonlarının bir dizi kontrol etmek en iyisidir. </li><li> Kültür tüpü içinde LB üst agar 4,5 ml karıştırın ve LB agar plakası üzerine dökün. Eşit plaka üzerinde dağıtılması ve yaklaşık 15 dakika boyunca katılaşması üst agar sağlamak için hafifçe karıştırılır. Not: Agar büyük gruplar (250 mi) ve ilave üst agar hazırlanabilir en oda sıcaklığında depolanır ve en az iki hafta, daha sonraki deneyler için yeniden kullanılabilir. Üst ağarı yapmak için kullanılan çeşitli formüller vardır, ancak biz LB ile karıştırılır agar 7 gr / L kullanılan agar üst yapmak Bir 60 ° C su banyosu içinde yer üst agar bir deney sırasında, sıvı durumunu korumak için. </li><li> Haricindekileri to plaka ve istenilen sıcaklıkta 48 saat için (sıcaklık 30 ° C) göre O / N inkübe edin. Mevcut fajlarla için optimize etmek bu koşullar Vary. En çok izole fajlar 24 saatlik bir kuluçka döneminden sonra 1-2.5 mM CaCl2 konsantrasyonunda 30 ° C &#39;de kuluçkaya yatırılmıştır gelir. </li></ol> 4. Faj Arıtma <ol><li> Plakalar üzerinde plak oluşumu için 24 saat inkübasyon kontrolünden sonra. Hiçbir plaklar belirgin eğer kadar 72 saat inkübasyon devam veya ek plaklar görünüp görünmeyeceğini belirlemek için. Bir haftaya kadar 4 ° C&#39;de önce usul için varsayılan plaklarla, mağaza plakaları Gerekirse. NOT: Bu bir plaka üzerinde farklı plak morfolojileri çeşitli bulmak için nadir değildir. 3 gün inkübasyondan sonra, plaklar bulmak mümkündür, ancak çok nadirdir. </li><li> Çizgi plak yöntemi kullanılarak açık bir şekilde izole plaklardan istenen fajları arındırın. Steril tahta sopa ile bir plaket dokunun. Streak, bir alev ile bir tahta sopa çalışan allowin tarafından plakg sopa kısaca serin ve Şekil 1&#39;de gösterildiği gibi daha sonra yumuşak hareket agar plaka üzerinde sopa ovuşturarak. Her geçişte için temiz bir sopa kullanarak bu işlemi tekrarlayın için. </li><li> Seçenek olarak ise, tek tek faj plakları izole edilmesi için geleneksel bir plak titre deneyi kullanmaktadır. Plak titre deneyi, faj plak izolasyonu için Petri plakaları gibi LB agar ve LB üst agar ve malzeme daha fazla reaktifler kullanılmasını gerektirir. Biz çizgi plak yöntemi kullanılarak son derece başarılı olmuştur ama o plak titre testi kullanılarak bireysel plaklar izole etmek teknik olarak daha kolaydır. </li><li> LB agar plakası üzerine en az üç kez çizgili sonra, farazi faj partiküllerinin en düşük konsantrasyon alanı işareti ve yavaşça üst levha, kalsiyum klorür, aynı konsantrasyonunu içeren (Arthrobacter, 0.5 ml erimiş LB üst agar 4,5 ml uygulamak ) ve plaka boyunca eşit yaymak için izin verir. </li><li> Ters çevirin ve / N plaka O kuluçkaya yatmaktadır. Çizgi plak tekrarlayınsaf faj türü sağlamak üzere en az üç tekrarı için teknik izole edilir. Faj canlılığı kaybı olmadan çizgiler arasında gerekli bir haftaya kadar olduğu gibi 4 ° C&#39;de muhafaza plakalar. </li><li> İstenilen plaklar son çizgi plaka üzerinde yetiştirilen ve izole edilmiş, bir mikropipet ucu ile bir iyi izole plak dokunun ve 100 ul PB yeniden askıya. Seri taze PB 180 ul içine numune 20 ul geçerek 10 -8 seyreltme bu 10 0 çözüm sulandırmak. </li><li> Faj izole etmek için kullanılan CaCI2 aynı konsantrasyonunu içeren erimiş LB üst agar 4.5 ml ile enfekte bakteri karıştırarak 10 dakika oda sıcaklığında ve plaka 5 için, bir kültür tübü içinde yetiştirilen Arthrobacter 0.5 ml için seyreltilerin her birine, 10 ul ekle Orijinal toprak örneğinden. Ertesi güne kadar 4 ° C &#39;de yapılan tüm seyreltiler kaydedin. </li><li> Geleneksel plak titresi olarak kullanarak bir web desen yapmak için gerekli faj seri seyreltme belirleyinsöylüyorlar. Plak titre Bu analizin amacı bir web model plakası elde etmek üzere gerekli olan, faj titresi belirlemektir. Çoğunlukla bakteri yoksun ancak henüz fajlarla tarafından parçalanır henüz bakteri kalıntıları içeren bir web desen plaka tanımlama. Steril bir 50 ml kültür şişesine geç / erken üslü sabit Arthrobacter kültürünün 5 ml ilave edilir. <ol><li> Kültür şişesi içinde seyreltilmesi (yani Arthrobacter kültürü için bir web deseni veren) 100 ul ilave edin ve faj, oda sıcaklığında 5-10 dakika boyunca bakteri hücreleri enfekte etmek için izin verir. Başlangıçta faj belirlemek ve 10 taze LB plakaları üzerine Bu karışımın 5 ml levha için kullanılan CaCI2 aynı konsantrasyonunu içeren erimiş LB üst agar ile karıştırılır. Katılaşmaya ve 30 ° C&#39;de ters plakaları inkübe izin verin. </li></ol></li><li> Bir sonraki gün, 4 ° C&#39;de PB ve mağaza O / N, 5 ml ya da oda sıcaklığında 4 saat web model plakalarını sel. </li><li> 0.22 um&#39;lik bir şırınga filtreden geçirilmiştir faj lizatı Filtreve 4 ° C&#39;de depolayın. Bu faj partiküllerinin elektron mikroskobu görüntüler, standart prosedürler kullanılarak, DNA kısıtlama enzimi analizi ve genomik dizileme için faj genomik DNA izolasyonu gibi ek deneyler için bu son faj stoku kullanın. Faj stokunun uzun süreli depolama için -80 ° C &#39;de hazır arşivleme için küçük şişelerde steril gliserol faj lizatının eşit miktarını ekleyin. NOT: Bu faj teknikleri ile deneyimli olanlar için büyük bir kaynak çevrimiçi erişilebilir www.phagesdb.org sitesidir. </li></ol>

<ol>
	<li>Shapir, N., Mongodin, E. F., Sadowsky, M. J., Daugherty, S. C., Nelson, K. E., Wackett, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Catabolic+Pathways:+Genomic+Insights+into+Microbial+s-Triazine+Metabolism.">Evolution of Catabolic Pathways: Genomic Insights into Microbial s-Triazine Metabolism.</a> J Bacteriol. 189 (3), 674-682 (2007).</li><li>Qingyan, L., Ying, L., Xikun, Z., Baoli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Atrazine+Degrading+Arthrobacter+sp.+AD26+and+Use+of+this+Strain+in+Bioremediation+of+Contaminated+Soil.">Isolation and Characterization of Atrazine Degrading Arthrobacter sp. AD26 and Use of this Strain in Bioremediation of Contaminated Soil.</a> J Environ Sci. 20, 1226-1230 (2008).</li><li>Wang, J., Zhu, L., Liu, A., Ma, T., Wang, Q., Xie, H., Wang, J., Jiang, T., Zhao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+an+Arthrobacter+sp.+Strain+HB-5+that+Transforms+Atrazine.">Isolation and characterization of an Arthrobacter sp. Strain HB-5 that Transforms Atrazine.</a> Environ Geochem Hlth. 33, 259-266 (2011).</li><li>Mages, I. S., Frodl, R., Bernard, K. A., Funke, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identities+of+Arthrobacter+spp.+And+Arthrobacter-Like+Bacteria+Encountered+in+Human+Clinical+Specimens.">Identities of Arthrobacter spp. And Arthrobacter-Like Bacteria Encountered in Human Clinical Specimens.</a> J Clin Microbiol. 46 (9), 2980-2986 (2008).</li><li>Brown, D. R., Holt, J. G., Pattee, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Arthrobacter+Bacteriophages+and+Their+Application+to+Phage+Typing+of+Soil+Arthrobacters.">Isolation and Characterization of Arthrobacter Bacteriophages and Their Application to Phage Typing of Soil Arthrobacters.</a> Appl Environ Microbiol. 35 (1), 185-191 (1978).</li><li>Petrovski, S., Seviour, R. J., Tillett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Gordonia+and+Nocardia+Stabilized+Foam+Formation+by+Using+Bacteriophage+GTE7.">Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7.</a> Appl Environ Microbiol. 77 (21), 7864-7867 (2011).</li><li>Le Marrec, C., Moreau, S., Loury, S., Blanco, C., Trautwetter, a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Characterization+of+Site-specific+Integration+Functions+of+phi+AAU2+infecting+&#8220;Arthrobacter+aureus&#8221;+C70.">Genetic Characterization of Site-specific Integration Functions of phi AAU2 infecting &#8220;Arthrobacter aureus&#8221; C70.</a> J Bacteriology. 178 (7), 1996-2004 (1996).</li><li>Casida, L. E., Liu, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthrobacter+globiformis+and+Its+Bacteriophage+in+Soil.">Arthrobacter globiformis and Its Bacteriophage in Soil.</a> J Appl Microbiol. 28 (6), 951-959 (1974).</li><li>Einck, K. H., Pattee, P. A., Holt, J. G., Hagedorn, C., Miller, J. A., Berryhill, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+a+Bacteriophage+of+Arthrobacter+globiformis.">Isolation and Characterization of a Bacteriophage of Arthrobacter globiformis.</a> J Virol. 12 (5), 1031-1033 (1973).</li><li>Twest, R., Kropinski, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+Enrichment+from+Soil+and+Water.">Bacteriophage Enrichment from Soil and Water.</a> Methods Mol Bio. 501, 15-21 (2009).</li><li>Fullner, K. J., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacteriophage+L5+Infection+of+Mycobacterium+bovis+BCG:+Implications+for+Phage+Genetics+in+the+Slow-growing+Mycobacteria.">Mycobacteriophage L5 Infection of Mycobacterium bovis BCG: Implications for Phage Genetics in the Slow-growing Mycobacteria.</a> Mol Microbiol. 26 (4), 755-766 (1997).</li><li>Robb, S. M., Woods, D. R., Robb, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Growth+Characteristics+on+Stationary+Phase+Achromobacter+cells.">Phage Growth Characteristics on Stationary Phase Achromobacter cells.</a> J Gen Virol. 41 (2), 265-272 (1978).</li><li>Bolger-Munro, M., Cheung, K., Fang, A., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T4+Bacteriophage+Average+Burst+Size+Varies+with+Escherichia+coli+B23+Cell+Culture+Age.">T4 Bacteriophage Average Burst Size Varies with Escherichia coli B23 Cell Culture Age.</a> Journal of Experimental Microbiology and Immunology. 17, 115-119 (2013).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarını beyan ederim.

Birçok önceki girişimleri Arthrobacter ana enfekte edebilen fajları izole etmek rağmen, biz standart zenginleştirme prosedürleri kullanarak küçük bir başarı vardı. geliştirilmiş ve çevresel örneklerden fajları zenginleştirmek için van Twest ve Kropinski 10 tarafından uyarlanan bakteriyel zenginleşme genelleştirilmiş yöntem zenginleştirme prosedürleri çoğunluğu için temel kalır. Önceki çalışmalarda elde edilen kanıtlar doğrudan kaplama yöntemleri izolasyonu ...

Toprak ortamlarında Arthrobacter türlerinin yaygınlığı konak bakteri bu türün izole edilebilen fajların geniş bir dizi ve çeşitlilik sunuyor. Acintobacteriaceae ailesinin Bakteriyel üyeleri atrazin ve diğer çeşitli pestisitler ve herbisitler 1,2,3 gibi inatçı bileşiklerin aşağılayıcı onların katabolik yollar için en önemli olan. Çoğu araştırma Arthrobacter çevresel suşları kullanılarak yapılmıştır olsa, bu cinsin klinik izolatları kan, idrar, göz, ve tüm filogenetik heterojeniteye 4 görüntülendiği diğer birçok insan kaynaklar bulunur. Arthrobacter bakteriler üzerine bir araştırma oldukça geniş gövde varken, sadece bir kaç çalışmalar bu farklı cinsin üyelerine enfekte edebilen fajlarla rapor. İlginçtir ama, Arthrobacter daha önce yapılan çalışmalar &lt;tür toprak yazarak gibi çeşitli anahtar farklı konularda dokunur Enterobacteria fajıem&gt; Arthrobacter türleri aktif çamur arıtma tesislerinde 6 zararlı köpük azaltılması, ve şantiye özgü rekombinasyon ve integraz genleri 7 vurgulayarak amacıyla 5, endüstriyel kullanımlar. Çeşitli zenginleştirme tekniği protokolleri saf faj Arthrobacter türlerinde izolatlarının oluşturmak için istihdam edilmiştir. Erken prosedürler sadece enfekte A. yeteneğine fajlarla 8 yıl bir aşkın veren yükseliş dönemleri için nikotin tuzları gibi ilave toksik ajanlar ile toprak inkubasyon dahil globiformis. Kararsız organik plak tahlil teknikleri ile tespit fajları üretmek için ortaya ile toprak kullanılarak yapılan çalışmalar uzun kuluçka dönemleri 8 atlayarak, percolated. İlginçtir ama, direkt kaplama benzeyen bir teknik düşük başarı oranları ile geçmiş çalışmaları gerekçe, hala araştırmacılar 5 tarafından özellikle düşük başarı oranına sahip olurken birkaç fajlarla sebebiyet veren geçmişte kullanılan <s&gt; 8 kadar. Genel olarak, geçmişte kullanılan izolasyon teknikleri, en sık aerobik toprak temsil Arthrobacter cins rağmen pratikte çok az etkinlik olması için önemli olan doğada 4,9 yılında izole, su ve topraktan fajları izole Van Twest ve Kropinski 10 mevcut zenginleştirme yöntemleri Çevre bakteriyel izolatları ancak bu zenginleştirme teknikleri zenginleştirmek için kullanılan teknikler daha önceki adapte Arthrobacter fajları izole verimsiz kanıtladı. Burada anlatılan yöntemin amacı, erken zenginleştirme yöntemleri tutarlı ve etkin bir şekilde bu bakteriyel cinsten fajları izole ilişkili önceki teknik zorlukların üstesinden, Arthrobacter fajları izole adapte edilebilir &quot;kavramının kanıtı&quot; göstermektir.

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

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

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

Arthrobacter fajlar için geliştirilmiş zenginleştirme tekniği tekrarlanabilirlik göstermek için, 30 farklı toprak örnekleri eşsiz Arthrobacter fajlar, bu zenginleştirme kullanılarak toplanan toprak örneklerinin 22 elde edilen bu 30 toprak örneklerinin Of 2014 yılı ilkbahar ve yaz aylarında farklı zamanlarda ve yerlerde kullanılan prosedür. Standart zenginleştirme işlemi aynı toprak örneklerinin 3 benzersiz fajları vermiştir. Zenginleştirme örnekleri plaklar veya plaklar baş...

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

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

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

Yalıtım için bir optimize Zenginleştirme Tekniği<em&gt; Arthrobacter</em&gt; Bakteriyofaj Türlerinin Toprak Numune İzolatlarının gelen

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

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Research

JoVE Journal

Environment

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

19.3K Görüntüleme.  University of the Sciences. We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

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

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

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

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

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

Sürekli Tasarımı ve İşletilmesi<sup&gt; 13</sup&gt; C ve<sup&gt; 15</supÜniformalı veya Diferansiyel için&gt; N Etiketleme Odası, Metabolik ve Yapısal, Bitki İzotop Etiketleme

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

Çevre

Design and Construction of an Urban Runoff Research Facility

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

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

Kentsel Akış Araştırma Tesisi Tasarımı ve İnşaat

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

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

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

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

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

Toprak Örnekleme ve Entomopatojenik Nematodlar İzolasyonu (Steinernematidae, Heterorhabditidae)

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

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

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

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

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

A CO<sub&gt; 2</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 ...

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

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

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

Yalıtım ve mikrobiyal topluluklarda toprak, rizosferde ve kökleri çok yıllık çim deneylerde Analizi

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

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

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

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

Kök büyüme ve yerelleştirilmiş besin kavramaları görselleştirmek için en iyi duruma getirilmiş bir Rhizobox Protokolü

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

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

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

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

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

Toxicants Trans ve çok jenerasyonel etkileri okumak için Caenorhabditis elegans kullanma

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

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

15Azot Analizi için Mikroplot Tasarımı ve Bitki ve Toprak Numune Hazırlama

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Electrostatic Method to Remove Particulate Organic Matter from Soil

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Removing recently deposited and incompletely decomposed plant material from soil samples reduces the influence of temporary seasonal inputs on soil organic carbon measurements. Attraction to an electrostatically charged surface can be used to quickly remove a substantial amount of particulate organic matter.

Partikül Organik Maddeyi Topraktan Çıkarmak için Elektrostatik Yöntem

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of ...

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Updegraff Yöntemi Kullanılarak Bitki Biyokütlesinin Kristal Selüloz İçeriğinin Tahmini

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...