このプロトコルの開発のための資金は、高等教育のための南東ペンシルベニア州コンソーシアムとカブリーニカレッジ科学部によって提供されました。追加の資金と支援はアルカディア大学とImmaculata大学から来ました。我々は、特に親切に私たちの孤立したファージの電子顕微鏡画像を撮影するために、セントジョセフ大学で博士カレンSnetselaarに感謝。追加のサポートは、ゲノミクスと進化科学（SEA-のファージ）プログラムを推進ハワード·ヒューズ医学研究所の科学教育連盟ファージハンターによって提供された。

ファージを単離するためのArthobacter細胞の作製<ul><li>文化アースロバクター·エスピー KY3901コロニーは2〜3日間30℃でインキュベートルリアBertaini（LB）寒天プレート上にストリーク。コロニーを採取し、バッフル培養フラスコ中のLBブロス250ミリリットルに追加し、30℃で225rpmで振盪インキュベーター中でインキュベートし、滅菌ループを使用。 </li><li>ファージ感染実験のために後半早い/指数関数定常期の細胞を得るための成長の約24時間を許可します。後半定常成長状態に真ん中に入るから細胞を防ぐために密接に細菌増殖の状態を監視します。 注：細胞増殖は、後述するファージの単離および精製手順/ステップのために0.5-.0.7間の光学濃度OD 600で構成する必要があります。 </li></ul>土壌サンプルからのファージの2コレクション<ol><li>目的の場所から土壌の200〜400グラムを収集します。注：Arthroba中の細菌以来CTER属は、それらを感染させるファージに見出され、土壌のほとんどの種類に普及していることができる一般的な土壌細菌である。 </li><li>土壌へのファージバッファ（PB）[68 mMの塩化ナトリウム、10mMのMgSO 4を、10mMのTris-CL液（pH7.5）]の少なくとも400 mlを加えPBの少なくとも200ミリリットルになるように十分な大きさのフラスコ内で混合上清中および抽出することが可能である。 </li><li>土壌が中断されるまで、静かに撹拌または旋回により混合し、土壌の堆積物は、典型的には30分を決済することができます。 </li><li>土壌堆積物の破片を除去する重力濾過により濾紙を通して「土壌抽出」を渡す。 </li><li> LB粉末を追加して、2％溶液（200ミリリットル土壌抽出物中の4グラムLB粉末）を作るために混合する。 </li><li>無菌の濾液を得、真空濾過により0.22μmのフィルターを通して溶液を通過させる。可能な場合は、すぐに2.7のステップに進みます。必要性は、4℃で、店舗フィルタリングされたサンプルは、しかし、生存可能なファージ粒子の数を進める前に、一週間までのためである場合削減されます。 </li><li>一定分量の複数の無菌技術を使用してバッフル培養フラスコを振っ滅菌個々の250ミリリットルにフィルター滅菌LB /土壌抽出液の50ミリリットル部分。異なるアースロファージが異なるのCaCl 2の条件で最適に成長するので、最終的な所望の濃度（0-50 mM）を滅菌2.0 M CaCl 2溶液を追加します。 注：私達は私達によって識別アースロファージの大半が1 MM-2.5のCaCl 2の範囲内であることを見つける。しかし、単離されたファージのいくつかは非常に高いのCaCl 2濃度で0のCaCl 2 .ORで独占的に我々が成長した。 3.1すぐにステップ進んでください。 </li></ol> 3.ファージの単離<ol><li>フラスコのそれぞれに1後半早い/指数関数定常期の細菌培養物を2.5ミリリットルに追加します。 0.5から0.7 OD 600のために、細胞の1ミリリットルを使用しています。 0.7のODよりも高いOD 600の場合の2.5ミリリットルを使用細胞。 注：指数関数的増殖期の細胞は、通常より多くのファージは、固定相成長の細胞よりも高い力価が得られる。 </li><li>振盪インキュベーター中で約24時間、30℃、250rpmで振盪フラスコ。 </li><li> 24時間のインキュベーション期間の後、振盪インキュベーターから濃縮フラスコを取り除き、濃縮サンプルをファージ緩衝液中で10倍に希釈する。 </li><li>後半早い/指数関数定常期の細菌培養物0.5ミリリットルで培養管を設定し、培養管に集積培養の様々な量（PBで10 -1希釈液500μlに5μl）を追加します。 注：これは、それぞれの土壌サンプルを濃縮した後に別のファージ力価を生成するので広範囲の濃度をテストすることをお勧めします。 </li><li>オリジナルの濃縮フラスコに存在する濃度に最終濃度が等しくなるように培養管に塩化カルシウムを追加します。 W多くのファージを選択するために異なるのCaCl 2濃度の範囲を使用してくださいi番目のCaCl 2の依存関係を変化させる。 注：ほとんどのファージは1〜2.5 mMとのCaCl 2の範囲内で分離されますが、0〜50のCaCl 2濃度からどこでも最も最適に分離されたファージがある。このためには、ユニークな孤立したアースロファージの数を最大化するのCaCl 2濃度の範囲を確認するのが最善です。 </li><li>培養管にLBトップアガーの4.5ミリリットルを混合し、LB寒天プレート上に混合物を注ぐ。均等にプレート全体に分散し、約15分間固化するトップアガーを可能にするために穏やかに渦巻。 注：トップ寒天は、より大きなバッチ（250ml）中で調製する​​ことができ、余分なトップ寒天を室温で保存し、少なくとも2週間、その後の実験のために再利用することができる。トップ寒天を作るために使用される様々な式がありますが、私たちは、LBと混合した寒天の7グラム/ Lを使用して寒天トップを作る実験の過程で液体状態を保持するための60°Cの水浴中でトップアガーを置く。 </li><li>反転トン彼プレートと48時間に（温度30℃）でO / N所望の温度でインキュベートする。これらの条件が存在するファージのために最適化することが異なります。ほとんどの単離されたファージを、24時間のインキュベーション期間の後に1〜2.5のCaCl 2濃度で30℃でインキュベートしたプレートから来る。 </li></ol> 4.ファージ精製<ol><li>プレート上のプラーク形成のための24時間のインキュベーションのチェックの後。全くプラークが明らかでない場合には最大72時間のインキュベーションを続行するか、追加のプラークが表示されますかどうかを判断する。週間まで4℃で前に進むのための推定プラークと、店のプレートを必要に応じて。 注：それは一つのプレート上の異なるプラークの形態の多様を見つけることは珍しいことではありません。インキュベーションの3日後にプラークを見つけることは可能ですが、まれである。 </li><li>ストリークプラーク法を用いて明らかに単離されたプラークから、所望のファージを精製する。無菌木製の棒でプラークをタッチします。炎を通して木の棒を実行することにより、ストリークプラークを、allowin簡潔に冷却した後、 図1に示すように、穏やかな運動で寒天プレート上の棒をこすりするGスティック。各パスのためのクリーンスティックを使用してこれを繰り返します。 </li><li>また、個々のファージプラークを分離するために、伝統的なプラーク力価アッセイを使用しています。プラーク力価アッセイは、LB寒天LB上層寒天と、ファージプラークを単離するためのペトリ皿のような材料として、複数の試薬の使用を必要とする。我々は、ストリークプラーク法を用いて非常に成功しているが、それは、プラーク力価アッセイを用いて、個々のプラークを単離することが技術的に容易である。 </li><li> LB寒天プレートを少なくとも3回に画線すると、推定上のファージ粒子の最低濃度領域をマークし、GENTLY母板として塩化カルシウムの同じ濃度を含む（ アルスロ 0.5mlの溶融LBトップアガー4.5mlの適用）、それは、プレート全体に均等に分散することができます。 </li><li>プレートO / Nを反転し、インキュベートする。ストリークプラークを繰り返します純粋なファージ種を確保するために、少なくとも3回の反復のための技術が単離される。ファージの生存率の有意な損失なしにすじの間で、必要に応じて週まで4℃で保存プレート。 </li><li>一度希望するプラークが、最終的なストリークプレート上で増殖させ、単離されている、マイクロピペットの先端で1よく孤立したプラークをタッチし、100μlのPBで再サスペンド。シリアルに新鮮なPBの180μLにサンプルを20μlを渡すことで、10 -8希釈にこの10 0ソリューションを希釈する。 </li><li> CaCl 2を同じ濃度で含有する溶融LBトップアガー4.5 mlの感染細菌を混合して5〜10室温で分およびプレート培養チューブ中で増殖させたアルスロ 0.5mlの各希釈物10μlを追加するファージを単離するために使用元の土壌サンプルから。次の日まで4℃で行われたすべての希釈液を保存します。 </li><li>伝統的なプラーク力価としてを使用してWebパターンを作るために必要なファージの連続希釈を決定します言う。プラーク力価アッセイの目的は、ウェブパターン板を得るために必要なファージ力価を決定することである。として主に細菌を欠いたが、まだのファージによって溶解されていない細菌の残骸を含むWebパターンプレートを識別します。無菌の50ミリリットル培養フラスコに後半早い/指数関数的に静止アルスロ文化の 5ミリリットルを追加します。 <ol><li>培養フラスコ内（ アースロ文化にウェブパターンを与えた）希釈液100μlを加え、ファージを室温で5から10分間細菌細胞に感染することができます。最初に10新鮮なLBプレート上にプレートし、ファージをこの混合物を5mlを識別するために使用されるのCaCl 2の同じ濃度を含む溶融LB上層寒天と混合する。固化し、30℃で反転したプレートをインキュベートすることを許可する。 </li></ol></li><li>次の日は、4℃または室温で4時間後にPBや店舗O / N 5mlのウェブパターンプレートをあふれさせる。 </li><li> 0.22μmのシリンジフィルターを通してファージ溶解物をフィルターそして4℃で保存する。ファージ粒子の電子顕微鏡像、標準的な手順を用いてDNAの制限酵素分析およびゲノム配列決定のためのファージゲノムDNAの分離のような追加の実験のために、この最後のファージストックを使用する。ファージストックの長期保存のため、-80℃でストック·アーカイブ用の小さなバイアルに無菌グリセロールにファージ溶解等量のを追加します。 注：これらのファージ技術で経験していない人のために、偉大なリソースがオンラインでアクセスwww.phagesdb.orgサイトです。 </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>

著者は競合する経済的利益を宣言していない。

アルスロホストに感染することができるファージを単離するための多くの以前の試みにもかかわらず、私たちは、標準的な濃縮手順を使用してほとんど成功していた。環境サンプルからファージを豊かにするバンTwestとKropinski 10によって開発され、適合した細菌の濃縮の一般化された方法は、濃縮手順の大多数のための基礎のまま。以前の研究からの証拠は、ダイレクトプレ?...

土壌環境でのロバクター種の普遍性は、宿主細菌のこの種から単離されることができるファージの膨大な数と多様性を提供しています。 Acintobacteriaceaeファミリーの細菌メンバーは、アトラジン、様々な他の殺虫剤や除草剤の1,2,3のような扱いにくい化合物を分解することが彼らの異化経路のための最も注目すべきである。ほとんどの研究は、 アルスロバクターの環境の株を用いて行われたが、この属の臨床分離株は、血液、尿、目、すべての系統発生異質4を表示する他の多くのヒト供給源において見出される。 アースロバクター菌の研究のかなり大規模なボディがありますが、唯一の少数の研究は、この多様な属のメンバーに感染することができるのファージについて報告する。興味深いことにしかし、 アルスロで以前に行われた作業は、土壌のタイピングなど、いくつかの重要な個別のトピックにタッチをファージを&lt;em&gt;のアルスロバクター種5、活性汚泥処理プラント6、及び作業強調部位特異的組換えおよびインテグラーゼ遺伝子7において有害な泡の低減を目的と工業用途。 様々な濃縮技術プロトコルは、純粋なファージは、 アルスロバクター種において単離生成するために使用されてきた。初期の手順は、1年以上8の期間のためのニコチン塩のような追加された有毒物質しかA.に感染することができるのファージを生じさせるとともに、土壌のインキュベーションを含むスロバクター 。不安定な有機物が長い潜伏期間を8を省略し 、プラークアッセイ技術により検出可能なファージを生成するために登場して土を使用して行われた研究は、パーコレート。興味深いことに、ダイレクトプレーティングに似た手法は、低い成功率の過去の研究を引用して、まだ研究者5によって著しく低い成功率を有しながら、いくつかのファージを生じる過去に使用した<s&gt; 8まで。 全体的に、過去に使用される分離技術は、最も一般的な好気性土壌を表すロバクター属にもかかわらず、実際にはほとんど効 ​​果を持つために注目すべきであったが、自然4,9に隔離する、水や土壌からファージを単離するためのヴァンTwestとKropinski 10現在の濃縮方法環境細菌分離株が、これらの濃縮技術を豊かにするために使用される以前の技術から適応は、 アルスロファージを単離する非効率的な証明した。ここで説明する方法の目的は、初期の濃縮方法は、この細菌属からファージを単離することに関連した以前の技術的な課題を克服し、一貫して効果的にアースロファージを分離するように適合させることができることを「コンセプトの証明」を示すことです。

<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.

アースロファージのための改善された濃縮技術の再現性を実証するために、30種類の土壌サンプルは、固有のアースロファージは、この濃縮を使用して採取した土壌サンプルの22から得られたこれらの30の土壌サンプルの2014年の春と夏の間、異なる時間と場所で使用した手順。標準濃縮手順は、同じ土壌試料の3からユニークなファージを得た。濃縮サンプルは、プラークまたは?...

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.

の単離のための最適化された濃縮技術<em&gt;アルスロ</em&gt;バクテリオファージ種土壌サンプルの分離株から

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

an-optimized-enrichment-technique-for-isolation-arthrobacter

Research

JoVE Journal

Environment

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

19.3K ビュー.  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.

ビデオ: 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.

連続の設計および動作<sup&gt; 13</sup&gt; Cと<sup&gt; 15</sup均一または差動、代謝および構造·&gt; N標識化会議所、工場同位体ラベル化

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

環境学

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.

アーバン流出研究施設の設計と建設

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.

土壌のサンプリングと昆虫病原線虫の分離（Steine​​rnematidae、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.

CO<sub&gt; 2</subテスト用のCO&gt;濃度勾配施設<sub&gt; 2</sub&gt;濃縮および草地生態系の機能に及ぼす土壌の影響

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.

実験で多年生の草の根、根、土壌微生物の分離と解析

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.

根の生長およびローカライズされた栄養素に対する反応性を可視化する最適化された Rhizobox プロトコル

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.

毒性物質のトランスおよび多世代効果の研究のためのカエナルハブ炎エレガンスの使用

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.

15窒素分析のためのマイクロプロット設計と植物および土壌サンプル調製

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.

土壌から粒子状有機物を除去する静電法

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法による植物バイオマスの結晶セルロース含有量の推定

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 ...