研究はAdvanDxによって後援されています。

1。標本の収集と準備<ol><li>敗血症が疑われる患者から静脈血を採取し、一緒に1つ血液培養のセットを含む二バクテック（BD、スパークス、MD）血液培養ボトル、一つ好気性と嫌気性のいずれかに入れ。 </li><li> 35バクテック9240楽器にボトルを置きます℃を</li><li>楽器は血液培養ボトル中の生物の成長のためにアラームにトリガされるまでインキュベートする。 </li><li>血液培養器からボトルを取り外します。 </li></ol> 2。染色の前に試薬の調製<ol><li>染色皿に直接新鮮な脱イオン水または蒸留水240 mLを続いて60倍のWash Solutionを4mLを追加することにより、洗浄溶液を準備します。 </li><li>暖めて55℃の水浴中で染色皿を置く。 </li><li> 2〜8の残りの濃縮液℃に保管してください</li><li>冷蔵庫から封入剤を取り外し、室温まで昇温。 </li></ol> 3。グラム染色<ol><li>優しくスワール血液培養ボトルを。 </li><li>針と注射器や継代培養装置を用いて、滅菌スクリューキャップ付きチューブのボトルと場所から血液（約5mL）を取り外します。 </li><li>滅菌ピペットを用いて、ガラスの顕微鏡スライド上に血液中の一つの大きな低下を置きます。 </li><li>空気は、スライドを乾燥し、10秒間メタノールに修正。 </li><li>スライドを風乾します。 </li><li>血液培養ボトルに成長する有機体の形態を決定するために血液のフィルムでグラム染色を行います。 </li><li>油浸レンズを使用して、スライドを表示します。ペアとグラム染色で観察された短鎖のグラム陽性球菌は、エンテロコッカス種と最も一致している。 </li><li>このグラム染色の結果は、細菌の同定に使用する適切なPNAのFISHプローブキットの選択を駆動します。この血液培養は、E.を用いて染色されます。 球菌 / OEのPNA FISH染色。 </li></ol> 4。 PNAのFISHは、染色<ol><li>塗抹標本の前に渦巻くことにより穏やかに血液を含むチューブを混ぜる。 </li><li> PNAのFISHの顕微鏡用スライドのよく上の固定液の一滴を置きます。 </li><li> 10μLまたは固定液に血液培養が陽性とチューブから一つの小さなドロップを転送して乳化するために静かに混ぜる。 </li><li> 20分のためにそれらを加熱することにより、スメアを修正。 55 ° Cの加熱プレート上に。 </li></ol> 5。品質管理材料つの正と負の品質管理のスライドは、染色用スライドの各バッチでテストする必要があります。 <ol><li> E.を使用してください球菌 / OEコントロールのスライドは、この目的のためにAdvanDxから購入またはE.の参照株の液体培養から塗抹標本を準備するポジティブコントロールは別のスライド上で、または1つのスライドおよび陰性対照物質としてブドウ球菌属やストレプトコッカス属の混合のいずれかとしてfaecalisのとE.フェシウム。 </li></ol> PNAの方法論は、細胞壁の浸透を最適化するように設計されているので、QC結果は、特に適切な試験条件、ハイブリダイゼーションのストリンジェンシーと細胞壁の浸透に影響を及ぼすものを監視することができるはずです 6。交配<ol><li> E.の一滴を追加塗抹標本による顕微鏡スライド上のウェルにフェカリス / OE PNA。 </li><li>カバースリップを追加。空気の泡を避けてください。 </li><li> 30秒間インキュベートする± 5分。 55℃± 1℃の加熱プレート上に</li><li>インキュベーション後、スライドキャリアにスライドを配置する</li></ol> 7。厳しいウォッシュ<ol><li> 55℃予熱洗浄液に浸し、スライドキャリア° Cと慎重にカバースリップを削除する。多くの場合、カバースリップは優しく洗浄ソリューションでスライドを撹拌することによりオフスライド。時折、カバースリップを静かにピンセットでオフプッシュする必要があります。 </li><li> 30の洗浄液± 5分でインキュベートする。 55℃± 1℃ </li><li>洗浄液からスライドを削除する</li><li>空気乾燥したスライドを許可する</li></ol> 8。取り付け<ol><li>各スライドに封入剤の一滴を追加</li><li>カバースリップを追加。空気の泡を避けてください。 </li><li> 2時間以内に蛍光顕微鏡でスライドを調べます。 </li><li>これは蛍光消光につながる可能性があるので太陽の光などの強い光源を指示するためにスライドを公開しないでください。 </li></ol> 9。結果の解釈スライドの検討に用いる蛍光顕微鏡はAdvanDxデュアルバンドフィルタと60倍または100X油浸対物装備されている必要があります。 QCのスライドは、そのハイブリダイゼーションが起こるか確認するために最初に調べる必要があります。E.は球菌は、ビューの複数のフィールドに明るい緑色の蛍光球菌のように表示されますとE.フェシウムは鮮やかな赤色球菌として表示されます。非腸球菌コントロールのスライドは非蛍光表示されます。コントロールにシステムを確認した後、患者のスライドを調べることができます。血液FIL最初はmは、赤みがかった表示されますが、明るい赤と緑球菌は非常に明らかであろう。 <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="図1" /> 図1。緑色の陽性E.の代表的な例としては、 球菌 （左）、赤のプラスE.フェシウム（真ん中）、そして負（右）のテスト結果。 10。代表的な結果三機関は、このPNAのFISH染色を評価し、ちょうど検討された短縮1.5時間のプロトコルに2.5時間のプロトコルを比較する多施設共同臨床試験に含まれていた。ペアとチェーンの152ルーチンのグラム陽性球菌の合計（GPC）陽性の血液培養ボトルは、研究に含まれていた。 100％（152分の152）は、E.のための修正と、元のアッセイ手順の結果の間に合意があった球菌 / OEのPNA FISH：41分の41 E.球菌 、33分の33、他の腸球菌と78分の78他のGPC（表1）。この染色法は、この臨床試験（表2）の間に絶妙な感度と特異性を持っていた。 <table border="1"><tbody><tr><td></td><td> ルーチン方法 </td><td> E. faecalisの / OEのPNA FISHの標準手順 </td><td> E. faecalisの / OEのPNA FISHショート手順 </td></tr><tr><td> E. faecalisの </td><td> 41 </td><td> 41（グリーン正） </td><td> 41（グリーン正） </td></tr><tr><td> E.フェシウム </td><td> 27 </td><td> 27（ポジティブレッド） </td><td> 27（ポジティブレッド） </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2（正レッド） </td><td> 2（正レッド） </td></tr><tr><td> E. gallinarum </td><td> 2 </td><td> 2（正レッド） </td><td> 2（正レッド） </td></tr><tr><td> 他の腸球菌属。 </td><td> 2 </td><td> 2（正レッド） </td><td> 2（正レッド） </td></tr><tr><td> 肺炎球菌 </td><td> 17 </td><td> 17（負） </td><td> 17（負） </td></tr><tr><td> S.ビリダンス </td><td> 33 </td><td> 33（負） </td><td> 33（負） </td></tr><tr><td> S.可鍛鋳鉄 </td><td> 1 </td><td> 1（負） </td><td> 1（負） </td></tr><tr><td> S.化膿連鎖 </td><td> 3 </td><td> 3（負） </td><td> 3（負） </td></tr><tr><td> S.ボビス </td><td> 2 </td><td> 2（負） </td><td> 2（負） </td></tr><tr><td> S.バリウス </td><td> 1 </td><td> 1（負） </td><td> 1（負） </td></tr><tr><td> S. sanguinis </td><td> 2 </td><td> 2（負） </td><td> 2（負） </td></tr><tr><td> S. agalagtae </td><td> 7 </td><td> 7（負） </td><td> 7（負） </td></tr><tr><td> 他のレンサ球菌属。 </td><td> 7 </td><td> 7（負） </td><td> 7（負） </td></tr><tr><td> Abiotrophia属。 </td><td> 3 </td><td> 3（負） </td><td> 3（負） </td></tr><tr><td> Peptostrepococcus属。 </td><td> 2 </td><td> 2（負） </td><td> 2（負） </td></tr><tr><td>合計</td><td> 152 </td><td> 152分の152 100％の契約</td><td> 152分の152 100％の契約</td></tr></tbody></table>細菌の表1。リストは、標準（2.5時間）と急速に（1.5時間）プロトコルの両方を使用してテスト。 <table border="1"><tbody><tr><td> 感度E.faecalis </td><td> 感度その他のエンテロコッカス属。 </td><td> 特異 </td></tr><tr><td> 100％（41分の41） 95％CI（93.0〜100） </td><td> 100％（33分の33）33分の33 95％CI（91.3〜100） </td><td> 100％（78分の78） 95％CI（96.2〜100） </td></tr></tbody></table> 表2。1.5時間のPNA FISHプロトコルの感度と特異性。

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

このビデオ記事の生産はAdvanDxが後援した。ベンジャミンクリスタルは、この記事で使用するツールと試薬を作るAdvanDx、の従業員です。

E.腸球菌用球菌 / OEのPNA FISHアッセイは、標準的な培養法よりも2〜3日以前の識別を提供することができます。アッセイのための短縮手順（1.5 hourr）は抗菌薬治療と患者の転帰への良いアプローチに使用することができる迅速な識別を提供します。これをサポートするために一つの研究は、フォレスト、 ら （6）で実施した。 2005年から2年連続にわたって微生物学研究室では、従来の微生物学的方法による血液培養ボトル中で成長し、2006年にE.を追加してペアとチェーンでグラム陽性球菌を同定した球菌 / OEのPNA FISH。さらに、機関抗菌チーム（AMT）が開発した治療アルゴリズムは効果的にタイムリーに実験室で生成されたPNAのFISHのデータを使用する。評価主要転帰は、血液培養からの時間だったのPNA FISHの実験室のワークフローに制定された前と後の効果的な抗菌薬治療の実装に描く。病気、患者の位置と経験的抗菌療法の重症度を測定した。腸球菌による菌血症を取得した病院で224例の合計は、介入前の期間に129とPNAのFISHの期間で95と評価した。 E.を特定のPNA FISH 3日前の従来の文化をよりフェカリス （1.1対4.1日、P &lt;0.001）。 PNAの魚は中央値2.3日以前のE.フェシウムを同定した（1.1対3.4日、p &lt;0.001）および効果的な治療を開始するまでの期間で統計的に有意な減少（1.3対3.1日、P &lt;0.001）に関連付けられており、30日に減少した死亡率（26％対45％、P = 0.04）。このグループは結論づけているE. AMT治療アルゴリズムと組み合わせてフェカリス / OEのPNA FISHアッセイは、菌血症E.フェシウムを取得した病院の患者のための適切な経験的抗菌薬治療の前の開始となりました。 <ol><li>ラベルと試薬に印刷されている有効期限が何らかの方法で変更しないでください後の試薬は使用しないでください。彼らは、使用しないときは、冷蔵庫に残ってくださいまたは、彼らは効力を失う可能性があります。 </li><li>血液培養ボトルは前のサンプリングに混合する必要があります。 </li><li>これはスライドの間に材料のクロスコンタミネーションを引き起こす、または試薬の汚染を引き起こす可能性があるため、血液塗抹標本に触れることドロッパーボトルの先端を許可しないことが重要です。 </li><li>水浴および加熱ブロックは一定の55℃に維持されていることを確認します</li><li>デュアルバンドフィルタまたはカラーを明確に識別することがない以外のフィルタを使用しないでください。 </li><li>顕微鏡スライドまたはハイブリダイゼーション発生しません以外の顕微鏡スライドを使用しないでください。 </li><li>顕微鏡が正常に機能していることが重要です。顕微鏡の電球は、使用のための時間の承認数の範囲内であることを確認してください。 </li></ol><ul><li>いくつかのまれな発性腸球菌種は、E. asinii、E.dispar、E. haemoperoxidus、E. cecorum、E.鳩類、大腸菌saccharolyticus、E.孤やEなどの他のエンテロコッカス種にハイブリダイズするPNAプローブによって検出されていません。亜硫酸。 </li><li>大腸菌moraviensisはE.として識別され配列類似性に起因するフェカーリス 。 </li><li> 連鎖球菌 anginosusの一部の菌株は、配列類似性に起因する偽の緑のポジティブの蛍光を生じる。 </li><li> VersaTREKの血液培養ボトルは、臨床試験中に評価されていません。 E.を検出するためにパフォーマンス球菌と他の腸球菌属。 VersaTREKの血液培養ボトルでは不明です。 </li><li>標準FITCフィルターはデュアルバンドフィルタの代わりに使用されている場合、偽陽性緑色自家蛍光が発生する可能性があります。 </li><li>偽陰性の結果は、まれに混在成長に起因またはアッセイ法におけるエラーが原因で発生する可能性があります。 </li><li>使用する計測機器の種類と条件が得られる画像の視覚的外観に影響を与えます。蛍光は、雇用顕微鏡の種類、光源と細胞内のrRNAのレベルにvarydueこと</li><li>固体培地上に分離は他の生物との混合の成長を区別すると否定的な結果をもたらす血液培養陽性を識別するために必要とされる。 </li><li>製品は、血液培養以外の標本で検証されていません。 </li></ul>

試薬 E. faecalisの / OEのPNA FISHは、次のキットのコンポーネントで構成されています。 <ul><li>固定ソリューションの固定液は、界面活性剤とリン酸緩衝食塩水3mLの。 </li><li> E. faecalisの / OE PNA E.ハイブリダイゼーションの球菌 / OE PNA 1.5 mLのPNAプローブ。ソリューション。 30％ホルムアミドが含まれています。 </li><li>洗剤と60倍のWash Solutionを60倍洗浄液は、50 mLのトリス緩衝生理食塩水</li><li>グリセリンで封入剤封入剤3mLの光退色抑制剤</li><li>品質管理のスライド</li><li> <a href="http://www.advandx.com">AdvanDX</a> </li></ul>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

腸球菌はE.による菌血症の一般的な原因です。 E.フェシウムが続く主な種であることフェカーリス 。理由E.のアンピシリン及びバンコマイシン耐性球菌は E.フェシウムの抵抗に比べて、まだ珍しいです、腸球菌種の間で差別化を可能にする迅速検査の開発は、適切な治療と耐性サーベイランスのために重要です。 E.カリス OEのPNA FISHアッセイ（AdvanDx、ウォーバーン、マサチューセッツ州）は、in situハイブリダイゼーションの形式で蛍光で種特異的なペプチド核酸（PNA）プローブを使用し、1.5時間と固有種にとって重要な情報を提供する可能性の結果に時間を提供しています治療。多施設共同研究は1.5時間E.の性能を評価するために行った球菌 / OEのPNA FISHの手順では、元の2.5時間のアッセイ手順にと血液培養陽性ボトルから直接、腸球菌の同定のための標準的な細菌学の方法に比べて。

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

in situハイブリダイゼーションアッセイの蛍光ペプチド核酸（PNAのFISH）を用いた血液培養陽性の腸球菌や他のエンテロコッカス種の直接同定するための迅速なプロトコル。

の識別のための1.5時間の手順腸球菌</em直接血液培養から>種

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

a-15-hour-procedure-for-identification-enterococcus-species-directly

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Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

20.7K ビュー.  Cedars-Sinai Medical Cente. in situハイブリダイゼーションアッセイの蛍光ペプチド核酸（PNAのFISH）を用いた血液培養陽性の腸球菌や他のエンテロコッカス種の直接同定するための迅速なプロトコル。

ビデオ: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

免疫・感染学

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

血液培養から細菌性病原体の検出および抗菌剤耐性テストのための一日のワークフロースキーム

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

臍帯血から前駆B細胞サブセットの分離

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

全タンパク質の抽出および2次元ゲル電気泳動法のための<em&gt;バークホルデリア</em&gt;種

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

鳥類の消化器系を通過した後の感染プリオンを識別するための手順

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

単離、同定、およびマウス胸腺上皮細胞の精製

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

迅速な細菌同定および抗生物質感受性試験のための血液培養ペレットの調製

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Fcγ受容体媒介食作用を評価するために、単球単層アッセイの使用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

の識別のための1.5時間の手順腸球菌種