This project was supported by Midwestern University, IL Office of Research and Sponsored Programs (ORSP) and Midwestern University College of Dental Medicine-Illinois (CDMI).

1. HSV株及び取扱い注：組換え非拡散HSV-1（KOS）gL86およびHSV-2（KOS）333gJを- V. Twiari 36,37によって提供された使用β-ガラクトシダーゼレポーター活性を有します。 <ol><li> 1で-80℃での単一のロットとストアからウイルスを使用してください：20％ウシ胎児血清（FBS）とダルベッコ改変イーグル培地（DMEM）の1の比率、使用するまでスキムミルク。ウイルスロット保存前、 入出力 -nitrophenyl-β-D-ガラクトピラノシド（ONPG）および5-ブロモ-4-クロロ-3-インドリルβ-D-ガラクトピラノシド（X-GAL）アッセイによりウイルス濃度を決定します。 </li><li>前述のように、レポーターウイルス侵入アッセイを使用して各実験アッセイの実行のためのX-Gal染色により、感染のウイルス生存率および多重度（MOI）を決定する（ 図1）14。 </li><li>希望のMOIに（オプトMEM）のウイルスを希釈します。染色の前に、単層（/ウェル0.5ミリリットルパラホルムアルデヒド）を修正しました。別々のmicroti中のウイルスの生存能力のコントロールを配置複数菌アッセイプレートと平行にタープレート。 </li></ol> 2.のHeLa 299細胞の取扱い<ol><li> 4.5g / Lグルコース、10％熱不活化ウシ胎児血清（FBS）、ゲンタマイシン（50μg/ ml）およびL-グルタミンを含むDMEM中で37℃、5％CO 2で成長します。トリプシン溶液で80％コンフルエンスで継代細胞（エチレンジアミン四酢酸、0.53 M EDTA、0.05％トリプシン; 5ミリリットル/フラスコ）。 </li></ol> 3. C.取り扱いアルビカンス 注：C.臨床検査源から得アルビカンス媒体約2倍のスキムミルクRemmelに-80℃で保存します。 <ol><li>文化はサブローデキストロース培地（37℃）に株式を凍結しました。 24時間のサブカルチャーC.後Fungisel媒体へアルビカンス （37°C、48時間）の使用のため。 </li><li>発芽管（GT）の形を生成（; 3ミリリットルFBSを、代表的なコロニーを選ぶ3時間、37℃、腹筋600、0.3）。インキュベーション後、GT洗浄（HBSSを、2倍、4,000×gでの）。 （37＆温めHBSSに洗浄GTを追加します。＃176; C; 0.32のAbs 600）。 GT形態は、血球計数によって決定されるように観察された細胞の99％であるべきです。 </li><li>代表Fungiselコロニーピッキングによって酵母形（YF）株式懸濁液を作る（HBSS、3ミリリットルを、0.32のAbs 600）。 /微視的に血球計数器を使用して、mlのYFのフォームの数をカウントします。 YF形態は、血球計数によって決定されるように観察された細胞の99％であるべきです。 </li><li>作業の真菌株（; 37℃; 10 5 CFU / mlの25ミリリットルHBSS中のGTまたはYF株式の250μl）を作ります</li></ol> 4. S.球菌取り扱い<ol><li>ストアS.黄色ブドウ球菌 ATCC 25923（-80°C; Remmelは牛乳の2倍を脱脂）。ヒツジ血液寒天培地（; 37℃、24時間、5％）上に培養。代表的なコロニーを選択し、株式のために2日以内に培地塩をマンニトールへの転送（37℃、18時間）。 </li><li> S.を作ります黄色ブドウ球菌ストック懸濁液（3ミリリットルHBSS; 1.32腹筋600; 10 8 CFU / ml）を<ol><li> S.作業してください黄色ブドウ球菌株（100μlの25ミリリットルのHBSSの株式の。 10 5 CFU / mlで）。 </li></ol></li></ol> 5. カンジダとS.球菌懸濁液<ol><li>メイク混合C.アルビカンスおよびS.黄色ブドウ球菌懸濁液（250μlのYFまたはGTの株式と25ミリリットルHBSS中の100μlの黄色ブドウ球菌株 ）。 </li></ol> 6.複数菌バイオフィルムアッセイ<ol><li> 2×10 5 HeLa細胞/ mlの200μlの（85％のコンフルエンスのレベル）でシード96ウェルプレート。ロックプレート（30から45分、37℃）インキュベーション前（37℃、5％CO 2インキュベーター; 18時間）。ウォッシュ単層（1×;オプトMEM）をHSVにシード（HSV-1（KOS）gL86またはHSV-2（KOS）33 GJ - 100μl中のOpt-MEM; MOI 50及び10）。プレートをインキュベート（3時間、37℃、5％CO 2）。 1日に1回だけウイルス株を使用してください。 </li><li>感染した単層（; Mgの2及びCa 2を含むリン酸緩衝生理食塩水（PBS）;100μlの1×）で洗浄します。 25＆＃残し暖かいHBSSでPBSを交換してください181;各ウェルのリットル。 <ol><li> YF、GTおよび/ ​​またはSを追加します。 表1に示されるように平板培養する（静的に、30分間、37°C、5％CO 2） 黄色ブドウ球菌ワーキング懸濁液（N = 16; 1、100μlの細胞比の目標= 5）。 </li></ol></li><li>インキュベーション後、一度に吸引1列には、すぐにマグネシウム2およびCa +2で300μlのPBSで再充填します。ラジオ免疫沈降アッセイ溶解バッファー（;濾過滅菌; 1:50希釈の200μlのRIPA）を追加し二回、この手順を繰り返します。 </li><li>急速にHeLa細胞溶解物は、次いでマンニトール塩（MS）及び/又はFungisel（F）培地（ 図2）の上に50μlのを配置し粉砕します。 90°の角度でガラス棒を曲げを使用して溶解液を広げます。 （37℃で18時間）培養します。手動でプレートあたりのコロニー数をカウントします。コントロールは、Sから成り黄色ブドウ球菌および/ ​​またはC. HSV-非感染HeLa細胞へアルビカンス付着 。 </li></ol> 7.イメージング研究&lt;/ P&gt; <ol><li>各ラウンドガラスカバースリップを洗ってください（12ミリメートル、50ミリリットルのアセトンミリリットルビーカー100中）。乾燥及び滅菌カバースリップ（キムワイプ;ガラスシャーレ）。アルコール火炎滅菌鉗子で滅菌24ウェルプレートのウェルに、乾燥、滅菌カバースリップを置きます。 </li><li>洗浄滅菌ラウンドガラスカバースリップを含む24ウェルプレートのウェルに、HeLa細胞（96ウェルプレートに使用される5倍の体積1ミリリットル）を追加します。上記の手順6.4に6.2.1で説明したように、その後インキュベートし、プロセス、5×96ウェルプレートに使用する音量でテンプレート（表2）によれば、ウイルス、細菌および真菌を追加します。 </li><li>最終洗浄後、メタノールでスライドをフラッディングし、それを蒸発させることにより、顕微鏡検査のために細胞を固定します。染色まで室温でプレートを保管してください。 </li><li>明視野顕微鏡（1,000倍、最終的な元の倍率）のために脱イオン水でメタノール固定カバースリップを含むウェルを埋めます。すぐに水を吸引。グラムクリスタルバイオレットで各カバースリップをカバー。ウォッシュは、非結合染色（脱イオン水）の自由カバースリップ。 その場で乾燥したカバースリップは、その後、標識されたスライドにメディアをマウントハードセット（図3）とそれらを接着します。 </li><li>蛍光顕微鏡（100倍の対物; 1,000倍、最終的な元の倍率）のためにステップ7.4で説明したように、洗浄は、基本的にメタノールの自由カバースリップ。ウェル中のカバースリップを乾燥させます。 <ol><li>乾燥させた後、カバースリップを削除し、ラベルされたスライド上に置きます。 （ - 5μL1）次に、フルオレセインイソチオシアネート（FITC）結合単純ヘルペスウイルスのタイプの1：20希釈のカバースリップをカバーするために1 + 2のgD抗体を十分な量を追加します。 </li><li> 30分間、37℃で湿気チャンバー内でスライドをインキュベートします。インキュベーション後、PBSの4変化​​でカバースリップを洗います。 </li><li>最終洗浄後、標識されたスライドにカバースリップを返します。 4 &#39;、6-ジアミジノ-2-フェニルインドール（;湿度室、30分間、37°C​​ DAPI）で染色。インキュベーション後4ちゃんにカバースリップを洗いますPBSのGESは、その後、メディアをマウントハードセットで標識されたスライドに貼り付けます。 </li><li>マウンティング培地は暗所で室温で24時間硬化させます。 FITCおよびDAPIカットオフフィルターで明視野顕微鏡や蛍光顕微鏡のいずれかで油浸対物レンズの下にカバースリップを調べます。共局在（ 図4）のために少なくとも50フィールド（生物ごとに最低100個の細胞）の写真を調べます。 </li></ol></li></ol>

The authors have nothing to disclose.

現在のところ情報は、原核生物、真核生物およびウイルスの複数の分類学上のドメイン、 すなわち 、交差ホストmicrobiomeの半永久的なメンバーに永久の間の複雑な相互作用に利用できません。したがって、我々はSでバイオフィルムの開始を研究するためのインビトロモデルシステムで小説を開発しましたaureusおよびC. HSV-1またはHSV-2上アルビカンス?...

<html>人間microbiomeは、体内の地理的領域を共有する複数の分類学上の王国からの多様な生物が含まれます。細胞表面への付着はmicrobiomeの植民地化プロセスの一部であるバイオフィルム形成に不可欠な最初のステップです。 microbiomeに含まれては、慢性かつ持続的な感染症を引き起こすウイルスであることができます。これらのウイルスによる慢性細胞感染は推定受容体の可用性における変化を引き起こす可能性があります。また1,2、細胞内病原体による細胞エントリも順番に細菌や真菌などの他のmicrobiomeメンバーの添付ファイルを、変更することができるホスト膜流動性/疎水性に影響を与える可能性があります。ヒト宿主の同じ地域に共局在これらの複数の病原体との間で発生する可能性があります相互作用を理解するために、我々は、粘膜表面に存在する分類学上の王国のスペクトルを表し、病原体の相互作用を研究することができなければなりません。 tは&quot;&gt;ヘルペスウイルスはmicrobiome 3,4の永久的なメンバーとしてのヒトの100％に存在する微生物のファミリーである。加えて、それらはまた、永続的両方の症状の存在下および非存在下で流すことができる。具体的には、単純ヘルペスウイルス1および単純ヘルペスウイルス2（HSV-1およびHSV-2、それぞれ）HSV-1およびHSV-2の原因の歯肉口内炎の両方、免疫適格個体で。oronasopharynx及び生殖管におけるmicrobiomeの永久的なメンバーであるだけでなく、性器ヘルペス5-8。これらのサイトでは、HSVは、慢性持続nectins、ヘパラン硫酸、脂質ラフトとヘルペスウイルスエントリーメディエーターの表面発現の変化における細胞の結果にHSVの9。エントリを流しウイルス無症候性/腫瘍壊死によって特徴づけ潜伏感染を引き起こします因子受容体（HVEM / TNFR）10月25日 。これらは、潜在的に日和見病原体ながら、いくつかの細菌および真菌のための共有の受容体を表す例えば黄色ブドウ球菌およびカンジダ・アルビカンス、これ、またoronasopharynx 26,27の粘膜microbiomeのメンバーとして常駐することができます。 oronasopharynx S.内aureusおよびC.アルビカンスは植民地化のの2つの異なる部位を占めます。天然歯を持つホストでは、口腔粘膜は、HSV-1およびCによって共有されていますアルビカンス、前部鼻鼻孔はS.によって占有されている間黄色ブドウ球菌 28。しかし、にもかかわらず、in vitroでの知見そのS.黄色ブドウ球菌は、口腔上皮細胞に付着し、29,30 S.正常組織29,30存在する場合球菌はまれ経口標本から単離されます。リトルは、臨床所見ものを超える生殖管の共同植民地化のニッチについて知られているS.黄色ブドウ球菌は C.ながら、性器の炎症、放電および性交疼痛症によって特徴づけられる、好気性膣炎に関連していますアルビカンスは、口腔31〜35で観察されたものと同様の粘膜の病変を生成します。このように、経口および生殖器microbiのこれらのメンバーが、分類学的王国少しOMEクロスは、宿主細胞表面5への接着を介してバイオフィルムの形成を開始することが影響能力としてのそれらの相互作用について知られています。このプロトコルは、効果的に、共コロニー形成/感染の機能的結果を判定するために適用されています。

<table border="0" cellpadding="0" cellspacing="0" width="1185">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">C.albicans</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">BBL Sabouraud Dextrose</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;">211584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Fungisel Agar</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">7205A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">S.aureus</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Mannitol Salt Agar</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">7143B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Sheep blood agar</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">221239</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hela cells</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">1xDMEM (Dubelcco&#39;s Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gentamicin 50mg/ml</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">1397</td>
			<td>50&#181;g/ml final concentration in the complete DMEM</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:391px;">Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X</td>
			<td style="width:195px;">Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Fetal Bovine Serum</td>
			<td style="width:195px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>10% final concentration in the complete DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Other medium and reagents</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">ONPG</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">34055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Ultra-Pure X gal</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:119px;">15520-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">1x HBSS (Hanks&#39; Balanced Salt Solution)</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">1XPBS</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">30042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">RIPA Lysis</td>
			<td style="width:195px;">Life Technologies</td>
			<td style="width:119px;">89901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Staining</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Methanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A433P-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">HSV 1&#38;2, specific for gD</td>
			<td style="width:195px;">ViroStat</td>
			<td style="width:119px;">196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">DAPI</td>
			<td style="width:195px;">SIGMA</td>
			<td style="width:119px;">D8417-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gram Crystal Violet</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">212527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Supplies</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:391px;">Petri dish 100X15</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">229693&#160;</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:391px;">Petri dish 150X15</td>
			<td style="width:195px;">Kord Valmark</td>
			<td style="width:119px;">2902</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:391px;">96-Well plates</td>
			<td style="width:195px;">Evergreen Scientific</td>
			<td style="width:119px;">222-8030-01F</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:391px;">24-well plates</td>
			<td style="width:195px;">Evergreen Scientific</td>
			<td style="width:119px;">222-8044-01F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Culture tubes 100x13</td>
			<td style="width:195px;">Thomas Scientific</td>
			<td style="width:119px;">9187L61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Cover slip circles, 12mm</td>
			<td style="width:195px;">Deckglaser</td>
			<td style="width:119px;">CB00120RA1</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of biofilm initiation on virus-infected cells by bacteria and fungi

真菌、細菌やウイルスを含む分類学上の王国全体で複数菌相互作用の研究は、以前microbiomeのウイルスのメンバーは、これらのウイルスに感染した宿主細胞とそれに続く微生物相互作用をどのように影響するかに関して検討されていません。細菌および真菌とのウイルスの共居住は、主に口腔及び生殖管の粘膜表面上に存在します。粘膜細胞、持続性の慢性または持続性ウイルス潜伏感染症、特にものは、発現された受容体の数とタイプでウイルス改変を通じてmicrobiomeのメンバーに重大な影響を与える可能性があります。宿主細胞膜のアーキテクチャの変更は、バイオフィルム形成の最初のステップを開始するための正常細菌叢と日和見病原体のその後のメンバーの変更された能力、 すなわち 、密着性をもたらすであろう。この研究は、biofilの開始時にHSVの効果の定量化および目視検査のための方法を説明しS.のm個の形成（付着） aureusおよびC.アルビカンス 。

このレポートに記載されているシステムから得られるデータの堅牢性のレベルは、 図2のaf 38に示されています。このシステムを使用することによりウイルス感染細胞や互いの密着性に及ぼす影響とブドウ球菌と真菌の相互作用の変調が描写することができます。多菌相互作用が同じ細胞で発生しているかどうかを決定するために、 図3</stron...

Watch this Scientific Journal Video about Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi at JoVE.com

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

細菌や真菌によってウイルス感染細胞上のバイオフィルムの開始の決意

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

determination-biofilm-initiation-on-virus-infected-cells-bacteria

Research

JoVE Journal

Immunology and Infection

9.6K ビュー.  Midwestern University. A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

ビデオ: Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

フローセル内の緑膿菌と出芽酵母のバイオフィルム

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

免疫・感染学

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

マイクロタイター皿のバイオフィルム形成アッセイ

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

宿主細胞に関連して病原性真菌の定量化のための画像サイトメトリーの使用

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

細胞内細菌によって遠藤-リソソームシステムの再構築を検討するために、蛍光ナノ粒子の応用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表現型および慢性リンパ球性脈絡髄膜炎ウイルスに感染したマウスから単離された活性化制御性T細胞の機能解析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

共焦点レーザー Endomicroscopy Ex Vivoひと肺組織の新規細菌固有の光学検査プローブします。

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...