著者は、この原稿の編集上の助けのために李張、西安志李、アーロン・ヒンツ、クレイトン・ホールに感謝したいと思います。このアッセイは当初、ダートマスのガイゼル医学部ジョージ・オトゥールの研究室で開発されました。博士マーの研究室での研究は、カナダの自然科学工学研究評議会と嚢胞性線維症カナダからの助成金によって支えられている。

1. MBC-B
<ol><li>
バイオフィルムの成長 (オトゥール 9 から適応).
	<ol>
<li>37°Cで富の培地で16時間の目的および変異株の野生型株の培養を成長させる。</li>
		<li>飽和した一晩培養物1:100を抗生物質耐性アッセイ用の新鮮な培地に希釈する。 P. 緑素吸塩 の標準培地は、M63最小限の培地で、硫酸マグネシウムとアルギニンを添加した( 表1参照)。この培地は、より堅牢なバイオフィルムの形成を刺激する。</li>
		<li>96ウェルマイクロティター皿にウェルあたり100μlの希釈液を加える( 表1参照)。これらのアッセイは、通常、各株の三重で行われるので、各株の24のウェルがあるはずです。</li>
		<li>マイクロタイタープレートを37°Cで24時間インキュベートします。</li>
	</ol>
</li>
	<li>
前形バイオフィルムを抗生物質の濃度勾配にさらす
	<ol>
<li>7つの井戸のための抗生物質の10x希釈シリーズを準備する。例: 抗生物質トブラマイシンの場合、ウェル内の最終濃度は 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.006 mg/ml 5-8. 25 mg/mlのストックから、4、2、1、0.5、0.25、0.125、および0.06 mg/mlの10x希釈液を調製する。氷の上に置いておきなさい。</li>
		<li>マルチチャンネルピペットを使用して、使用済み上清(プランクトニックセルを含む)を取り除く( 表1参照)。</li>
		<li>すべてのウェルに90 μl M63(Mg/Arg)を加えます。</li>
		<li>所望の最終濃度を達成するために、各10倍の抗生物質濃度の10 μlを加えます。反復および株ごとに最終ウェルに10μl水(抗生物質制御なし)を加えます。</li>
		<li>マイクロタイタープレートを37°Cで24時間インキュベートします。</li>
	</ol>
</li>
	<li>
メディアをリフレッシュし、生きた細胞の剥離を可能にする。
	<ol>
<li>マルチチャンネルピペットを使用して、使用済み上清(プランクトン細胞を含む)を取り除きます。</li>
		<li>すべてのウェルに115 μl M63(Mg/Arg)を加えます。</li>
		<li>マイクロタイタープレートを37°Cで24時間インキュベートします。</li>
	</ol>
</li>
	<li>
生細胞のアッセイ。
	<ol>
<li>96ウェルマイクロティタープレートごとに2枚のLB寒天プレートにラベルを付けます。</li>
		<li>100%エタノールにプロングを浸し、ブンゼンバーナーの開いた炎を横切って突起を通過させることによって、マルチプロング装置( 表1を参照)を殺菌します。繰り返す。プロングを少し冷まします。マルチプロング装置を用いて、マイクロタイタープレートの各ウェルからLB寒天板の表面にプランクトン培養液の〜3μl(通常は突起の先端に保持される量)を移す。</li>
		<li>LB寒天プレートを37°Cで16時間インキュベートします。</li>
		<li>細菌増殖のためのカットオフを目で同定することにより、抗生物質の最小殺菌濃度を決定する(図1)。</li>
	</ol>
</li>
</ol>2. MBC-P
<ol><li>
細菌株の準備
	<ol>
<li>37°Cで富の培地で16時間の目的および変異株の野生型株の培養を成長させる。</li>
		<li>飽和した一晩培養物1:100を抗生物質耐性アッセイ用の新鮮な培地に希釈する。 P. 緑素吸塩 の標準培地は、M63最小限の培地で、硫酸マグネシウムとアルギニンを添加した( 表1参照)。</li>
		<li>96ウェルマイクロティター皿にウェルあたり90μlの希釈液を加える( 表1参照)。これらのアッセイは、通常、各株の三重で行われるので、各株の24のウェルがあるはずです。</li>
	</ol>
</li>
	<li>
抗生物質の濃度勾配にプランクトニック細胞を曝露する
	<ol>
<li>7つの井戸のための抗生物質の10x希釈シリーズを準備する。例: 抗生物質トブラマイシンの場合、ウェルの最終濃度は 0.032、0.016、0.008、0.004、0.002、0.001、および 0.0005 mg/ml です。25 mg/ml の在庫から、0.32、0.16、0.08、0.04、0.02、0.01、および 0.005 mg/ml の希釈液を準備します。氷の上に置いておきなさい。</li>
		<li>所望の最終濃度を達成するために、各10倍の抗生物質濃度の10 μlを加えます。反復および株ごとに最終ウェルに10μl水(抗生物質制御なし)を加えます。</li>
		<li>マイクロタイタープレートを37°Cで24時間インキュベートします。</li>
	</ol>
</li>
	<li>
生細胞のアッセイ。
	<ol>
<li>96ウェルマイクロティタープレートごとに2枚のLB寒天プレートにラベルを付けます。</li>
		<li>100%エタノールにプロングを浸し、ブンゼンバーナーの開いた炎を横切って突起を通過させることによって、マルチプロング装置( 表1を参照)を殺菌します。繰り返す。プロングを少し冷まします。マルチプロング装置を用いて、マイクロタイタープレートの各ウェルからLB寒天板の表面にプランクトン培養液の〜3μl(通常は突起の先端に保持される量)を移す。</li>
		<li>LB寒天プレートを37°Cで16時間インキュベートします。</li>
		<li>細菌増殖のために遮断を同定することにより、抗生物質の殺菌濃度を最小限に抑える(図2)。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int. J. Antimicrob. Agents. 35 (4), 322-332 (2010).</li><li>Mah, T. F., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Zhang, L., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Involvement+of+a+Novel+Efflux+System+in+Biofilm-Specific+Resistance+to+Antibiotics.">The Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics.</a> J. Bacteriol. 190 (13), 4447-4452 (2008).</li><li>Zhang, L., Hinz, A. J., Nadeau, J. P., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+tssC1+Links+Type+VI+Secretion+and+Biofilm-specific+Antibiotic+Resistance.">Pseudomonas aeruginosa tssC1 Links Type VI Secretion and Biofilm-specific Antibiotic Resistance.</a> J. Bacteriol. 193 (19), 5510-5513 (2011).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biofilm-Specific+Antibiotic+Resistance+Gene,+ndvB,+is+Important+for+Expression+of+Ethanol+Oxidation+Genes+in+Pseudomonas+aeruginosa+Biofilms.">The Biofilm-Specific Antibiotic Resistance Gene, ndvB, is Important for Expression of Ethanol Oxidation Genes in Pseudomonas aeruginosa Biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+Dish+Biofilm+Formation+Assay.">Microtiter Dish Biofilm Formation Assay.</a> J. Vis. Exp. (47), e2437 (2011).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+tolerance+of+biofilms+and+persister+cells.">Multidrug tolerance of biofilms and persister cells.</a> Curr. Top. Microbiol. Immunol. 322, 107-131 (2008).</li><li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+Analyzing+Static+Biofilms.">Growing and Analyzing Static Biofilms.</a> Curr. Protoc. Microbiol. 1, 1B.1.1-1B.1.17 (2005).</li><li>Ramey, B. E., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.+Growing+and+analyzing+biofilms+in+fermenters.">Chapter 1. Growing and analyzing biofilms in fermenters.</a> Curr. Protoc. Microbiol. 1, 1B.3.1-1B.3.14 (2005).</li></ol>

著者は、彼女が競合する財政的利益を持っていないと宣言します。

プランクトニック細胞における抗生物質耐性は、細胞の永久的な変化(例えば 突然変異)による抗生物質の最小阻害濃度(MIC)の増加と定義される。これまでに同定されたバイオフィルム特異的耐性または耐性のメカニズムは、バイオフィルム内の野生型遺伝子の発現の結果である。したがって、従来の抵抗の定義はバイオフィルムには適用されません。しかし、別の定義のセットが提示?...

抗生物質耐性アッセイは、当初、細菌のプランクトニック(遊泳)培養物の耐性をアッセイするために開発されました。多くの細菌感染はバイオフィルム(表面付着細胞)を含むため、バイオフィルム特異的な抗生物質耐性をアッセイする方法の開発に興味を持っていました。しかし、ほとんどの抗生物質耐性アッセイは、バイオフィルムの耐性を測定するためには不十分です。例えば、最小阻害濃度(MIC)を決定することは、プランクトニック細菌培養 物1の抗生物質耐性を決定するためのゴールドスタンダードである。このアッセイは、希釈されたプランクトニック培養物と希釈系の抗生物質を混合することを伴う。 プランクトニック細胞の目に見える成長を阻害する抗生物質の濃度は、MICです。このアッセイは成長の阻害に依存しているため、定義上、それは、プレウジョンバイオフィルムにおける細胞の抗生物質感受性を調べる必要があるバイオフィルム培養物では動作できません。ここで説明するMBC-Bアッセイは、増殖阻害を測定する代わりに、バイオフィルムに存在する細胞を殺す抗生物質の濃度を決定する。従って、このアッセイは、確立されたバイオフィルム感染症の抗生物質治療を模倣し、 生体内での細菌抗生物質耐性のより関連性の高い見解を提供することを目的とする。
バイオフィルムは一般にプランクトニック培養 2-4 よりも抗生物質耐性が高いため、バイオフィルムの抗生物質耐性をプランクトニック培養物に直接関連付ける方法を考案する必要があった。したがって、この方法のもう一つの目標は、プランクトニック細胞とバイオフィルム細胞間の抗生物質耐性のレベルを直接比較できることである。ここで説明する MBC-P および MBC-B アッセイは、細胞が同様の条件で培養されるため、これを可能にします。我々は、バイオフィルム特異的抗生物質耐性 5-8 に重要ないくつかの遺伝子を研究するために、この方法を利用している。

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:160px;">
			1x&#160;M63
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare as a 5x&#160;M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.&#160; Dilute 5x&#160;stock 1:5, autoclave, cool, then add the desired components.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			KH2PO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P285-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			K2HPO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P288-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			(NH4)2SO4
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5132
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Magnesium sulfate
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			M63-500
			</td>
			<td style="width:160px;">
			Add to 1 mM final concentration.&#160; Prepare as a 1 M stock in water and autoclave.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tobramycin
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare 50 mg/m&#160;stock. Aliquot and store at -20&#176;C.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Arginine
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5131
			</td>
			<td style="width:160px;">
			Add to 0.4% final concentration.&#160; Prepare as a 20% stock in water and filter sterilize.&#160; This alternative carbon/energy source can replace glucose and casamino acids
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			96-well microtiter plates
			</td>
			<td style="width:160px;">
			Corning
			</td>
			<td style="width:160px;">
			3595
			</td>
			<td style="width:160px;">
			Sterile, flat-bottom, low evaporation
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tranferpette (multichannel pipette)
			</td>
			<td style="width:160px;">
			BrandTech
			</td>
			<td style="width:160px;">
			2703610
			</td>
			<td style="width:160px;">
			8-channel, 20-200 &#956;l
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Multiprong device
			</td>
			<td style="width:160px;">
			Dan-Kar
			</td>
			<td style="width:160px;">
			MC48
			</td>
			<td style="width:160px;">
			48 prongs fit into &#189; of a 96-well microtiter plate
			</td>
		</tr>
	</tbody>
</table>

establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b)

このプロトコルは、 インビトロでバイオフィルムを形成することができる細菌株のプランクトニックとバイオフィルム耐性の間の直接比較を可能にする。細菌は96ウェルマイクロティタープレートの井戸に接種される。プランクトニックアッセイの場合、選択した抗菌剤の連続希釈が細菌懸濁液に添加される。バイオフィルムアッセイでは、一度接種すると、細菌は一定期間にわたってバイオフィルムを形成するために残される。未接続の細胞は、ウェルから除去され、培地が補充され、選択した抗菌剤の連続希釈が加え加える。抗菌剤に曝された後、プランクトニック細胞は成長のためにアッセイされる。バイオフィルムアッセイの場合、培地は、抗菌剤を欠いた新鮮な培地でリフレッシュされ、バイオフィルム細胞は回復するために残されます。バイオフィルム細胞の生存率は、回復期間後にアッセイされる。抗菌剤のMBC-Pは、プランクトン培養中の細胞を殺す薬物の最も低い濃度として定義される。 対照的に、株に対するMBC-Bは、24時間の抗菌剤の濃度を増加させるために、プレフォー形成バイオフィルムを曝露することによって決定される。MBC-Bは、バイオフィルム内の細胞を殺す抗菌剤の最も低い濃度として定義されます。

MBC-PおよびMBC-Bアッセイを実施し、PA14野生型の感度をPA14∆ndvBと比較した。トブラマイシンは抗生物質として使用された。ステップ 1.4.4 (図 1) とステップ 2.3.4 (図 2) に対応する結果が表示されます。PA14および∆ndvBは、三重化でMBC-PおよびMBC-Bアッセイに接種された。MBC-B プロトコルのステップ 1.0-1.4 および MBC-P プロトコルのステップ 2.0-2.3 を完了した後?...

Watch this Scientific Journal Video about Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B at JoVE.com

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

このプロトコルは、96ウェルマイクロタイタープレートを使用して インビトロで バイオフィルムを形成することができる細菌株のプランクトニックとバイオフィルム耐性の間の直接比較を可能にする。プランクトニックまたはバイオフィルム細菌は、選択した抗菌剤の連続希釈液に曝露される。生存率は寒天プレート上の成長によってアッセイされる。

プランクトン細胞(MBC-P)およびバイオフィルム細胞(MBC-B)の抗菌剤の最小殺菌濃度の確立

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

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JoVE Journal

Immunology and Infection

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

19.8K ビュー.  University of Ottawa. このプロトコルは、96ウェルマイクロタイタープレートを使用して インビトロで バイオフィルムを形成することができる細菌株のプランクトニックとバイオフィルム耐性の間の直接比較を可能にする。プランクトニックまたはバイオフィルム細菌は、選択した抗菌剤の連続希釈液に曝露される。生存率は寒天プレート上の成長によってアッセイさ?...

ビデオ: Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8ウェルチャンバースライドを用いたin vitro のバイオフィルム形成における

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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 ...

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

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

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 ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

超高感度単一分子の開発と検証配列人間のインターフェロン-α のデジタルの酵素免疫測定法

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

ミトコンドリア、リソソーム T 細胞のマルチカラー フロー フローサイトメトリーを用いた定量化

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

高スループット赤痢菌-特定の殺菌試験

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

多能性幹細胞からのヒトマクロファージの生成と特性

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

細菌性角膜炎に対する薬物治療を研究するためのブタEx Vivoコルネアモデルの確立

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.