シェリークチマ、ピートニューウェルと図1の画像を提供するためのロバートシャンクスに感謝します。この作品は、GAOにNIHの助成金R01AI083256によってサポートされていました

1。バイオフィルムの成長<ol><li>野生型の緑膿菌や豊富な培地で一晩変異株（すなわち、LB）の文化を育てる</li><li>バイオアッセイのための新鮮な培地に一晩培養1:100に希釈する。 Pの標準的なバイオアッセイ培地緑膿菌は、硫酸マグネシウム、グルコースとカザミノ酸（表を参照）を添加したM63最少培地です。小さいプランクトンの成長と、より強固なバイオフィルムを刺激する媒体を代替の促進バイオフィルムとして、グルコースとカザミノ酸を唯一の炭素およびエネルギー源としてのアルギニンに置き換えることができます。 </li><li> 96ウェルディッシュに1ウェルあたり希釈液100μLを加える。定量的なアッセイでは、我々は通常、それぞれの治療のために井戸を複製4-8使用。 </li><li> 37 4から24時間のためのマイクロタイタープレートをインキュベート℃に</li></ol> 2。バイオフィルムを染色<ol><li>インキュベーション後、プレートを裏返して液体を振とうして細胞をダンプする。 </li><li>水の小さな浴槽に静かに沈めるプレート（すなわち、浴槽などのP1000 pipetmen用ピペットチップボックスの底部を使用）。水を払います。このプロセスをもう一度繰り返します。この手順は、次のステップで染色することができますアタッチされていない細胞と培地成分を除去する手助けを、と大幅にバックグラウンド染色を低減します。 </li><li>マイクロタイタープレートの各ウェルに水のクリスタルバイオレットの0.1％溶液125μLを加える。解決しながら手袋と白衣を着用してください。粉体が吸湿性と容易に汚れの衣類、皮膚、等であるとしてCVを計量するときは注意してください</li><li> 10〜15分間室温でマイクロタイタープレートをインキュベートします。 </li><li>上記のように水の浴槽に沈めることによって水でプレートを3〜4回​​すすぎ、振ると、すべての余分な細胞と色素のプレートを取り除くためペーパータオルのスタック上に精力的に汚点。 </li><li>逆さにマイクロタイタープレートを回して、数時間または一晩乾燥させます。 </li><li>乾燥時に定性的なアッセイでは、井戸を撮影することができます。 </li></ol> 3。バイオフィルムの定量化<ol><li> CVを可溶化するマイクロタイタープレートの各ウェルに水に30％酢酸125μLを加える。 </li><li> 10〜15分間室温でマイクロタイタープレートをインキュベートします。 </li><li>新しい平底マイクロタイター皿に可溶化したCVの125μLを転送する。 </li><li>ブランクとして水に30％酢酸を用いて550nmでプレートリーダーで吸光度を定量化する。 </li></ol> 4。代表的な結果： <img src="/files/ftp_upload/2437/2437fig1.jpg" alt="図1" /> 図1は、。 緑膿菌 、 蛍光菌や黄色ブドウ球菌に対して実行バイオフィルム形成アッセイのための代表的な結果を示しています。 （A）、 緑膿菌のバイオフィルム（8時間、37 ° C）と同様の側面図を。 P.のバイオフィルムと同様の（B）側面図蛍光菌 （6時間、30 ° C）。 S.によって形成されたバイオフィルムの（C）トップダウンビュー平底マイクロタイタープレートの黄色ブドウ球菌 （二つの井戸、24時間、37 ° C）。P.緑膿菌とP.蛍光菌は 、両方の運動性の生物であり、空気-液体界面でのバイオフィルムを形成します。黄色ブドウ球菌は、非運動性であり、井戸の底にバイオフィルムを形成する。

<ol>
	<li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signalling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis.</a> Mol. Microbiol. 28, 449-461 (1998).</li><li>O'Toole, G. A., Doyle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. , 91-109 (1999).</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=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426, 306-310 (2003).</li><li>Kuchma, S. L., Connolly, J. P., 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+three-component+regulatory+system+regulates+biofilm+maturation+and+type+III+secretion+in+Pseudomonas+aeruginosa.">A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa.</a> J Bacteriol. 187, 1441-1454 (2005).</li><li>Caiazza, N. C., 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=SadB+is+required+for+the+transition+from+reversible+to+irreversible+attachment+during+biofilm+formation+by+Pseudomonas+aeruginosa+PA14.">SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14.</a> J Bacteriol. 186, 4476-4485 (2004).</li><li>Shanks, R. M., Sargent, J. L., Martinez, R. M., Graber, M. L., 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=Catheter+lock+solutions+influence+staphylococcal+biofilm+formation+on+abiotic+surfaces.">Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces.</a> Nephrol Dial Transplant. 21, 2247-2255 (2006).</li><li>Caiazza, N. C., Merritt, J. H., Brothers, K. M., 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=Inverse+regulation+of+biofilm+formation+and+swarming+motility+by+Pseudomonas+aeruginosa+PA14.">Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14.</a> J. Bacteriol. 189, 3603-3612 (2007).</li><li>Hinsa, S. M., Espinosa-Urgel, M., Ramos, J. L., 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=Transition+from+reversible+to+irreversible+attachment+during+biofilm+formation+by+Pseudomonas+fluorescens+WCS365+requires+an+ABC+transporter+and+a+large+secreted+protein.">Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein.</a> Mol Microbiol. 49, 905-918 (2003).</li><li>Mack, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+transposon+mutants+of+biofilm-producing+Staphylococcus+epidermidis+impaired+in+the+accumulative+phase+of+biofilm+production:+genetic+identification+of+a+hexosamine-containing+polysaccharide+intracellular+adhesin.">Characterization of transposon mutants of biofilm-producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: genetic identification of a hexosamine-containing polysaccharide intracellular adhesin.</a> Infect. Immun. 62, 3244-3253 (1994).</li><li>Vidal, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+an+Escherichia+coli+K-12+mutant+strain+able+to+form+biofilms+on+inert+surfaces:+involvement+of+a+new+ompR+allele+that+increases+curli+expression.">Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.</a> J. Bacteriol. 180, 2442-2449 (1998).</li><li>Junker, L. M., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+for+small-molecule+inhibitors+of+Pseudomonas+aeruginosa+biofilm+development.">High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.</a> Antimicrob Agents Chemother. 51, 3582-3590 (2007).</li></ol>

利害の衝突は宣言されません。

このメソッドは、微生物種の多様なで使用するように変更することができます。非運動性の微生物は、通常、ウェルの底に付着しながら運動性微生物は、一般的に、壁及び/または井戸の底に付着する。バイオフィルム形成（すなわち、増殖培地、温度、インキュベーションの時間）の最適条件は、それぞれの微生物に対して経験的に決定する必要があります。私はそれぞれの菌株または条件...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 X M63</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Prepare as a 5X 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. Dilute 5X stock 1:5, autoclave, cool, then add the desired components.</td>
</tr>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P285-500</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P288-500</td>
<td>&nbsp;</td>
<tr>
<td>(NH4)2SO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5132</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M63-500</td>
<td>Add to 1 mM final concentration. Prepare as a 1 M stock in water and autoclave.</td>
</tr>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>D16-3</td>
<td>Add to 0.2% final concentration. Prepare as a 20% stock in water and autoclave.</td>
</tr>
<tr>
<td>Casamino acids</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>223050</td>
<td>Add to 0.5% final concentration. Prepare as a 20% stock in water and autoclave.</td>
</tr>
<tr>
<td>Arginine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5131</td>
<td>Add to 0.4% final concentration. Prepare as a 20% stock in water and filter sterilize. This alternative carbon/energy source can replace glucose and casamino acids</td>
</tr>
<tr>
<td>Microtiter plates</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>353911</td>
<td>Falcon 3911, Microtest III, Flexible assay plates, 96 well, U-bottom, non-sterile, non-tissue-culture treated.</td>
</tr>
<tr>
<td>Microtiter plate lids</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>353913</td>
<td>The lids can be reused by cleaning with 95% ethanol in water.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>229641000</td>
<td>Prepare as a 0.1% solution in water.</td>
</tr>		</tbody>
		</table>
		</div>

microtiter dish biofilm formation assay

バイオフィルムは、医療、産業および自然な設定で見つけることができる表面に付着した微生物のコミュニティです。実際には、バイオフィルムでの生活は、おそらくほとんどの環境での微生物の成長の支配的なモードを表します。成熟したバイオフィルムは、いくつかの異なった特徴を持っている。バイオフィルムの微生物は、通常、地域社会への構造と保護を提供する細胞外マトリックスに囲まれています。バイオフィルムで成長している微生物はまた、一般的に液体で満たされたチャンネルに囲まれたmacrocolonies（細胞の数千を含む）で構成される特徴的なアーキテクチャを持っている。バイオフィルム成長微生物はまた、臨床的に適切な抗生物質を含む抗菌薬の範囲への抵抗性は周知の事実です。 マイクロタイター皿のアッセイは、バイオフィルム形成の初期段階の研究のための重要なツールであり、このアッセイはまた、真菌バイオフィルム形成を研究するために使用されているが、細菌のバイオフィルムの研究のために主に適用されています。このアッセイは、静的、バッチ成長の条件を使用しているので、それは通常、フローセルシステムに関連付けられている成熟したバイオフィルムの形成のために許可されていません。しかし、このアッセイは、バイオフィルム形成の開始に必要な多くの因子を同定するのに有効であった（す​​なわち、鞭毛、繊毛、アドヘシン、サイクリック - ジ - GMPの結合や代謝に関与する酵素）と同様に細胞外多糖の生産に関与する遺伝子いる。さらに、出版された仕事は、マイクロタイター皿で増殖したバイオフィルムは、免疫系のエフェクターのような抗生物質耐性と耐性を成熟バイオフィルムのいくつかのプロパティを開発しないことを示します。 このシンプルなマイクロタイター皿のアッセイは、壁および/またはマイクロタイター皿の底部にバイオフィルムを形成することができます。アッセイのハイスループットな性質は、遺伝子スクリーニングだけでなく、様々な成長条件の下で、複数の菌株によってバイオフィルム形成のテストに役立ちます。このアッセイの亜種がこれに限定されないが、シュードモナス、 コレラ菌 、 大腸菌、staphylocci、腸球菌 、 マイコバクテリアおよび真菌を含む微生物の様々な、のために初期のバイオフィルム形成を評価するために使用されています。 プロトコルここで説明するには、我々はモデル生物、緑膿菌によるバイオフィルム形成を研究するために、このアッセイの使用に焦点を当てます。このアッセイでは、バイオフィルム形成の程度は、染料クリスタルバイオレット（CV）を用いて測定されます。しかし、他の比色及び代謝汚れの数は、マイクロタイタープレートアッセイを用いてバイオフィルム形成の定量化のために報告されている。マイクロタイタープレートアッセイの容易さ、低コストと柔軟性がバイオフィルムの研究のための重要なツールきました。

Watch this Scientific Journal Video about Microtiter Dish Biofilm Formation Assay at JoVE.com

Microtiter Dish Biofilm Formation Assay

アッセイは、細菌および真菌の早期のバイオフィルム形成を測定する迅速な手段を説明します。このメソッドは、微生物のバイオフィルム形成のための基層としてのマイクロタイタープレートを使用し、バイオフィルムは、クリスタルバイオレットの菌株を用いて可視化です。アッセイは、どちらかの初期のバイオフィルム形成のための定性的または定量的なアッセイを提供する。

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 ...

Research

JoVE Journal

Immunology and Infection

106.7K ビュー.  Dartmouth Medical School. アッセイは、細菌および真菌の早期のバイオフィルム形成を測定する迅速な手段を説明します。このメソッドは、微生物のバイオフィルム形成のための基層としてのマイクロタイタープレートを使用し、バイオフィルムは、クリスタルバイオレットの菌株を用いて可視化です。アッセイは、どちらかの初期のバイオフィルム形成のための定性的または定?...

ビデオ: Microtiter Dish Biofilm Formation Assay

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

監視形成との抗真菌薬感受性試験のための96ウェルマイクロタイタープレートベースの方法カンジダアルビカンスバイオフィルム

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

免疫・感染学

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 ...

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. ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

マイクロタイタープレート形式で液体希釈法により、第一、第二ラインの薬のための結核菌の複合体の抗菌薬感受性試験

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

イメージング病因とのコーディングを解読し、<em&gt;マイコバクテリウム膿瘍</em&gt;ゼブラフィッシュ胚で

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

バイオフィルム関連をターゲット<em&gt;黄色ブドウ球菌</em&gt;レサズリン基づく薬剤感受性アッセイを使用して

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

「ファゴソーム閉鎖アッセイ」全反射蛍光顕微鏡（TIRFM）を用いて、三次元的にファゴソーム形成を可視化します

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

自動マイクロ流体デバイスを使用してカンジダ菌バイオ フィルム形成の可視化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

腸管病原体の胆汁酸塩によるバイオ フィルム形成: 同定及び定量化の手法

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

高スループット アッセイを行い好中球細胞外トラップ形成の定量化

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...