Name: visualizing the effects of sputum on biofilm development using a chambered coverglass model
Uploaded: 2016-12-14T14:00:00.000+00:00
Duration: 5 min 3 s
Description: Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

TBは、嚢胞性線維症、カナダからの研究フェローシップを認めています。

研究倫理委員会（REB）は、ヒト被験者から痰サンプルを収集し、保存するために必要なことに注意してください。これらの研究は、病気の子供REBの＃1000019444のために病院によって承認されました。 1.準備CF痰試料<ol><li>嚢胞性線維症の診療所へのルーチンの訪問中に、患者からの痰のサンプルを収集し、氷上に保ちます。 </li><li>処理を受けるために、研究室に、コレクションの最初の一時間以内に氷上に喀痰試料を輸送。 </li></ol> 2.喀痰処理<ol><li>得られた痰試料の体積を記録します。サンプル（ すなわち、2部PBS、1部の試料）の体積を2倍のリン酸緩衝生理食塩水（PBS）を加えます。 </li><li>トランスファーピペットでよくサンプルを混ぜます。 1分が完全に混合するための最高の設定にボルテックスのサンプルを。 </li><li>適切な数の1.5ミリリットルのマイクロチューブに小分けし、上記混合物の1ミリリットルをし、5,000×gでスピンダウン4℃で20分間。 </li><li>遠心分離後、上清を除去し、ペレットを捨てます。 </li><li>フィルタは、0.22μmフィルターを通して上清を殺菌し、清潔なマイクロ遠心チューブに集めます。 注：ろ液の無菌性は、LB寒天上にプレーティングし、液体培地に接種することによってテストされています。 </li><li>将来の使用のために-80℃で痰上清を保管してください。 注：複数の患者からの喀痰もろ過以下のプールすることができます。 </li><li>使用前に、所望のメディアに喀痰ろ液1/10 V / V（痰を100μl、メディアの900μl）を希釈します。 注記：ここでは、標準のLB培地（LB）培地を用いました。 </li></ol>バイオフィルム形成のための3チェンカバーガラス法<ol><li>振盪（200rpm）しながら37℃で、所望のメディアの関心を一晩の細菌分離株を成長させます。 注：別の細菌数を使用した、 緑膿菌 、 黄色ブドウ球菌の臨床分離株を含みます、バークホルデリア・セパシア錯体とアクロのxylosoxidans。媒体の選択は、目的の株および条件に依存し、しかしLB培地は、最初の実験のために使用することができます。 </li><li>一晩培養物から、新鮮な培地の4ミリリットル中に文化の40μlのを配置し、約0.5の600nmで（OD 600）での光学密度と文化を得るために、振盪（200rpm）しながら37℃で3-4時間成長-0.6。 </li><li> 10％の喀痰ろ液または（対照として）痰ろ液をせずに所望のメディアで1/5にステップ3.2からの培養を希釈します。痰ろ液の他の濃度を試験することができる（ すなわち、50％または100％）。 </li><li>スライドチャンバーのウェルを播種するために希釈液200μlを使用してください。 </li><li>細菌は振盪せずに37℃で4時間アタッチすることができます。 </li><li> 4時間後、メディアを取り出し、軽く1xの新鮮な培地でバイオフィルムを洗います。 200μlの新鮮な培地と交換してください。 注：バイオフィルム上の痰の効果を研究するために、FRES時間メディアは、痰上清を含める必要があります。 </li><li>バイオフィルムは、顕微鏡検査のための時間になるまで洗浄することなく、メディアごとに12時間を置き換え、振盪せずに37℃で、所望の時間のために成長することを可能にします。 注：バイオフィルムの抗生物質感受性に痰上清の効果を研究するためには、抗生物質は、バイオフィルムの成長の24時間後の培地に添加され、染色およびバイオフィルムの撮影まで培地中で維持されています。 </li></ol> 4.染色バイオフィルムと共焦点顕微鏡<ol><li>所望の成長時間（24〜48時間が最高の作品）に続いて、チャンバーウェルから培地を除去し、静かに無菌PBS300μlで2回チャンバーを洗います。 </li><li>必要なソリューションの各ミリリットルのための（生存率キットで提供される）各色素の1μLを混合することにより、バイオフィルムに対する染色混合物を準備します。水やメディア溶液中の色素を作ります。 注：水は、製造業者によって推奨されています。 </li><li>チャンバ型coverglaの各ウェルに染料混合物の200μLを追加ssは暗所で45分間、室温でインキュベートし、。 </li><li>室から染色混合物を削除し、滅菌PBS300μlで各ウェルを洗浄します。 PBSを削除し、新鮮な水やメディアと交換してください。 </li><li>共焦点顕微鏡を介したバイオフィルムの可視化を進めます。 </li></ol> 5.共焦点顕微鏡を用いてバイオフィルムの可視化<ol><li>すぐに（1時間以内）染色した後、チャンバ内の染色されたバイオフィルムをお読みください。一度に2 8ウェルチャンバーに1を染色することによって、スライドの可視化の遅れを最小限に抑えます。 </li><li>取得のための励起とフィルターのセットのためのレーザーで共焦点顕微鏡を用いて撮影を行います。 注：ここでは、スペクトルボレアリスレーザーとスピニングディスク共焦点システム（緑：491nm、赤：561 nm）と励起のために使用しました。 40分の515と40分の624の発光フィルターセットは、バイオフィルムの生存率キットから汚れを視覚化するために使用されました。 </li><li>カメラと共焦点顕微鏡で25X水対物レンズを用いて画像を撮影します。 </li><li&gt;を使用してZ-スタックバイオフィルムをモデル化します。 （一般的に48時間のバイオフィルムのための30から80ミクロンに及ぶ）、バイオフィルムの最後の合焦フレームに最初の合焦面から出発した画像ごとに0.5から1ミクロンを取ります</li><li>各ウェルから3-5画像を撮影します。 注：このように8よくチャンバーカバーガラスのために、24から40の画像が生成されます。 </li><li>分析のために画像を保存します。 注：画像は、犯罪統計システム18,19を用いて分析するOME-TIFFファイルとして保存する必要があります。バイオフィルムの画像解析の手順については、http://www.comstat.dk/で見つけることができます。画像がインポートされると、そのような各チャネル（赤と緑）の平均厚さは、バイオマスおよび表面被覆率などのパラメータを分析することができます。 </li></ol>

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本明細書に記載される方法は、外因性生成物の存在下で増殖させた細菌性バイオフィルムの可視化を可能にします。このタイプのシステムを使用する場合に当然のことながら、exoproductsの生産が重要です。例えば、ジチオスレイトール（DTT）は、多くの場合、サンプルを液化するために役立つ人間の唾液サンプルに使用されています。しかし、単独でDTTの効果は、バイオフィルムの発達およ?...

細菌バイオフィルムは、互いに結合し、自己分泌マトリックスに包まれている微生物の基です。 1,2古典的には、それらが物理的に流れの条件の下で形成された非生物または生物の表面に付着した細菌を表します。バイオフィルムはまた、試験管内で形成された熱プールまたはペリクルの空気 - 液体界面でのような表面からの静的条件（流れの欠如）および遠位で成長することが示されています。これらのバイオフィルムは、長い環境で認識され、彼らは生物付着、腐食や閉塞が生じ、貯水池内またはパイプで形成することができるよう、産業プロセスへの主要な不利益であるされています。 3,4 彼らはカテーテル関連感染症、嚢胞性線維症の患者では、だけでなく、多数の他の感染症における肺感染症に関与することが示されているようにバイオフィルムは、また、医療現場で重要です。 5,6バイオフィルム感染症の特徴の一つは、デです抗生物質に対する細菌の感受性を折り目と自然免疫系によるクリアランスを損ないます。バイオフィルムベースの感染を伴う7-9で最もよく研究され、臨床的に関連するシナリオが慢性的に緑膿菌バイオフィルムに感染している嚢胞性線維症（CF）の患者に起こります。 緑膿菌は、それが非常に困難治療することを可能にする慢性感染症の確立中に変更の数を受けることができます。 10,11バイオフィルムは、示差的に先天性免疫を活性化し、炎症を駆動することができます。 12-14これらの感染症は、CF患者において増加罹患率および死亡率につながるように、この文脈では、バイオフィルム発達に影響を与えることができる因子を理解することが重要です。 最近の研究は、宿主因子が緑膿菌バイオフィルムの凝集体の形成に重要であることを示唆しています。 15これらのバイオフィルムは、抗生物質および宿主防御機構に対する感受性の低下に貢献しています。 presaの複数形CF肺中に存在する微生物から、このような好中球エラスターゼなどの宿主由来因子、ならびに分泌物のNCEは、大幅にバイオフィルム形成と発展を調節する可能性を秘めています。 16はまた、バイオフィルムは、多数の経路の発現を調節し、炎症を開始するためにホストと対話します。標準的なクリスタルバイオレットアッセイ等のハイスループット方法が、バイオフィルムの処理に関していくつかの情報を提供することができ、これらの因子に応答したバイオフィルムの可視化は、より詳細な情報を提供します。 本稿では、 インビトロでのバイオフィルムの発達を研究するためにCF患者の痰からの係数を使用する方法を記載しています。この方法は、市販のバイオフィルムの生存率キットを使用して痰を含む宿主因子に曝露されたバイオフィルムの迅速な可視化を可能にします。この技術は、視覚的にexogenoの存在下でのバイオフィルムの成長中に発生する変化を同定するために使用され得ます私たちの製品、および様々な条件下でのバイオフィルムの開発の変化を分析するための改良された方法を表しています。

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

バイオフィルムは、自己分泌マトリックスに包まれた細菌のグループで構成されています。彼らは、工業汚染だけでなく、多くの健康に関連する感染症の発症と持続に重要な役割を果たしています。ヒトの疾患の中で最もよく説明し、研究バイオフィルムの一つは、嚢胞性線維症患者の慢性肺感染症で発生します。ホストのコンテキストでバイオフィルムを研究すると、多くの要因は、バイオフィルム形成と発展に影響を与えることができます。宿主因子はバイオフィルムの形成及び発達に影響を与えることができる方法を同定するために、我々は、痰上清の形で宿主由来因子の存在下でバイオフィルムを成長させるために、静的チェンカバーガラスの方法を使用しました。細菌は、チャンバーに播種し、痰ろ液にさらされています。成長の48時間後に、バイオフィルムは、共焦点顕微鏡および分析の前に商業バイオフィルムの生存率キットで染色されています。画像取得後、バイオフィルムの特性は、異なるソフトウェア・プラットフォームを用いて評価することができます。この方法は、私たちは、抗生物質などの異なる物質の存在下でのバイオフィルムの成長の重要な特性を視覚化することができます。

実験の全体的な設計は、図1に示されています。このプロトコルの使用は、異なる時間（ 例えば、24、48または72時間）増殖させたバイオフィルムの変化を視覚化するための便利な方法を提供します。重要なことには、例えば、痰濾液ような外因性シグナルは、バイオフィルムの発達の変化を視覚化するために添加することができます。 図2</str...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

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

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 ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Research

JoVE Journal

Immunology and Infection

8.3K ビュー.  Hospital for Sick Children. 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.

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

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培養モデルの緑膿菌</emライブヒト気道細胞上に成長した>バイオフィルム

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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 ...

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 ...

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. ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

ハイスループットの使用<em&gt;インビトロ</emオーラル多種バイオフィルムを開発するために&gt;マイクロ流体システム

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

生物工学

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

研究のための方法論<em&gt; B。枯草菌</em小分子バイオフィルム阻害剤を特徴付けるためのモデルとしての&gt;バイオフィルム

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

WinCFモデル - 肺感染症の微生物学を研究するための粘液で閉鎖された細気管支の安価で収縮可能なマイクロコスモス

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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. ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

嚢胞性線維症患者の喀痰中の 緑膿菌 の可視化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

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.

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

要約

さらに動画を探す

この動画の章

共培養モデルの緑膿菌バイオフィルム

フローセル内の緑膿菌と出芽酵母のバイオフィルム

マイクロタイター皿のバイオフィルム形成アッセイ

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

ハイスループットの使用

研究のための方法論

WinCFモデル - 肺感染症の微生物学を研究するための粘液で閉鎖された細気管支の安価で収縮可能なマイクロコスモス

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

嚢胞性線維症患者の喀痰中の緑膿菌の可視化

COMSTATソフトウェアを使用した In vitro バイオフィルムアーキテクチャに対する抗菌剤の影響の定量化

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

要約

さらに動画を探す

この動画の章

共培養モデルの緑膿菌バイオフィルム

フローセル内の緑膿菌と出芽酵母のバイオフィルム

マイクロタイター皿のバイオフィルム形成アッセイ

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

ハイスループットの使用

研究のための方法論

WinCFモデル - 肺感染症の微生物学を研究するための粘液で閉鎖された細気管支の安価で収縮可能なマイクロコスモス

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

嚢胞性線維症患者の喀痰中の 緑膿菌 の可視化

COMSTATソフトウェアを使用した In vitro バイオフィルムアーキテクチャに対する抗菌剤の影響の定量化

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

嚢胞性線維症患者の喀痰中の緑膿菌の可視化