JWVのグループでの作業は、EUのマリーキュリー復帰フェローシップ、Sysmo2グラント（NWO-ALW/ERASysBio）、ホライゾン助成金（ZonMW）でとVENIフェローシップ（NWO - ALW）によってサポートされています。 OPKのグループは、いくつかのSTWの助成金（NWO）、SYSMO1（IGdeJ）とSYSMO2助成金、ESF Eurocoresシンバイオ助成金（SynMod）でと工業的発酵食品と栄養のトップ研究所のゲノミクスのためのKluyverセンターによってサポートされています。

<ol>
	<li>Veening, J. W., Smits, W. K., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistability,+epigenetics,+and+bet-hedging+in+bacteria.">Bistability, epigenetics, and bet-hedging in bacteria.</a> Annu. Rev Microbiol. 62, 193-193 (2008).</li><li>Dubnau, D., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistability+in+bacteria.">Bistability in bacteria.</a> Mol Microbiol. 61, 564-564 (2006).</li><li>Locke, J. C., Elowitz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+movies+to+analyse+gene+circuit+dynamics+in+single+cells.">Using movies to analyse gene circuit dynamics in single cells.</a> Nat. Rev. Microbiol. 7 (5), 383-383 (2009).</li><li>Veening, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bet-hedging+and+epigenetic+inheritance+in+bacterial+cell+development.">Bet-hedging and epigenetic inheritance in bacterial cell development.</a> Proc. Natl. Acad. Sci. U. S. A. 105 (11), 4393-4393 (2008).</li><li>de Jong, I. G., Veening, J. W., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochronic+phosphorelay+gene+expression+as+a+source+of+heterogeneity+in+Bacillus+subtilis+spore+formation.">Heterochronic phosphorelay gene expression as a source of heterogeneity in Bacillus subtilis spore formation.</a> J. Bacteriol. 192 (8), 2053-2053 (2010).</li><li>Veening, J. W., Murray, H., Errington, 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+mechanism+for+cell+cycle+regulation+of+sporulation+initiation+in+Bacillus+subtilis.">A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis.</a> Genes Dev. 23 (16), 1959-1959 (2009).</li><li>Eberhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+localization+of+choline-utilization+proteins+in+Streptococcus+pneumoniae+using+novel+fluorescent+reporter+systems.">Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems.</a> Mol Microbiol. 74 (2), 395-395 (2009).</li><li>Vasantha, N., Freese, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+changes+during+Bacillus+subtilis+sporulation+caused+by+deprivation+of+guanine+nucleotides.">Enzyme changes during Bacillus subtilis sporulation caused by deprivation of guanine nucleotides.</a> J Bacteriol. 144 (3), 1119-1119 (1980).</li><li>Stewart, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+death+in+an+organism+that+reproduces+by+morphologically+symmetric+division.">Aging and death in an organism that reproduces by morphologically symmetric division.</a> PLoS. Biol. 3, 45-45 (2005).</li><li>Rosenfeld, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+at+the+single-cell+level.">Gene regulation at the single-cell level.</a> Science. 307 (5717), 1962-1962 (2005).</li><li>Guberman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSICIC:+noise+and+asymmetry+in+bacterial+division+revealed+by+computational+image+analysis+at+sub-pixel+resolution.">PSICIC: noise and asymmetry in bacterial division revealed by computational image analysis at sub-pixel resolution.</a> PLoS. Comput. Biol. 4 (11), 1000233-1000233 (2008).</li><li>Montero, L. l. o. p. i. s. . P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+of+the+flow+of+genetic+information+in+bacteria.">Spatial organization of the flow of genetic information in bacteria.</a> Nature. 466, 77-77 (2010).</li><li>Avery, O. T., Macleod, C. M., McCarty, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+chemical+nature+of+the+substance+inducing+transformation+of+pneumococcal+types:+Induction+of+transformation+by+a+desoxyribonucleic+acid+fraction+isolated+from+pneumococcus+type+III.">Studies on the chemical nature of the substance inducing transformation of pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III.</a> J Exp. Med. 79, 137-137 (1944).</li><li>Hoskins, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+of+the+bacterium+Streptococcus+pneumoniae+strain+R6.">Genome of the bacterium Streptococcus pneumoniae strain R6.</a> J Bacteriol. 183, 5709-5709 (2001).</li><li>Martin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recA+gene+of+Streptococcus+pneumoniae+is+part+of+a+competence-induced+operon+and+controls+lysogenic+induction.">The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction.</a> Mol Microbiol. 15, 367-367 (1995).</li></ol>

利害の衝突は宣言されません。

他の多くの単一のセル技術とは対照的に、ここで説明するタイムラプス蛍光顕微鏡法は、その祖先、その動作、および除算イベントに関する特定のセルの履歴をたどるために使用することができます。蛍光標識した標的プロモーターまたはタンパク質と組み合わせることで、特定の発生経路の活性化は、時間とタンパク質の局在ならびにタンパク質のダイナミクスは、細菌の開発時に監視することができるで追跡することができます。 上記に示したように、別の細菌種に集​​中して研究は特定の細菌の要件に応じて増殖条件を適応することによって行うことができます。我々が遭遇した唯一の制限は、成長条件とサンプルサイズに関連しています。密閉された環境のために、培地条件が実験中に変更することはできません。また、実験ごとに4つの系統の最大は、効率的に監視することができます。 いくつかの重要なステップを考えると、ここで説明する単一細胞解析の手法は、容易に自動化された顕微鏡を使用して適用することができます。以下では、これらの重要な手順の概要が与えられます。詳細情報は、メインテキストに記載されて一般的な準備が：それは前の実験への特定の細菌のために必要なオートフォーカスの設定を確認するのが賢明です。可能であれば同様に、蛍光の可視化のためのおおよその最適な設定は、事前に決定されるべきである。また、準備したタイムラインは時間ですぐに使用できるすべての材料を持ってするのに役立つ、以下の（スライド1時間の準備を、顕微鏡の設定をプログラミング、顕微鏡室を事前に温めて細胞は、目的の増殖期になる前に、図1を参照） B.の 。 成長TLMとCDMの枯草菌 ：TLMとCDMが化学的に定義されている飢餓の媒体Bの枯草菌はゆっくりと成長。細胞を培地で栽培される時間期間は、特定の菌株によって延長する必要がある場合があります。低成長がお互いに積み重ねから細胞を防止顕微鏡の試料の調製は：。遺伝子のフレームの間に空気の泡を、スライドガラスとカバーガラスは、アガロースベースの媒体の広範囲な乾燥を防ぐために防止する必要があります。同じことはミディアム/カバースリップのインタフェースにも当てはまります。それは水泳および/ ​​または複数の層の成長を防ぐために、細胞が十分に乾燥させることが重要ですタイムラプス蛍光顕微鏡：。スライドだけでなく、環境室の前の温暖化が主要なオートフォーカスの問題を防ぐために重要である。これらは（サンプルが十分に乾燥させて提供）実験中にフィールドとフォーカスに滞在する最高のチャンスがあるので、細胞は、寒天のパッドの中央に選択する必要があります。実験ごとに10カ所の最大は、まだ正しく動作します。興味のある最初のセルを選択した後のみ（詳細は本文を参照）フォーカスを調整するためのソフトウェアを使用しています。 。細胞は30分間隔での実験分析の最初の数時間中にフォーカス可能かどうかを確認してください：それは媒体の背景が蛍光チャンネルで同じような値を持っているかどうかを事前に拡張された分析手順に確認することが重要です。小さ ​​な塵の粒子が、培地成分、汚れたレンズまたは小型アガロースの塊は、 トラブルシューティングを分析するために映画が困難または不可能に、局所的に増大した蛍光に貢献することができます。細胞は互いの上に成長した場合、これはどちらかのカバースリップがあったことを示すかもしれません早すぎるか、そのメディアが微小コロニー単分子膜の成長に適していない添付。スライド上の他の細胞が喜んで分割するのに対し、関心の細胞は連続して、途中で死亡した場合、あなたは位置にUVフィルターを置くかどうかをチェックすることがあります。また、長時間の実験中に、露光時間や光強度を減少させるために役立つかもしれない。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>遺伝子のフレーム</td><td> ABgene </td><td> AB - 0578 </td><td> 1.7 × 2.8センチメートル</td></tr><tr><td>高解像度の低融点アガロース</td><td>シグマ</td><td> A4718 </td><td></td></tr><tr><td>大きなカバースリップ</td><td>いくつか</td><td></td><td> 24 × 50ミリメートル</td></tr><tr><td>必要に応じて、膜色素、例えば、FM 5から95 </td><td>インビトロジェン</td><td> T23360 </td><td>他の膜の染料も使用可能です。 <a href="http://probes.invitrogen.com/media/pis/mp34653.pdf">http://probes.invitrogen.com/media/pis/mp34653.pdf</a> </td></tr><tr><td>環境室とのタイムラプス顕微鏡</td><td>いくつか</td><td></td><td>対応するセクションで我々のデバイスの詳細を参照してください。 </td></tr></tbody></table>

live cell imaging of bacillus subtilis and streptococcus pneumoniae using automated time-lapse microscopy

ここ数年の間に科学者は微生物集団ベースの実験から得られた平均的なデータが単一の細胞の挙動、状態や表現型の代表ではないことをますます認識するようになりました。この新しい洞察による単一細胞の研究の数は、（最近のレビューのために1,2,3を参照）継続的に上昇する。しかし、適用される単一のセル技術の多くは、時間のある特定の単一のセル（例えばフローサイトメトリーまたは標準顕微鏡）の開発と動作を監視することはできません。 ここで、我々は多くの世代のための成長と分裂を介して枯草菌と肺炎球菌の個々の細菌細胞を以下のと（の蛍光）を記録できるようにいくつかの最近の研究4、5で使用される顕微鏡法、6、7、の詳細な説明を提供しています。結果として得られる動画は、1つの共通祖先に由​​来する集団内の単一セルの歴史をさかのぼることによって系統学的系統樹を構築するために使用することができます。このタイムラプス蛍光顕微鏡法は、個々の細胞の増殖、分裂と分化を調査するだけでなく、特定の細胞挙動のセルの歴史と祖先の影響を分析するために使用することはできません。また、タイムラプス顕微鏡は、理想的には細菌の細胞周期の間に遺伝子発現のダイナミクスとタンパク質の局在を調べるために適しています。方法は、細菌の細胞を調製し、微小コロニーに単一細胞の伸長を有効にするために顕微鏡スライドを構築する方法を説明します。一言で言えば、単一の細胞は、彼らが成長し、温度制御された環境室内で蛍光顕微鏡下で割っているアガロースを添加した増殖培地から成る半固体表面上にスポットされています。画像は特定の間隔でキャプチャされ、後にオープンソースソフトウェアImageJを用いて解析している。

Watch this Scientific Journal Video about Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy at JoVE.com

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

このプロトコルは、自動化された蛍光タイムラプス顕微鏡を用いて時間の異なる細菌の単セルの動作を監視するためにステップバイステップ手順を提供します。さらに、我々は、顕微鏡画像を分析する方法のガイドラインを提供します。

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

live-cell-imaging-bacillus-subtilis-streptococcus-pneumoniae-using

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

42.2K ビュー.  University of Groningen. このプロトコルは、自動化された蛍光タイムラプス顕微鏡を用いて時間の異なる細菌の単セルの動作を監視するためにステップバイステップ手順を提供します。さらに、我々は、顕微鏡画像を分析する方法のガイドラインを提供します。

ビデオ: Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

免疫・感染学

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

の単一細胞解析枯草菌</em蛍光顕微鏡とフローサイトメトリーを用いて>バイオフィルム

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

ライブセル蛍光顕微鏡を用いた細菌毒素誘導される応答の可視化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

真菌病原体の食作用の生細胞ビデオ顕微鏡

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更<em&gt;肺炎球菌</em&gt;蛍光染料を使用

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

の莢血清型<em&gt;肺炎球菌</em&gt;膨張反応を使用して、

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

の莢膜血清型<em&gt;肺炎球菌</em&gt;ラテックス凝集による

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

の生理反応を測定します<em&gt;ショウジョウバエ</em&gt;ライブカルシウムイメージングを使用して脂質フェロモンの感覚ニューロン

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

コンプリートの蛍光タイムラプスイメージング<em&gt; S。ベネズエラ</emマイクロ流体デバイスの使用&gt;ライフサイクル

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

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の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

の単一細胞解析枯草菌バイオフィルム

ライブセル蛍光顕微鏡を用いた細菌毒素誘導される応答の可視化

真菌病原体の食作用の生細胞ビデオ顕微鏡

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更

の莢血清型

の莢膜血清型

の生理反応を測定します

コンプリートの蛍光タイムラプスイメージング