JWV의 그룹에 작업이 EU 마리 퀴리 합체 원정대, Sysmo2 그랜트 (NWO-ALW/ERASysBio), 호라이즌 부여 (ZonMW)와 VENI 친목 (NWO - ALW)에 의해 지원됩니다. OPK의 그룹은 여러 STW 보조금 (NWO), SYSMO1 (IGdeJ)와 SYSMO2 부여, ESF Eurocores SynBio 부여 (SynMod)와 산업 발효 및 식품 영양에 대한 최고 연구소의 게놈에 대한 Kluyver 센터에 의해 지원됩니다.

<ol>
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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. 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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>

관심 없음 충돌 선언하지 않습니다.

많은 다른 단일 세포 기술에 대조적으로, 여기에서 설명한 시간 경과 형광 현미경 방법은 그 조상의 행동, 그리고 분열 이벤트에 관한 특정 세포의 역사를 수행하는 데 사용할 수 있습니다. 찬란 표시 대상 발기인 또는 단백질와 함께 구체적인 발달 경로의 활성화는 시간과 단백질 지방화뿐만 아니라 단백질 역학이 박테리아 개발되는 동안 모니터링할 수 있습니다에 따라 수 있습니다. 위에서 표시된 다른 세균 종에 집중 연구는 특정 세균에 대한 요구 사항에 따라 성장 조건을 적응하여 수행할 수 있습니다. 우리가 발생한 유일한 제한은 성장 조건과 샘플의 크기와 관련이 있습니다. 밀폐 환경으로 인해 중간 조건은 실험 기간 동안 변경할 수 없습니다. 또한, 실험 당 4 개의 변종이 최대가 효율적으로 모니터링할 수 있습니다. 몇 가지 중요한 단계를 고려, 단일 세포 분석 방법은 여기에 설명된 쉽게 어떤 자동화된 현미경을 사용하여 적용할 수 있습니다. 다음에서 이러한 중요한 단계의 개요가 주어집니다. 자세한 정보는 주요 텍스트에서 찾을 수 있습니다 일반 준비 사항 :. 그것은 이전 실험 특정 박테리아에 필요한 자동 초점 설정을 확인하는 것이 현명합니다. 가능하면 마찬가지로, 형광의 시각화에 대한 대략적인 최적의 설정은, 사전에 결정되어야합니다. 또한, 준비 시간 라인을 따라하는 것은 시간에 사용할 준비가 모든 자료 (사전 온난, 현미경 챔버를 현미경 설정을 프로그래밍, 세포가 원하는 성장 단계에되기 전에 슬라이드 한 시간 준비를 참조 그림 1)을하는 데 도움이 B.니다. 성장 TLM와 CDM에 subtilis : TLM과 CDM은 화학적으로 기아 미디어를 정의하는 B. subtilis은 천천히 자랍니다. 세포가 언론에서 재배되는 시간 기간은 특정 변형에 따라 연장 될 수도 있습니다. 느린 성장은 서로에 말뚝의 세포를 방지 현미경 샘플의 준비 :. 유전자 프레임, 유리 슬라이드와 커버 슬립 사이의 공기 방울은 아가로 오스 기반 매체의 광범위한 건조를 방지하기 위해 예방해야합니다. 동일 매체 / 커버 슬립 인터페이스 보유하고 있습니다. 그것은 수영 및 / 또는 여러 계층의 성장을 방지하기 위해, 세포가 충분히 건조하게하는 데있어 매우 중요합니다 시간 경과 형광 현미경 :. 슬라이드뿐만 아니라 환경 챔버 사전 온난 주요 오토포커스 문제를 방지하기 위해 중요합니다. 이들은 실험 (시료가 충분히 건조했습니다 제공) 중 분야와 초점을 유지하기 위해 최고의 기회를 갖고 있기 때문에 세포는 한천 패드의 중간에 선택되어야합니다. 실험 당 10의 위치 최대 여전히 제대로 작동합니다. 선택하는 데 후 관심의 첫 번째 셀은 (자세한 내용은 텍스트를 참조) 초점을 조정하는 소프트웨어를 사용합니다. . 세포가 30 분 간격의 실험 분석의 첫 번째 시간 동안 초점이 아직 있는지 확인하십시오 : 이것은 매체의 배경 형광 채널에서 유사한 가치를 가지고 있는지 사전에 연장 분석 절차를 확인하는 것이 중요합니다. . 작은 먼지 입자, 중간 부품, 더러운 렌즈 또는 작은 아가로 오스의 대단히 짧은 시간 문제 해결을 분석할 수있는 동영상이 어렵거나 불가능하게 만들었 로컬 증가 형광에 기여할 수 세포가 서로의 위에 성장하는 경우,이 중 coverslip가되었음을 나타내고 있습니다 너무 빨리 또는 매체가 microcolony monolayers의 성장에 적합하지 않습니다 첨부. 슬라이드에 다른 세포 행복 나누기 반면 관심 세포가 지속적으로, 일찍 죽으면, 당신은 위치에서 UV - 필터를 넣어 여부를 확인 할 수 있습니다. 또한 오랜 실험 중에 노출 시간이나 빛의 강도를 감소하는 데 도움이 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 진 프레임 </td><td> ABgene </td><td> AB - 0578 </td><td> 1.7 X 2.8 cm </td></tr><tr><td> 고해상도 낮은 용융 아가로 오스 </td><td> 시그마 </td><td> A4718 </td><td></td></tr><tr><td> 큰 커버 슬립 </td><td> 여러 </td><td></td><td> 24 X 50mm </td></tr><tr><td> 원하는 경우, 멤브레인 염료 예 : FM 5-95 </td><td> Invitrogen </td><td> T23360 </td><td> 다른 멤브레인 염료도 사용할 수 있습니다 : <a href="http://probes.invitrogen.com/media/pis/mp34653.pdf">http://probes.invitrogen.com/media/pis/mp34653.pdf</a> </td></tr><tr><td> 환경 챔버와 함께 시간 경과 현미경 </td><td> 여러 </td><td></td><td> 해당 섹션에있는 장치에 대한 세부 정보를 볼 </td></tr></tbody></table>

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

지난 몇 년 동안 과학자들은 미생물의 인구 기반의 실험에서 얻은 평균 데이터는 단일 세포의 동작, 상태 또는 표현형 대표하지 않는 점차 알게되었습니다. 때문에이 새로운 통찰력 단일 세포 연구의 번호는 (최근 리뷰를 1,2,3 참조) 지속적으로 상승합니다. 그러나, 적용된 단일 세포 기술을 많은 시간에 한 구체적인 하나의 셀 (예 : 유동세포계측법 또는 표준 현미경)의 개발 및 동작을 모니터링 허용하지 않습니다. 여기서 우리는 다음과 녹음 (의 형광) 바실러스 subtilis 많은 세대를위한 성장과 분열을 통해 연쇄상 구균의 pneumoniae의 개별 세균 세포 수있는 몇 가지 최근의 연구 4, 5, 6, 7,에 사용되는 현미경 방법에 대한 자세한 설명을 제공합니다. 그 결과 영화는 하나의 공통된 조상에서 유래 인구 이내에 단세포의 역사를 다시 추적하여 phylogenetic 가계 나무를 구성하는 데 사용할 수 있습니다. 이 시간 - 저속 형광 현미경의 방법뿐만 아니라 성장, 분할 및 개별 세포의 분화를 조사뿐만 아니라, 특정 세포의 행동에 세포의 역사와 조상의 효과를 분석하는 데 사용할 수 있습니다. 또한, 시간 경과 현미경은 이상적으로 세균 세포주기 동안 유전자 발현 역학 및 단백질 지방화를 검사 적합합니다. 방법은 박테리아 세포를 준비하고 microcolony에 단일 세포의 가지를 사용하는 현미경 슬라이드를 구성하는 방법에 대해 설명합니다. 즉, 하나의 세포들은 성장 온도 제어 환경 챔버 내에서 형광 현미경으로 분리하는 아가로 오스와 보충 성장 매체로 구성된 반 고체 표면에 발견됩니다. 이미지는 특정 간격으로 캡처하고 나중에 오픈 소스 소프트웨어 ImageJ를 사용하여 분석하고 있습니다.

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

이 프로토콜은 자동 형광 시간 경과 현미경을 사용하여 시간에 다른 박테리아의 단일 세포의 행동을 모니터하기위한 단계별 절차를 제공합니다. 또한, 우리는 어떻게 현미경 이미지를 분석하는 지침을 제공합니다.

라이브 셀 이미징 바실러스 subtilis과 연쇄상 구균 pneumoniae 자동 시간 저속 현미경을 사용하여

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 조회수.  University of Groningen. 이 프로토콜은 자동 형광 시간 경과 현미경을 사용하여 시간에 다른 박테리아의 단일 세포의 행동을 모니터하기위한 단계별 절차를 제공합니다. 또한, 우리는 어떻게 현미경 이미지를 분석하는 지침을 제공합니다.

비디오: 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.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

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

연구

면역학 및 감염병학

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.

의 단일 세포 분석 바실러스 subtilis</em형광 현미경 및 유동세포계측법 사용> Biofilms

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.

라이브 세포 형광 현미경을 사용하여 박테리아 독소 유도 응답의 시각화

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.

곰팡이 병원체 Phagocytosis의 라이브 셀 비디오 현미경

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.

연쇄상 구균 폐렴으로 비강 내 식민지 화 중 염증 반응의 특성화

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.

막 극성, 멤브레인 무결성 및 세포 내 이온 농도의 모니터링 변경<em&gt; 폐렴 구균</em&gt; 형광 염료를 사용

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.

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; Quellung 반응을 이용하여

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.

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; 라텍스 응집에 의해

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.

의 생리적 반응을 측정<em&gt; 초파리</em&gt; 라이브 칼슘 이미징을 사용하여 지질 페로몬에 감각 뉴런

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.

전체의 형광 시간 경과 영상<em&gt; S. venezuelae</em미세 유체 장치를 사용&gt; 수명주기

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