这项工作是由比尔和梅琳达·盖茨基金会（PneuCarriage项目，资助52099）和维多利亚州政府的业务基础架构支持计划资助项目。由于艾琳·邓恩，珍妮特·斯特拉坎和金野兔的手稿的批判性阅读。

1。用于制备打字细胞悬液<ol><li>使用一次性无菌1微升环，取一小扫肺炎球菌生长在固体非选择性的马血琼脂的新鲜过夜纯培养，接种100微升心浸液（HI）的肉汤。悬挂应该看起来只是明显混浊。轻轻搅动管至乳化。 注：其他媒体，例如脑心浸液肉汤也可能是合适的。除了 ​​血清（10％，例如终浓度（体积/体积）的马血清），并温育于37℃下在使用前约1小时内可用于提高反应。 </li></ol> 2。检查细胞悬液密度在步骤1.1中制备的细胞悬浮液也将被用作阴性对照，并加入分型血清前应检查的细胞密度。 <ol><li>使用1微升循环，转移跌幅在步骤1.1制备的接种物到GLASs显微镜幻灯片。 </li><li>使用镊子，放置一个小的圆形盖玻片上悬挂。 </li><li>采用相位对比度设置，观察准备在显微镜放大400倍，检查悬挂的密度（ 图1A）。 <ol><li>如果接种物是过重（ 图2A），添加更多的液体培养基在细胞悬浮液，在步骤1.1以冲淡制成。再次轻轻混匀。 </li><li>如果接种物过轻，例如，有&lt;50细胞中可见的微观领域（ 图2B）中，添加更多的细菌在步骤1.1制成细胞悬浮液制剂，并轻轻混合。 注：随着经验的积累，细胞悬浮液的密度可以通过摇动和举起来的光，它应该看起来只是明显浑浊来判断。 </li></ol></li><li>重复步骤2.1-2.3，直到细胞的合适数量是在微观领域可见。 注：玻璃幻灯片和coverslIPS呈现锐器危险。使用后，载玻片和盖玻片被污染的传染性物质，必须配置成一个生物危害锐器容器。 </li></ol> 3。打字<ol><li>转移（ 例如使用1微升环）到玻璃载玻片〜1微升的准备接种。丢弃循环进入生物危害锐器容器。 </li><li>添加的室温抗血清等体积（1微升菌环）到幻灯片和混合都与循环下降好。 </li><li>使用镊子，放置一个小的圆形盖玻片上的悬挂，小心不要触碰下降。放置在悬浮液中加入抗血清后，立即盖玻片，以防止在滑动流体干燥。 </li><li>利用显微镜，观察下用400X放大率相衬的悬浮液。负荚膜肿胀反应观察时很少或没有肺炎球菌的细胞“肿胀”中可以看出，在400X倍率，类似的是，在肺炎球菌的细胞悬浮液用无血清观察到添加（阴性对照）。当细胞相比，细胞的阴性对照出现放大或“肿”和更明显的阳性荚膜肿胀反应被观察到。 注意：模棱两可的反应，应通过检查反应后5-10分钟，在室温下培养，或通过重复与更多的抗血清的反应进一步研究，例如。 </li><li>进行肺炎球菌血清型与个体的抗血清池的连续测试，直到一个积极的反应观察。另外，池可以在&#39;发&#39;取决于血清型的发病率局部应用。 注意：测试在“轮”保证含有抗体的最常见的血清型的池首先测试，最小化的努力和成本。几个池的并发测试还提供了额外的阴性对照，它可以在解译器是有用的看的旋转。如果在一个特定的&#39;圆&#39;所有的池为负，测试与随后的几轮池的分离。 </li><li>使用包含在反应池内适当的类型或组抗血清测试分离。在适当情况下，采用因子抗血清，以进一步区分血清型。 注：按键肺炎球菌血清制造商提供在适当的游泳池，类型，团体或抗血清因子和结果（包括任何交叉反应）的解释的选择使用。 </li><li>如果负的成绩与所有池获得，测试分离与Omniserum试剂（含有抗91种血清型21）。如果测试的肺炎球菌隔离与Omniserum试剂时的正荚膜肿胀反应仍然没有观察到的，这可能是因为菌株被表达很少或无包膜，或该抗体的血清中未Omniserum表示。后一类可能包括新的血清型。 </li> &lt;/ OL&gt;

<ol>
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作者什么都没有透露。

在该技术中的一个关键步骤是确保的纯培养物用于制备一起添加细菌细胞的适当数目的肉汤的悬浮液中。但应注意的是，集落生长和形态在琼脂平板上的不同肺炎球菌菌株之间变化。一些产生干燥和粗糙的殖民地，这可能很难用一个循环接机送机，不要在肉汤乳化很好。出于这个原因，制备细胞悬浮液时，可能需要细菌的较大扫描。在任何情况下，当务之急是单个细胞，并且不聚集体，是明显的负控制，使得它们的相对尺寸可以相比的细胞与抗血清中加入。此外，过多的细菌细胞，可能会导致假阴性反应，其中过量的荚膜抗原可以抑制荚膜肿胀反应10。出于这个原因，重要的是要创造一种细菌悬浮液与合适的细胞密度（ 图1）。技能提升计划退休金可以通过加入几滴浓甲醛水溶液的保存数月。这使得更容易批量测试，并在消毒液中减少了实验室获得性感染的风险;尽管这必须与福尔马林处理的安全风险，这是有毒的，腐蚀性，致癌，致敏和易燃加以权衡。此外，我们观察到一些菌株表现较弱的反应在福尔马林（数据未显示）的存在。 荚膜肿胀反应是用显微镜检查。使用相位对比增强了胶囊的可见性。在一些实验室中，反应是使用油镜在1000倍放大14,16被观看。此外，一些实验室使用一个计数器染色（亚甲蓝），以进一步增强了积极的反应10，16，22的可见性。这里介绍的方法无油我被看作mmersion下放大400倍15，17。这种方法快速，简单，比较容易阅读23。 重要的是要保持良好的内部质量控制程序，以确保抗血清和测试程序是顺序很重要。参与外部质量保证计划往往是有益的。 对肺炎球菌的血清型的荚膜肿胀反应的一个主要限制是必需的抗血清成本高。然而，这是最小化在上面的协议由于用于各反应的少量血清的。其它几种方法已经被描述为肺炎球菌血清型23和24。然而，荚膜肿胀仍然是金标准和新方法的发展需要标准化对荚膜肿胀反应23。使用适当的抗血清中，荚膜肿胀反应也可用于识别并输入其他上限ULE产生菌25。该荚膜肿胀反应是有助于当前和新兴的肺炎球菌血清型的鉴定，以及提供有关疫苗的疗效和预防接种对马车和疾病的长期影响的信息。

肺炎链球菌 （肺炎球菌）负责五岁以下的估计有80万儿童每年的死亡，疾病负担主要落在资源贫乏的国家1。肺炎球菌性疾病的局部感染，如急性中耳炎严重威胁生命的感染，如败血症，脑膜炎和肺炎2范围的严重程度。超过90荚膜肺炎球菌血清型已经描述了3，4。目前肺炎球菌疫苗可抵御负责大部分侵袭性疾病5，6的血清型。然而，广泛使用的疫苗已与取代非疫苗血清型包括在疫苗血清型的关联，无论是在鼻咽部和侵入性疾病7，8。这突出血清分型的流行病学的重要性监视和长期影响疫苗的研究9。 肺炎球菌分型的金标准方法是荚膜反应试验，被称为荚膜肿胀反应，首先通过罗芬德在1902年描述（参见奥10）。一致性程度高已经发现实验室之间，以及荚膜肿胀反应和其他血清分型方法11-13之间。还有的荚膜肿胀反应10，14-16的几个变化。这里所描述的方法并不需要使用一个计数器污点或油浸没透镜，而是一滴制备试样是与打字血清混合并在400倍放大立即检查。 通常进行使用市售的抗血清的荚膜肿胀反应。这里描述的方法使用较少的抗血清与某些其他方法16，17相比，使其更符合成本效益。一旦ISOL吃了被识别为肺炎双球菌，其依次用抗血清池测试，直到阳性反应是观察。每个池的抗血清含有抗血清的不同混合物提出对91肺炎球菌血清型18。一旦池中被建立时，个人类型和组的抗血清中所包含的反应性池按顺序逐个进行试验。型抗血清通常与单个血清型（ 例如，1型的抗血清反应与血清型1）进行反应，而基团的抗血清与所有血清型的反应，在一个特定的组（血清型22F和22A，例如 22组的抗血清起反应）。组内的某些血清型采用因子抗血清（ 如血清型22F反应与22b的因素，但不是22C）被进一步分化。在这种情况下测试是继续与所有相关因素，抗血清，直到血清型确定19，20。该荚膜肿胀反应是简单快捷14，可以在任意的l进行aboratory是配备了优质的显微镜和相应的一组抗血清。

<table border="0" cellpadding="0" cellspacing="0" width="1305">
	<colgroup>
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		<col />
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	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Neufeld) antisera</td>
			<td>SSI Diagnostica</td>
			<td></td>
			<td><a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a> 
			Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Omni) antiserum</td>
			<td>SSI Diagnostica</td>
			<td>2438</td>
			<td>Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical Microscope</td>
			<td>Leica Microsystems</td>
			<td>Leica DML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated Disposable Inoculating Loops 1 &#181;l</td>
			<td>Copan</td>
			<td>CD175S01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse Blood Agar (HBA) plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>PP2001</td>
			<td><a href="http://www.thermofisher.com.au/">http://www.thermofisher.com.au/</a> 
			Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heart Infusion (HI) broth</td>
			<td>Media Preparation Unit (MPU), The University of Melbourne</td>
			<td>40HIN</td>
			<td>Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass microscope slides 76 mm x 26 mm</td>
			<td>Lomb Scientific</td>
			<td>7101</td>
			<td>Sharps hazard: Dispose in a biohazard sharps container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular glass coverslips</td>
			<td>NeuVitro&#160;</td>
			<td>GG-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">*Excluding the antisera, all other materials can be purchased from alternative suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">*Plates prepared from defibranated sheep blood are suitable but blood plates prepared from citrated blood or human blood are not</td>
		</tr>
	</tbody>
</table>

capsular serotyping of streptococcus pneumoniae using the quellung reaction

有肺炎链球菌超过90种不同的血清型囊（肺炎球菌）。除了作为了解肺炎球菌流行病学的工具，荚膜血清型可以提供有用的信息，疫苗效力和影响研究。该荚膜肿胀反应是肺炎球菌荚膜血清分型的金标准方法。该方法涉及测试一种肺炎球菌的细胞悬浮液与汇集和特异性抗血清针对荚膜多糖。抗原 - 抗体反应的显微观察。该协议具有三个主要步骤：1）制备的菌体悬浮液，将细胞与抗血清在载玻片上的2）的混合，和3）读出用显微镜荚膜肿胀反应。该荚膜肿胀反应是相当简单的执行并且可以应用于任何地方的合适显微镜和抗血清是可用的。

<html>当型特异性抗体结合的肺炎双球菌的胶囊，从而在其折射率10发生变化时的正荚膜肿胀反应。当在显微镜下观察，细菌出现&#39;肿&#39;和更明显14。 图1A显示肺炎球菌细胞在HI肉汤。当特定类型的抗血清加入到细菌悬浮液中，肺炎球菌出现“肿”，折射和更圆的（ 图1B）。 加到肉汤太多的细菌细胞，可能会导致细胞的聚集或假阴性反应。 图2显示了接种肺炎球菌细胞在HI肉汤是“太重”（ 图2A）或“过轻”（ 图2B）的。 08fig1highres.jpg“SRC =”/ files/ftp_upload/51208/51208fig1.jpg“/&gt; 图1。阴性和阳性荚膜肿胀反应。血清型肺炎球菌9V隔离的准备，没有抗血清（1A）和第9组抗血清（1B），在放大400倍进行观察。后者显示了“消肿”和舍入细菌细胞通常出现在一个积极的荚膜肿胀反应。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> 图2。过重和过轻接种。下放大400倍观察时，肺炎球菌细胞悬液过于沉重（2A）或过轻（2B），准备工作。

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction at JoVE.com

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

该荚膜肿胀反应是血清型肺炎链球菌的金标准技术，该技术利用显微镜和具体的肺炎球菌血清和参考和研究实验室常用全世界。

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

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

capsular-serotyping-streptococcus-pneumoniae-using-quellung

Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

45.2K 次观看.  Murdoch Childrens Research Institute. 该荚膜肿胀反应是血清型肺炎链球菌的金标准技术，该技术利用显微镜和具体的肺炎球菌血清和参考和研究实验室常用全世界。 

视频: Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

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.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

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

科研

免疫与感染

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

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

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

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

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

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.

肺炎链球菌在鼻内殖民期间炎症反应的特征

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.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

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 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.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

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

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.