This work was part of the PneuCarriage project, funded by the Bill and Melinda Gates Foundation with contribution by the Victorian Government&#8217;s Operational Infrastructure Support Program.

1，准备内部乳胶试剂<ol><li>甘氨酸缓冲液的制备（GBS） <ol><li>添加1.5克甘氨酸，11.7克氯化钠和100ml蒸馏水的200ml玻璃烧杯中。 </li><li>混合使用磁力搅拌器以使其溶解。 </li><li>转移混合物至500毫升的量筒中，加入蒸馏水，使总量为200ml。 </li><li>将溶液转移回200毫升烧杯中，检查pH值调整到8.2用氢氧化钠5酸钠。注：氢氧化钠有腐蚀性，穿戴个人防护装备。 </li><li>过滤消毒用0.22微米的过滤器和几个60毫升注射器将溶液倒入250毫升preautoclaved螺口玻璃瓶。 </li><li>储存在室温下。 </li></ol></li><li> GBS的含0.2％牛血清白蛋白的制备<ol><li>加入0.2克牛血清白蛋白（BSA）的粉末和100毫升GBS的一个200ml的玻璃烧杯中，并混合使用磁力搅拌器以使其溶解。 </li><li>过滤器使用多个60毫升注射器成250毫升preautoclaved螺口玻璃瓶消毒，通过0.22微米的过滤解决方案。 </li><li>保存在4℃。 </li></ol></li><li>乳胶试剂的配制<ol><li>标签2毫升圆底离心管中。 注：圆底试管用于减少颗粒沉降可能影响抗体附着。 </li><li>使用P1000的吸管无菌针头加975微升的GBS进入离心管中，再加入25微升合适的肺炎球菌血清的乳胶试剂正在准备（ 即池，组，类型或因素）。翻转5次混合。 </li><li>通过加入120微升的胶乳颗粒以1080微升的无菌生理盐水稀释的聚苯乙烯胶乳颗粒1:10。 注意：这可以在更大的体积制成，但新鲜每一胶乳试剂的制备时应当作出。 </li><li>增加1000微升1:10胶乳悬浮液（在步骤1.3.3的方法制备）向1，000微升一点40的抗血清悬浮液（在步骤1.3.2的方法制备）。翻转5次混合。 </li><li>孵育于37℃在缓慢旋转的车轮（ 例如，每分钟4转38.5厘米直径的一个车轮）2小时。 </li><li>离心15分钟，在1100×g下。弃去上清液，并轻轻重悬沉淀于2ml无菌生理盐水。 </li><li>离心15分钟，在1100×g下。弃去上清液，并加入1 ml的GBS，轻轻重悬沉淀。 </li><li>吸取乳胶悬浮到标签5毫升螺口管。添加另一个1毫升GBS的。 </li><li>加入含GBS 0.2％BSA（重量/体积）（pH值8.2）（在步骤1.2的方法制备）的2毫升添加10％的叠氮化钠作为防腐剂（重量/体积）溶液40微升。 注：叠氮化钠是危险的，如果吸入，并且是皮肤和眼睛有刺激性。希望购买一个准备好的10％的溶液，而不是粉末状的，因为这可以减少吸入暴露的风险。个人防护设备（包括手套的ð防溅护目镜）应该处理该试剂时，必须佩戴。请参阅材料安全数据资料。 </li><li>店内的乳胶试剂在4℃。 </li></ol></li><li>阴性对照乳胶试剂的制备<ol><li>标签2毫升圆底离心管中。 注：圆底试管用于减少颗粒沉降可能影响抗体附着。 </li><li>使用P1000的吸管无菌针头加975微升的GBS进入离心管中，再加入25微升的正常兔抗血清。翻转5次混合。 </li><li>请按步骤1.3.3至1.3.10以上</li></ol></li></ol> 2，质量控制乳胶试剂（QC） <ol><li>使胶乳试剂至室温。在即将使用前，轻轻颠倒离心管几次混合胶乳试剂。 </li><li>检查试剂自身凝集吹打15微升到显微镜载玻片上，并继续按照步骤4.7和4.8以下。应该有没有Agglutination的胶乳粒子。该试剂应出现白色，光滑。 </li><li>测试的低传肺炎球菌菌株的面板试剂。利用制成新生长的文化在步骤3中分离的面板应包括“目标”的血清型菌株（用于试剂的特异性血清型进行测试）和“非目标”血清型（其他血清型的通常不应越过株与试剂发生反应）。包括每个目标和非目标的血清型为每个胶乳试剂进行质量控制中的至少一个分离物。如果只有一个目标或非目标血清型，试验两种不同的特定血清型的菌株。 注：测试小组应包括几个隔离每个血清型，其中有些是罕见的侵袭性疾病和/或类型的文化藏品。最好是包括一些托架分离物，因为它们常常是更苛刻的血清型（数据未示出），从而进一步测试容量øF表示试剂，确保试剂有可能能够血清分型均创和分离运输的。 </li><li>继续进行的乳胶试剂与靶和非靶的肺炎球菌菌株的检测按照在步骤3和4下面描述的方法。 </li><li>质量控制试剂在生产的时间，然后以后每年最少。 注：如果试剂没有通过质量控制，批处理必须扔掉，新配制而成。中的重复QC故障的情况下，推荐使用的试剂使用Ortika 等人 ，2013 8，并在下面的讨论中所描述的其他方法来制备。 </li></ol> 3，准备培养肺炎球菌血清型为中<ol><li>用无菌环选择肺炎球菌的单个菌落 隔离和条纹这一点在固体非选择性的血液琼脂以使初级接种覆盖大约三分之一的板面。条痕为单菌落。 注：从脱纤维马或羊的血制备平板都适合;但准备与人类血液或柠檬酸盐动物血血板都没有。 </li><li>在5％CO 2气氛中孵育板在o / n 37℃。 </li><li>观察在板上纯度的生长，并评估是否有足够的生长对血清分型。 注：肺炎球菌菌株的纯培养所需的血清型。根据我们的经验，汇合，或近汇合生长覆盖大约三分之一的板表面的通常是足以完成血清分型。由于肺炎球菌的文化可能会有自溶，血清分型准备的文化应该在传代培养24小时进行测试。不要把培养皿出的孵化器超过15〜20分钟的血清分型开始前。 </li></ol> 4，开展乳胶凝集血清分型<ol><li>从冰箱中取出所有的乳胶试剂，使他们能够升温到室温约imately 30分钟。临用前，轻轻颠倒试管混合试剂。 </li><li>标签载玻片。 </li><li>将15微升阴性对照乳胶试剂到显微镜载玻片。 </li><li>使用一次性无菌1微升循环利用的肺炎球菌文化的横扫，这样的循环大约是半满的。 </li><li>轻轻涂抹接种到滑动接近阴性对照胶乳试剂的液滴的表面上。接种物应该是在载玻片上可见。 </li><li>快速，彻底混合接种到使用环阴性对照胶乳试剂。确保降不进一步蔓延比其初始大小。 </li><li>拿起滑板，拿着它在两端。摇动来回滑动，持续1分钟。注意保持液体缓慢地移动，并确保液滴的尺寸也不会增大。 注：或者使用板摇杆。 </li><li>观察的宏观凝集（ 即通过裸眼），并在黑色背景上悬挂的结算。 </li><li>如果阴性对照胶乳试剂不显示凝集或结算继续测试培养，在步骤4.1到4.8以上的取代试验胶乳试剂用于阴性对照胶乳试剂。清新的循环，必须每次都使用。着手开始与池乳胶试剂的测试。 </li><li>直到获得阳性结果连续测试每个&#39;池&#39;乳胶试剂。 注：可在&#39;轮&#39;做了池测试的，以反映特定血清型（局部发生，例如，通过测试的第一张幻灯片的三个最可能的游泳池如果没有得到积极的反应，然后进行测试接下来的三个最有可能在第二滑动其余池，等等，直到获得肯定的反应）。这确保了最常见的血清型第一检验，最大限度地减少所需的时间和试剂。 </li><li>使用抗血清制造商的主要决定哪些个人抗血清的代表在正池。 </li><li>测试每一个包含在正极池单独确定“组”或“类型”的组或类型的如在步骤4.1到4.8以上。 </li><li>确定的结果作为参考，以抗血清生产商的关键。如果一个“类型”被确定（ 例如，血清型5），然后不进行进一步的测试是必要的，这个结果被记录下来。 </li><li>如果一个“组”被确定（ 例如，组19），则指的是制造商密钥，以确定待测试的因素。继续测试与个体的因素“胶乳试剂，并确定最终的血清型（ 例如血清型19B）。 </li></ol>

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The authors have nothing to disclose.

乳胶凝集是肺炎球菌血清型的简单，快捷，廉价的方法。商业肺炎球菌乳胶凝集试剂可用，但他们目前还无法区分所有已知的肺炎球菌血清型10,12。然而，胶乳凝集试剂可以容易地在内部使用针对特定肺炎球菌荚膜抗原购抗血清制备。在这里所描述的方法中，抗体被被动地附着到胶乳颗粒，使一组的胶乳凝集试剂。 当结合到乳胶粒子的特异性抗体连接到抗原上的肺炎球菌的多糖胶囊隔离18,19所产生的正极胶乳反应。附着于抗体的胶乳颗粒允许在不放大到被可视化的特定抗体 - 抗原反应。 乳胶凝集方法适宜于高通量实验室的D还为资源贫乏。胶乳凝集法的主要优点是，没有专门的设备需要执行的测试，试剂具有至少2年的保质期储存于4℃下8时，并且它是便宜的，简单的和快速的执行。通过胶乳凝集血清型肺炎球菌菌株需要大约10分钟，和最多四个胶乳试剂可以在一个滑动并行进行测试。此外，如果血清型的流行率是已知的相关流行病学设定，测试可以在轮开始用含有最常见的血清型相似于荚膜肿胀试验17的池进行。这种方法可以减少所需要的，以确定血清型的测试次数。该方法也不同运营商之间的高度重现性（数据未示出）。乳胶凝集血清型的缺点是生产的试剂是费时，服用约4小时;虽然多个reage核苷酸可以很容易地在平行来制造。此外，一些抗原在不同的肺炎球菌血清型共同的，从而导致交叉反应与某些抗血清（ 例如 ，血清型29，42和35组）。然而，这些交叉反应是公特征在于使用市售的抗血清（通常是预吸附以除去更多的问题交叉反应的抗体）和额外的反应表由生产商以区分哪个血清型存在时提供。胶乳试剂也可交叉反应，如果抗体不上的胶乳颗粒适当地对准;这些被检测为QC故障。根据我们的经验，严格的质量控制是至关重要的这一方法的成功，即使市售的抗血清用于生产。 QC大约需要15分钟每试剂和依赖株上述一组适当的质量控制。 这里介绍的方法将导致试剂给予了积极的反应以及无线网络薄的测试周期。根据我们的经验，误报实际上很罕见的（数据未示出）。偶尔的假阴性反应的观察;通常这些涉及到已知的问题与特定的抗血清（ 如血清型6株可能与乙组抗血清反应），或胶囊的表达下调的分离。使用由制造商提供的血清分型algrorithm也是重要的，因为在大多数情况下，假阳性或阴性反应会导致一个“盲端​​”的结果。在这些情况下，当反应是很难解释，我们建议重复检验和/或测试用的另一种方法，如荚膜肿胀反应。 有些故障是偶尔需要作出令人满意的乳胶试剂。在这里所描述的方法中，该抗体通过被动吸附15,19附着到胶乳颗粒，所以关键的变量是抗体的使用浓度，它决定如何很好胶乳粒子的表面被覆盖和的抗体的活性位点15,20随后对准。因此，如果过量或不足的抗体附着到胶乳颗粒中，抗体-抗原反应，将不会是最佳的，并不能令人满意的试剂可产生15,20,21。作为抗体效价不同批次的抗血清之间变化，一套标准稀释液可能不会产生对执行好的8试剂。一个有用的出发点是用1:40稀释的抗血清，如果这个不成功，进一步稀释血清。根据我们的经验，这通常可以解决问题8。在少数情况下试剂可能会显示弱凝集未伴有暂停结算时进行测试，目标菌株。在这些情况下，稀释的抗血清，并不能改善该试剂的质量，但令人满意的试剂通常可以通过省略离心来制备和洗涤步骤8,22 </sup&gt;。 抗体包被的胶乳颗粒通常用在许多不同的微生物15,20的检测，鉴定或血清分型诊断微生物学。因此，这里所描述的方法可以是适合于制备胶乳试剂用于其他目的，提供适当的抗血清可与适当的质量控制程序被通过。

肺炎链球菌 （肺炎球菌）是发病率和死亡率在儿童的重要原因五岁以下的旧世界各地，特别是在资源贫乏1,2。从局部感染到危及生命的疾病如肺炎，败血症肺炎球菌疾病的范围和脑膜炎1,2。 超过90种血清型肺炎链球菌已根据其荚膜多糖3的差异识别。当前的疫苗针对荚膜多糖和从引起侵袭性疾病4的主要血清型提供保护。血清型肺炎球菌菌株是评估疫苗的运输和疾病的影响，并提供更广泛的流行病学资料5,6重要。目前的“金标准”方法，肺炎球菌血清型的荚膜肿胀试验，但它是耗时的，需要一些技巧来PERFORM 7。乳胶凝集是推荐世界卫生组织7的替代血清分型方法。乳胶凝集快速和简单的执行，并在全球范围内8-11应用在许多实验室。重要的是，乳胶凝集显示精度相媲美的荚膜肿胀试验对肺炎球菌血清型10,12-14。总体来说，这种方法非常适合于资源贫乏，以及高通量实验室。 胶乳凝集试剂（“胶乳试剂”）由抗体的附着到胶乳颗粒15产生。在积极的反应，这些标记颗粒凝集的特异性抗原的存在。商业乳胶试剂可用于血清型的范围限制。在内部使用市售的抗血清10胶乳试剂，也可制得。抗血清的混合物（“池”）以及特异性抗血清对肺炎球菌šerogroups（ 例如，组19），从个人的血清型（ 例如血清型5），并以特定的抗原（“因素”， 如 ，因子19C识别血清型19A），用于进一步定义内组的血清型可用16。 该原稿概述了使用被动附着市售的抗血清到胶乳颗粒在生产胶乳试剂的主要步骤。该方法的质量控制（QC）方面进行描述和协议的使用胶乳试剂的肺炎球菌血清型被包括在内。

<table border="0" cellpadding="0" cellspacing="0" width="1085">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:263px;">Comments/Description</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:299px;">Microparticles based on polystyrene, 800 nm</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:127px;">65984-10 ml-F</td>
			<td style="width:263px;">Or other volumes as required</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:299px;">Normal rabbit serum</td>
			<td style="width:180px;">Antibodies Australia&#160;</td>
			<td style="width:127px;">NRS-1ml</td>
			<td style="width:263px;">Or other volumes as required, bring to room temperature before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:299px;">Pneumococcus (Neufeld) antisera</td>
			<td style="width:180px;">SSI Diagnostica</td>
			<td style="width:127px;">Various</td>
			<td style="width:263px;">Bring to room temperature before use 
			<a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:299px;">Sterile Bovine Albumin&#160;</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:127px;">A-4503</td>
			<td style="width:263px;">Bring to room temperature before use</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:299px;">Sodium azide 10% (v/v) solution&#160;</td>
			<td style="width:180px;">VWR International</td>
			<td style="width:127px;">ROAC3902/100ml</td>
			<td style="width:263px;">Caution: hazardous if inhaled; skin and eye irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Eppendorf Safe-Lock tubes, 2 ml</td>
			<td style="width:180px;">Eppendorf</td>
			<td style="width:127px;">0030 120.094</td>
			<td style="width:263px;">Round bottom</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium chloride</td>
			<td style="width:180px;">Merck</td>
			<td style="width:127px;">10241.AP</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">Calibrated disposable inoculating loops 1 &#181;l</td>
			<td style="width:180px;">Copan</td>
			<td style="width:127px;">CD175SO1</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:299px;">Glass microscope slides 76 mm x 26 mm</td>
			<td style="width:180px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">LBS 2950RC</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">5 M NaOH</td>
			<td style="width:180px;">BDH</td>
			<td style="width:127px;">10252</td>
			<td style="width:263px;">Caution: highly corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glycine</td>
			<td style="width:180px;">Merck</td>
			<td style="width:127px;">10119.05</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Horse Blood Agar (HBA) plates</td>
			<td style="width:180px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">PP2001</td>
			<td>Bring to room temperature before use 
			<a href="http://www.thermofisher.com.au/">http://www.thermofisher.com.au</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Rotating wheel</td>
			<td style="width:180px;">Wyble Engineering Development Corporation</td>
			<td style="width:127px;">Not available</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:299px;">Polystyrene flat bottom screw cap tube, 5 ml</td>
			<td style="width:180px;">Technoplas</td>
			<td style="width:127px;">S5016SU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">60 ml syringes without needles</td>
			<td style="width:180px;">Terumo</td>
			<td style="width:127px;">SS-60L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Millex-GP syringe filter unit, 0.22 &#181;m</td>
			<td style="width:180px;">Merck Millipore</td>
			<td style="width:127px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Brochure on Neufeld antisera</td>
			<td style="width:180px;">Statens Serum Institut</td>
			<td style="width:127px;"></td>
			<td>Key to determining serotypes 
			<a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Key to pneumococcal factor serum</td>
			<td style="width:180px;">Statens Serum Institut</td>
			<td align="right" style="width:127px;">18058</td>
			<td style="width:263px;">For determining serotype within Groups 
			<a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:180px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:180px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">*alternative sources are available for most materials</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

当附连到乳胶粒子类型特异性抗体结合的肺炎球菌的胶囊，以及抗体标记颗粒的凝集随之而来15所产生的正极胶乳反应。 图1A示出了积极的反应，该反应的特征在于，所述背景的可见的凝集及结算悬挂。负反应的特点是胶乳凝集试剂悬浮其余光滑和白色（图1B）。所用试剂进行了优化以便产生积极的反应应在1分钟的时间间隔（通常在大约20秒可检测的）的端部之前被观察到。我们不建议阅读1分钟的时间间隔后的反应。很偶然，反应显示周围不伴有清除背景悬挂，或出现&#39;粘性&#39;没有背景清算下降的边缘弱凝集（数据未显示）。这是最像<html>立法院消极的反应。在这种情况下，建议重新测试和/或使用另一种血清型的方法（例如，荚膜肿胀反应17）进一步的调查。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51747/51747fig1highres.jpg" width="500" /> 图1：正面和负面的乳胶凝集反应。肺炎球菌的分离物制剂混合乳胶凝集试剂呈现阳性反应（A）或阴性反应（b）。凝集伴有背景的结算中的阳性反应（A）中可以看出。有不可见的凝集反应与阴性对照胶乳试剂或在负测试反应（B）中 。照片8使用许可。“目标=”_ blank将“&gt;请点击这里查看该图的放大版本。

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination at JoVE.com

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

乳胶凝集试验是血清型肺炎链球菌简单，快速，廉价的方法，并且也被广泛应用在诊断微生物。这份手稿描述了内部生产的乳胶凝集试剂的质量控制程序和该技术的肺炎球菌血清型的应用程序。

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 ...

capsular-serotyping-of-streptococcus-pneumoniae-by-latex-agglutination

Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

21.7K 次观看.  Murdoch Childrens Research Institute. 乳胶凝集试验是血清型肺炎链球菌简单，快速，廉价的方法，并且也被广泛应用在诊断微生物。这份手稿描述了内部生产的乳胶凝集试剂的质量控制程序和该技术的肺炎球菌血清型的应用程序。 

视频: Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

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

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

脊髓灰质炎病毒识别粒子凝集法

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

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 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.

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 ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

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, ...