Name: protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination
Uploaded: 2007-07-04T00:00:00
Duration: 14 min 10 s
Description: Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

A.配子体文化的制备： <ol><li>把新鲜的人体血液和血清37℃水浴中。 </li><li>在通风橱中，在无菌条件下，吸管1毫升热血到Eppendorf管，。 5分钟2200转，在离心机旋转的血液。红细胞应颗粒;删除和丢弃明确血清。与同等体积的一个新的血清3次清洗erythocytes获得的配子体的全血。 </li><li>检索的CO 2培养箱或蜡烛JAR的配子体的文化。应保持这种文化上的RPMI媒体和美联储文化的第16天的蚊子。要确定gametocytemia％（文化的第16天），使一滴血的载玻片上薄涂片染色执行Geimsa。在寄生虫文化领域的数量确定的配子。它通常范围为2 - 4％。全血，你将需要达到理想的最终％gametocytemia（0.3 -1％）计算量。 </li><li>除去大部分用吸管从您的配子体文化传媒，小心不要打扰或吸了寄生虫（寄生虫会显示为黑色，而媒体将浅粉色/红色）。吸取15毫升猎鹰管媒体和寄生虫，其余。在离心机中旋转5分钟2200转。寄生虫和红细胞沉淀，去除，并丢弃上清媒体，而不会干扰寄生虫。 </li><li>准备全血的数量（从步骤A2）相结合的寄生虫，给你所需的最终gametocytemia根据以前的计算。吸取到调匀。 （〜1分钟） </li></ol>现在，这是你养活你的蚊子样本。在水浴保持在37℃。 B.饲养蚊子 ： <ol><li> 按蚊 （30-50）放入蜡内衬纸板盖杯网状扣除橡皮筋或纸板盖杯提供担保。这一步，​​我们使用电池供电的抽吸。 </li><li>准备橡皮管插入喷嘴玻璃馈线两侧循环水浴。许多馈线连接，你需要一系列系列的一端应连接管的循环水浴（的一部分，推动水通过馈线），该系列的另一端应包括橡胶管淹没在洗澡，让循环水，空回浴。打开洗澡，直到水约37℃的水是循环通过馈线和洗澡。这是保持血液的温暖和保持寄生虫的生存至关重要。 </li><li>准备通过拉伸周围的玻璃膜料机，使用压力密封紧密接驳开放膜（拉伸封口膜，我们用小方块）。这种膜会模仿皮肤。 </li><li>将一个接驳膜侧到每个蚊子杯和安全使用夹具或磁带馈线。膜应该坐下来与净冲洗，使蚊子可以从内杯。 </li><li>仔细移液器寄生虫血粉到馈线的脖子（从步骤A5），确保血液经过的脖子一路进了水库，接触膜，使蚊子可以进入血液。我们使用约200μL每个饲养者的血液，但也存在变化。 </li><li>让蚊虫饲料。我们让她们能够养活≈30分钟（可变theexperiment;大多数实验，偶尔监测有多少人已经采取的一餐，在腹部的血液中的含量。） </li><li>喂奶后，去除蚊子杯进料器。关闭水浴，从管断开馈线。 </li><li>取出膜，浸泡在10％漂白，去污，用清水冲洗进料器。有何污染的材料（膜，纸巾，手套等），到一个小的生物危害袋和一个大的生物危害插座存款。 </li><li>放置在冰箱或冷藏室杯麻醉蚊子。一旦蚊子是完全麻醉，在冰桶中嵌入一个培养皿转移。排序的蚊子，仔细寻找任何一个血粉迹象的腹部，并丢弃那些没有不喂。有些蚊子会完全充血，而有些则表现出窄带摄入血，全都是“喂”可以接受。把喂蚊子放回纸板杯。 </li><li>蚊子提供一种糖餐（一棉片浸在10％蔗糖），在正常insectary条件下孵育7天。每天，蚊子，应监测的死亡率。此外，棉花应与蔗糖湿，让他们没有饭吃。这是更好，但添加一纸毛巾浸泡在蒸馏水中，以保持潮湿，蚊子。这些蚊子是P。 falcipraum感染，所以必须采取所有关心，所以没有蚊子亚太经社会E杯。这是最好的，把一个小cagefor双重保护杯。 </li></ol> C.解剖蚊子肠和估计卵囊负载： <ol style="margin-top: 0in"><li style="tab-stops: list .5in">吸蚊（步骤B）纸板杯使用电池供电的抽吸。麻醉蚊子，这里面可移动的抽吸水库，寒冷的房间，或埋在冰水库至少5分钟，或直至蚊子停止移动。 </li><li style="tab-stops: list .5in">麻醉蚊子从水库转移到玻璃培养皿中嵌入冰。蚊子传播的单层和覆盖用塑料培养皿盖子。不能上的菜可容纳的蚊子停留在水库，这是返回到冷室或冰。 </li><li style="tab-stops: list .5in">用细尖的镊子，从盘传输单一的蚊子100 UL PBS下​​安装一个解剖镜下玻片上。离开冷冻培养皿中所涉及的其他人员。 </li><li style="tab-stops: list .5in">用细尖的镊子，在胸部和腹部后举行的蚊子。除了拉的身体，直到肠暴露。使用镊子，去除gut.The肠道多余的组织，看上去就像一张白纸，SAC般的身体。删除多余的组织，从一个12孔的玻片上，沉​​浸在5-10微升0.2％红汞gut.Mount肠道。壁球的胴体和其他蚊子，直到12井的幻灯片潮湿的纸张towel.Continue丢弃已满。玻璃盖玻片覆盖安装的胆量。 </li><li style="tab-stops: list .5in">光显微镜下检查的胆量，为圆形的卵囊，对清除/淡淡的粉红色的肠道组织，明亮的粉红色污点。计数卵囊，记录，并继续下一个样品。继续为所有的蚊子，从所有的试验性治疗。 </li><li style="tab-stops: list .5in">完成后，用纸巾擦去幻灯片清洁。将与胆量的纸巾，纸与尸体毛巾，小生物危害袋，并丢弃在垃圾桶。戴手套是整个过程。护理是不要让任何受感染的蚊子逃生。 </li></ol>

<ol>
	<li>Avila, S. L., Ferreira, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+diagnosis:+a+review.">Malaria diagnosis: a review.</a> Braz J Med Biol Res. 29 (4), 431-443 (1996).</li><li>Bell, A. S., Ranford-Cartwright, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+PCR+assay+for+quantifying+Plasmodium+falciparum+infections+in+the+mosquito+vector.">A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.</a> Int J Parasitol. 34 (7), 795-802 (2004).</li><li>Collins, K. M., Hochberg, L. P., Ryan, P. R., Collins, W. E., Wirtz, R. A., Ryan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Plasmodium+malariae+infection+in+mosquito+vectors.">Quantification of Plasmodium malariae infection in mosquito vectors.</a> Ann Trop Med Parasitol. 98 (5), 469-472 (2004).</li><li>Czeher, C., Labbo, R., Djibrilla, A., Here, L., Arzika, I., Duchemin, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA+study+of+oocyst-sporozoite+transition+in+malaria+vectors.">ELISA study of oocyst-sporozoite transition in malaria vectors.</a> Parasite. 13 (3), 257-261 (2006).</li><li>Haghdoost, A. A., Mazhari, S., Bahadini, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+results+of+light+microscopy+with+the+results+of+PCR+method+in+the+diagnosis+of+Plasmodium+vivax.">Comparing the results of light microscopy with the results of PCR method in the diagnosis of Plasmodium vivax.</a> J Vector Borne Dis. 43 (2), 53-57 (2006).</li><li>Patsoula, E., Spanakos, G., Sofianatou, D., Parara, M., Vakalis, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-step,+PCR-based+method+for+the+detection+and+differentiation+of+Plasmodium+vivax.">A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax.</a> Ann Trop Med Parasitol. 97 (1), 15-21 (2003).</li><li>Osta, M. A., Christophides, G. K., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mosquito+genes+on+Plasmodium+development.">Effects of mosquito genes on Plasmodium development.</a> Science. 303 (5666), 2030-2032 (2004).</li><li>Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Dimopoulos, G., Kafatos, F. C., Adams, J. H., Fujioka, H., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Plasmodium+and+Anopheles+transcriptomes+during+oocyst+differentiation.">Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation.</a> J Biol Chem. 279 (7), 5581-5587 (2004).</li></ol>

可以在疟原虫感染及感染的表型测定的一些变化和不同的实验室可能遵循不同的技术。

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

一旦一个基因被确定潜在的疟疾耐火材料，必须进行评估，防止在疟原虫感染的蚊子的作用。该协议说明了如何程度上可以检测疟原虫感染的蚊子。准备配子体的文化，膜喂养蚊子人体血液，并化验在蚊子中肠病毒滴度的技术演示。

Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

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