噬菌体的分离纯化及其效价测定
分别将五份污水样品进行富集，次日经离心、过滤除菌进行噬菌体的分离，37℃放置过夜后，用来作无菌检查。用单斑法进一步证实噬菌体的存在。于牛肉膏蛋白胨琼脂平板上滴加一滴（150ul）大肠杆菌悬液，用高压蒸汽灭菌后的玻璃涂布器将滴加的菌液涂成一薄层，使其均匀，分散滴加几滴过滤后的滤液，并做标记，12h后经证实，只有校外污水河、水池样本有噬菌斑出现。进而制备高效价的噬菌体，进而进行效价测定。为了准确测定噬菌体效价，采用了两种方法，分别是试管法（以在液体试管中能引起溶菌现象的最高稀释度表示）和琼脂平板法（平板菌苔表面形成的噬菌斑数换算为每毫升样品中的噬菌体数表示）[1]。琼脂平板法分为双层法和单层法。本实验为噬菌体分离纯化的探索性实验，结果并不明显。双层琼脂平板法中，只有10-2稀释度的滤液中出现9个噬菌斑。39120
毕业论文关键字：噬菌体；大肠杆菌；分离；纯化；效价；琼脂平板
Purification and titer of phage
Abstract:This paper introduces the method of separating coli phage from dirty river ,the lake in the park ,ground , pool, and Off-campus sewage river. Illustrates the separation of e. coli phage significance and application value.
The five water samples were enriched ,the next day,after centrifugation,in order to separation phage ,I use the the method to filter sterilization 37℃ place overnight to check for sterility. After aseptic examination no bacterial growth of filtrate further confirmed the existence of phage.Dropping to beef extract peptone agar plate drop (150ul) E. coli suspension,then use sterilized glass coating machine to bacteria liquid coating into a thin layer evenly, add a few drops filtrate,and marking,12 h later proved that the only external sewage river, pond plaque samples occur.Further purification of bacteriophage were using double-plate method,just purified from phage titer is not high, the need for preparing high titer phage titer determination and then conduct. In order to accurately detect phage titer, adopted two kinds of methods, respectively is in vitro method (in the liquid can cause lysis phenomenon is the highest degree of dilution) and AGAR plate method (tablet bacterium moss on the surface of the formation of the plaque number conversion per ml of sample number of phage said).Agar plate method pided into double and single law. In this study, separation and purification of bacteriophage exploratory experiments, the results are not obvious.Double agar plate method, the filtrate was only 10-2 dilution occurs nine plaques.
Keywords:Phage;Escherichia coli;Separate;Purification;Titer;Agar plate
目 录
摘 要1
1 绪论2
1.1引言2
2 实验部分.2
2.1 实验器材.2
2.2 实验步骤.3
2.2.1材料.3
2.2.2噬菌体的分离...3
2.2.3生物测定法 4
2.2.4噬菌体的纯化 5
2.2.5高效价噬菌体的制备 6
2.2.6噬菌体效价的测定 6
3 结果与讨论 8
4 存在的问题及展望 9
5 总结 9
参考文献 11
致谢 12
噬菌体的分离纯化及其效价测定1 绪论
1.1 引言
1917年法国的Felix Herelle发现含有志贺疾杆菌的新鲜液体培养物能被某种无菌的污水滤液溶解，并且液体培养物由浑浊变澄清，他将这种能使浑浊液体培养物变澄清并使菌落形成透明的斑的溶菌因子定义为噬菌体[2]。
噬菌体具有体积微小、结构简单和严格的寄生性，在自然界分布极广，是一类侵害细菌和放线菌的病毒，凡有细菌的地方，都可能有其对应的噬菌体，并且噬菌体对宿主细胞具有严格的寄生性，且必须在活的敏感的细菌体内进行增殖，并能将菌体裂解[3]。了解噬菌体的这些特性后，能快速检查、分离，并进行效价的测定，对在生产或者科研工作中出现的噬菌体污染具有重要意义[4]。为了易于分离可先经富集培养，使样品中的噬菌体数量大量增加。采用生物测定法进行噬菌体检查，约需12h左右，该法能准确判断是否有噬菌体污染。本实验进行噬菌体的分离，并采用生物测定法来进行确证[5]。
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