贵州医科大学学报

2018, v.43;No.209(02) 160-163

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一种简便的幽门螺杆菌液体培养法的构建
A Simple Culture Method of Helicobacter pylori in Liquid Media

李雅洁;张晓怡;周建奖;赵艳;龙妮娅;陈学书;王琴容;程薇;谢渊;
LI Yajie;ZHANG Xiaoyi;ZHOU Jianjiang;ZHAO Yan;LONG Niya;CHEN Xueshu;WANG Qinrong;CHENG Wei;XIE Yuan;Key Laboratory of Endemic and Ethnic Diseases,Guizhou Medical University,Ministry of Education;Key Laboratory of Medical Molecular Biology,Guizhou Medical University;

摘要(Abstract):

目的:建立一种简便、有效的幽门螺杆菌(H.pylori)的液体培养法。方法:取脑心浸液9 m L、胎牛血清1 m L、H.pylori选择剂(万古霉素、头孢磺啶、甲氧氨苄嘧啶及两性霉素B)400μL制成H.pylori液体培养基,将H.pylori接种于H.pylori液体培养基,在厌氧罐中通过微需氧袋建立微需氧条件,于37℃振荡培养3 d,同时设立传统固体培养基培养的H.pylori作为对照;观察2种方法培养的H.pylori生长状况及H.pylori形态,比较2种方法培养的H.pylori的尿素酶、氧化酶及触酶试验结果。结果:固体培养到第3天时,H.pylori生长良好,哥伦比亚血琼脂平板上见透明、针尖样菌落形态;液体培养第3天时可见培养基明显浑浊,涂抹少量菌液于无抗生素的哥伦比亚血琼脂平板培养,肉眼未见污染菌生长,并有透明、针尖样的菌落长出;革兰染色后,固体培养基中的H.pylori为螺旋状、海鸥状、S状弯曲菌或短杆菌;液体培养基中的H.pylori则多为螺旋短杆状、亦见部分长丝状细菌,有的互相缠绕聚集成团,有的散在分布;取两种方法培养的H.pylori菌进行尿素酶、氧化酶及触酶试验,结果均为阳性。结论:成功建立一种简便、有效的H.pylori液体培养法,细菌生长良好,且H.pylori形态变长。
Objective:To develop a simple and effective liquid culture method of Helicobacter pylori(H.pylori).Methods:H.pylori was inoculated into a T25 cell culture flask with 10 m L of brain heart infusion containing 10% fetal bovine serum,and 0.4% H.pylori selection agent(including vancomycin,cefsulazidine,methicillin and amphotericin B),incubated at 37℃ for 3 days,where microaerophilic atmosphere was established by atmosphere generation systems of Thermo scientific.H.pyloris were identified by urease test,oxidase test,catalase test and Gram stain.H.pylori grew on classic solid media was as control and test results of urease,oxidase and catalase of the 2 groups were compared.Results:On the 3 rd day of culture,H.pylori grew well on solid media.On Golumbia sheep blood agar,there were needle tip-H.pylorilike and transparent bacterial colonies.Liquid culture showed obvious turbidity,and when smeared on Golumbia sheep blood agar,there were needle tip-H.pylorilike and transparent bacterial colonies growing out without contamination.Gram staining showed H.pylori on solid media appeared as heliciform,seagull-H.pylorishaped,and S-H.pylorishaped campylobacteria or brevibacteria.H.pylori in liquid media mostly showed as short screw,and some as long filaments,scattered or intertwining to cluster.The test results of enzymes from H.pylori cultured by the both 2 methods were positive.Conclusion:A simple and convenient method for H.pylori liquid culture is developed successfully.With this method H.pylori could grow well and some could have long-H.pylorishaped appearance.

关键词(KeyWords): 幽门螺杆菌;液体培养;脑心浸液;革兰氏染色;H.pylori选择剂;微需氧
Helicobacter pylori;liquid culture;brain heart infusion;Gram stain;H.pylori selective agent;microaerophilic

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31660031;31560326);; 贵阳市科技计划项目(20161001);; 贵州卫计委科技基金(gzwiki2015-1-010);; 贵州省科技合作计划项目[黔科合LH字(2015)7360]

作者(Author): 李雅洁;张晓怡;周建奖;赵艳;龙妮娅;陈学书;王琴容;程薇;谢渊;
LI Yajie;ZHANG Xiaoyi;ZHOU Jianjiang;ZHAO Yan;LONG Niya;CHEN Xueshu;WANG Qinrong;CHENG Wei;XIE Yuan;Key Laboratory of Endemic and Ethnic Diseases,Guizhou Medical University,Ministry of Education;Key Laboratory of Medical Molecular Biology,Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2018.02.006

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