幽门螺杆菌菌株存活能力及常温传递方法
1中国预防医学科学院流行病学微生物学研究所腹泻病研究室 102206
2上海医科大学华山医院消化内科 上海市 200040
张建中,男,1964-01-05生,山东省东营市人,汉族. 1984年山东医科大学医学系毕业,1992年中国预防医学科学院硕士,副主任,副研究员,主要从事幽门螺杆菌相关研究,承担全国幽门螺杆菌协作组菌株库的构建和管理工作.
项目负责人 张建中,102206,中国预防医学科学院流行病学微生物学研究所腹泻病研究室,北京市昌平县流字5号02室.
Correspondence to Dr. Jian-Zhong Zhang, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Changping, Beijing 102206, P.O.Box 5, China
, http://www.100md.com
Tel. +86·10·61739456,\ Fax. +86·10·61739456
E-mail: Helico@public.bta.net.cn
收稿日期 1998-07-06
Storage and transportation of Helicobacter pylori at room temperature
Jian-Zhong Zhang1, Xiu-Gao Jiang1, Jing-Jing Chen1, Zhao-Jun Sun1, Tao Sheng1 and Zhi-Fang Li2
, http://www.100md.com
1Diarrhea Disease Lab, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing 102206, China2Huashan Hospital, Shanghai Medical University, Shanghai 200040, China
Abstract
AIM To establish an easy and useful transporting method of H.pylori isolates.
METHODS Three newly cultured H.pylori strains (NCTC 11637, NCTC11639 and CAPMN62) were suspended with H.pylori storage solution (containing 100g/L sucrose and 500mL/L new bovine serum) to 1013cfu/L, put into 1.5mL bottles in half and full amount respectively and airproofed. All bottles were placed under 37℃ condition, and inoculated into culture media (Brucella agar with 100mL/L sheep blood) after 12, 24, 36, 48, 60, 72, 96, 120, 144 and 168 hours respectively. The H.pylori growth was observed 3 days after culture at microaerophilic environment (100mL/L CO2, 50mL/L O2 and 850mL/L N2). On the other hand, a bottle of H.pylori (NCTC 11637 strain, 1013 cfu/L) was transported by express mail service (EMS) from Beijing to Shanghai (room temperature 20℃-34℃). The organism was subcultured immediately after arrival and at various interval.
, http://www.100md.com
RESULTS At 37℃, H.pylori can survive at least 72 hours, and addition of full amount of storage solution can prolong the survival of Hp significantly. At room temperature (20℃-34℃), the H.pylori well survived after EMS transportation and in the period of 55 hours.
CONCLUSION With H.pylori storage solution (100g/L sucrose and 500mL/L new bovine serum), H.pylori isolates can be transported by EMS under room temperature successfully, and it is suitable for all hospitals and labs in our country.
, 百拇医药
Subject headings Helicobacter pylori; bacterial storage; bacterial transportation
Zhang JZ, Jiang XG, Chen JJ, Sun ZJ, Sheng T, Li ZF. Storage and transportation of Helicobacter pylori at room temperature.
Huaren Xiaohua Zazhi,1998;6(12):1077-1078
摘要
目的 观察幽门螺杆菌(Helicobacter pylori,Hp)在常温保存液中的存活能力及影响因素,通过实际菌株传递实验,为我国Hp菌株库建立中各地Hp的收集和供应,提供切实可行的菌株传递方案.
, 百拇医药
方法 取新鲜培养3d的Hp 3株(NCTC 11637,NCTC11639和CAPMN62),用Hp保存液(含10g/L蔗糖,500mL/L小牛血清)作成1013cfu/L浓度的菌悬液,分别以满管和半管形式装于1.5mL灭菌小管中,经密封后放于37℃恒温箱中,于12,24,36,48,60,72,96,120,144,和168h,按每种各取出一管,接种双份平皿(100mL/L羊血布氏琼脂)后,于微需氧环境培养3d后观察细菌生长情况. 另外,将一满管1013cfu/L浓度的Hp用特快专递(EMS)自北京传送到上海,收到后马上进行接种,并连续观察传递管中菌株在不同时间内的存活情况.
结果 在37℃保存液中Hp至少可存活72h,保存管中通过加满保存液减少空气残留,能明显延长Hp的存活时间;在气温20℃~34℃条件下,经EMS实际传递的菌株存活良好,且在传递到达后可继续存活至55h.
, 百拇医药 结论 使用含10%蔗糖的50%小牛血清细菌传送保护液,在常温下通过EMS进行国内Hp的菌株传递能满足我国各地菌株传递的需要,可被全国Hp菌株库采用.
主题词 螺杆菌,幽门;细菌保存;细菌传递
张建中, 蒋秀高, 陈晶晶, 孙兆军, 盛涛, 李志芳.幽门螺杆菌菌株存活能力及常温传递方法.华人消化杂志,1998;6(12):1077-1078
0 引言
幽门螺杆菌(Helicobacter pylori, Hp)感染与慢性胃炎、消化性溃疡和胃癌的关系日益受到国内外的关注,为深入开展Hp相关性研究工作,需建立一种简便易行的菌株传递方法. 我们对多年来开展的菌株传送方法进行简单总结,并将一种比较满意的传递方法介绍给大家.
, http://www.100md.com
1 材料和方法
1.1 材料 菌株:实验采用NCTC 11637,NCTC11639和CAPMN62三株Hp,其中NCTC 11637和NCTC11639为国际标准菌株,CAPMN62为国内分离株,实验前均经系统鉴定. 保存液配制:用蒸馏水配制200g/L的蔗糖溶液,经115℃,30min灭菌后,将蔗糖溶液与经灭活的小牛血清按1∶1比例混合即成为本实验所用菌株保存液,保存液平时可置普通冰箱冷冻备用.
1.2 方法 将三株实验用菌分别接种含100mL/L羊血的布氏琼脂平板后,置37℃混合气体(100mL/L CO2,50mL/L O2和850mL/L N2)环境培养. 取新鲜培养3d的Hp,用保存液(含100g/L蔗糖,500mL/L小牛血清)作成1013cfu/L的菌悬液,分别用满管和半管保存液量装于1.5mL灭菌小管中,经密封后放于37℃恒温箱中,于12,24,36,48,60,72,96,120,144和168h,按每种保存量各取出一管,接种双份平皿(100mL/L羊血布氏琼脂)后,于微需氧环境培养3d后观察细菌生长情况. 另外,将一满管1013cfu/L浓度的Hp用特快专递(EMS)自北京传送到上海(上海医科大学华山医院),收到后马上进行接种,并连续观察传递管中菌株在不同时间内的存活情况.
, http://www.100md.com
2 结果
在气温20℃~34℃条件下,经邮政特快专递(express mail service, EMS)传递的菌株存活良好,且在传递到达后可继续存活至55h. 在37℃保存液中不同条件下的存活情况见表1.
表1 Hp在37℃保存液中的存活能力
, 百拇医药
+:细菌存活; -:无活菌存在.
3 讨论
目前进行菌株传递的方法主要有冻干菌株传递、冷冻菌株传递和培养平皿(或培养小瓶)传递等几种方案[1-3]. 冻干菌株传递常被用来进行国际菌株传递,因冻干菌株可在室温条件下存活若干年,已被各国科研机构广泛采用,但菌株冻干(特别是Hp菌株的冻干)程序较繁琐,要求技术较高,要对国内大量用户采用冻干菌株供应存在一定困难,同时,因需冻干设备,此方案不适合各地向菌株库传送菌株. 鉴于以上情况,在近5a内,我们更多地采用了冷冻菌株传递方法,这种方法对菌株库的压力较小,已有几十家医院、大学和科研机构从菌株库成功获得菌株,但此方法一般需专人传递,而且在传递中要保持菌株一直处于冷冻状态,因而时有传递失败的情况发生.
, http://www.100md.com
我们在进行耗时较长的药敏实验中发现,Hp在布氏肉汤中能存活较长时间,但有研究资料表明,Hp在盐水、Stuart传送培养基、含200mL/L甘油的布氏汤、含170mL/L甘油的脱脂牛奶中,室温6h后活菌数即明显下降. 一般认为在最适保存液中,4℃至室温环境只能存活5h~48h[4-8]. 我们在实验中对Hp在以小牛血清蔗糖为主的保存液中的存活能力进行了观察,发现至少3d的存活能力已能利用遍布各地特快专递系统(EMS)进行有效传递;对此,我们在北京-上海间进行的实地传递实验也获得了成功.
本实验表明,使用液体保护液,在常温下通过EMS进行国内Hp的菌株传递能满足我国各地菌株传递的需要,此方法简单、实用,可被全国Hp菌株库及各临床、科研单位采用.
4 参考文献
, 百拇医药
1 Xia HX, Keane CT, Chen JJ, Zhang JZ, Walsh EJ, Moran AP et al. Transportation of Helicobacter pylori culture by optimal system.
J Clin Microbiol, 1994;32(12):3075-3077
2 Xia HX, Keane CT,O Morain CA. Determination of the optimal transport system for Helicobacter pylori culture.
J Med Microbiol, 1993;39(2):334-337
3 Han SW, Flamm R, Hachem CY, Kim HY, Clarridge JE, Evans DG et al.Transport and storage of Helicobacter pylori from gastric
, 百拇医药
mucosal biopsies and clinical isolates. Eur J Clin Microbiol Infect Dis, 1995;14(4):349-352
4 Goodwin CS, Blincow ED, Warren JR, Waters TE, Sanderson CR, Easton L. Evaluation of cultural techniques for
isolating Campylobacter pyloridis from endoscopic biopsies of gastric mucosa. J Clin Pathol,1985;389(10):1127-1131
5 Humphreys H, Bourke S, Dooley C, McKenna D, Power B, Keane CT et al. Effect of treatment on Campylobacter pylori in
, http://www.100md.com
peptic disease: a randomized prospective trial. Gut,1988;29(3):279-283
6 Megraud F, Bonnet F, Garnier M, Lamoulistte H. Characterization of “Campylobacter pyloridis” by culture, enzymatic profiles,and protein content. J Clin Microbiol, 1985;22(6):1007-1010
7 Queiroz DMM, Mendes EN, Rocha GA. Indicator medium for isolation of Campylobacter pylori. J Clin Microbiol, 1987;25(12):2378-2379
8 Soltesz V, Zeeberg B, Wadstrom T. Optimal survival of Helicobacter pyloriunder various transport conditions.
J Clin Microbiol,1992;30(6):1453-145, 百拇医药(张建中1 蒋秀高1 陈晶晶1 孙兆军1 盛涛1 李志芳2)
2上海医科大学华山医院消化内科 上海市 200040
张建中,男,1964-01-05生,山东省东营市人,汉族. 1984年山东医科大学医学系毕业,1992年中国预防医学科学院硕士,副主任,副研究员,主要从事幽门螺杆菌相关研究,承担全国幽门螺杆菌协作组菌株库的构建和管理工作.
项目负责人 张建中,102206,中国预防医学科学院流行病学微生物学研究所腹泻病研究室,北京市昌平县流字5号02室.
Correspondence to Dr. Jian-Zhong Zhang, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Changping, Beijing 102206, P.O.Box 5, China
, http://www.100md.com
Tel. +86·10·61739456,\ Fax. +86·10·61739456
E-mail: Helico@public.bta.net.cn
收稿日期 1998-07-06
Storage and transportation of Helicobacter pylori at room temperature
Jian-Zhong Zhang1, Xiu-Gao Jiang1, Jing-Jing Chen1, Zhao-Jun Sun1, Tao Sheng1 and Zhi-Fang Li2
, http://www.100md.com
1Diarrhea Disease Lab, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing 102206, China2Huashan Hospital, Shanghai Medical University, Shanghai 200040, China
Abstract
AIM To establish an easy and useful transporting method of H.pylori isolates.
METHODS Three newly cultured H.pylori strains (NCTC 11637, NCTC11639 and CAPMN62) were suspended with H.pylori storage solution (containing 100g/L sucrose and 500mL/L new bovine serum) to 1013cfu/L, put into 1.5mL bottles in half and full amount respectively and airproofed. All bottles were placed under 37℃ condition, and inoculated into culture media (Brucella agar with 100mL/L sheep blood) after 12, 24, 36, 48, 60, 72, 96, 120, 144 and 168 hours respectively. The H.pylori growth was observed 3 days after culture at microaerophilic environment (100mL/L CO2, 50mL/L O2 and 850mL/L N2). On the other hand, a bottle of H.pylori (NCTC 11637 strain, 1013 cfu/L) was transported by express mail service (EMS) from Beijing to Shanghai (room temperature 20℃-34℃). The organism was subcultured immediately after arrival and at various interval.
, http://www.100md.com
RESULTS At 37℃, H.pylori can survive at least 72 hours, and addition of full amount of storage solution can prolong the survival of Hp significantly. At room temperature (20℃-34℃), the H.pylori well survived after EMS transportation and in the period of 55 hours.
CONCLUSION With H.pylori storage solution (100g/L sucrose and 500mL/L new bovine serum), H.pylori isolates can be transported by EMS under room temperature successfully, and it is suitable for all hospitals and labs in our country.
, 百拇医药
Subject headings Helicobacter pylori; bacterial storage; bacterial transportation
Zhang JZ, Jiang XG, Chen JJ, Sun ZJ, Sheng T, Li ZF. Storage and transportation of Helicobacter pylori at room temperature.
Huaren Xiaohua Zazhi,1998;6(12):1077-1078
摘要
目的 观察幽门螺杆菌(Helicobacter pylori,Hp)在常温保存液中的存活能力及影响因素,通过实际菌株传递实验,为我国Hp菌株库建立中各地Hp的收集和供应,提供切实可行的菌株传递方案.
, 百拇医药
方法 取新鲜培养3d的Hp 3株(NCTC 11637,NCTC11639和CAPMN62),用Hp保存液(含10g/L蔗糖,500mL/L小牛血清)作成1013cfu/L浓度的菌悬液,分别以满管和半管形式装于1.5mL灭菌小管中,经密封后放于37℃恒温箱中,于12,24,36,48,60,72,96,120,144,和168h,按每种各取出一管,接种双份平皿(100mL/L羊血布氏琼脂)后,于微需氧环境培养3d后观察细菌生长情况. 另外,将一满管1013cfu/L浓度的Hp用特快专递(EMS)自北京传送到上海,收到后马上进行接种,并连续观察传递管中菌株在不同时间内的存活情况.
结果 在37℃保存液中Hp至少可存活72h,保存管中通过加满保存液减少空气残留,能明显延长Hp的存活时间;在气温20℃~34℃条件下,经EMS实际传递的菌株存活良好,且在传递到达后可继续存活至55h.
, 百拇医药 结论 使用含10%蔗糖的50%小牛血清细菌传送保护液,在常温下通过EMS进行国内Hp的菌株传递能满足我国各地菌株传递的需要,可被全国Hp菌株库采用.
主题词 螺杆菌,幽门;细菌保存;细菌传递
张建中, 蒋秀高, 陈晶晶, 孙兆军, 盛涛, 李志芳.幽门螺杆菌菌株存活能力及常温传递方法.华人消化杂志,1998;6(12):1077-1078
0 引言
幽门螺杆菌(Helicobacter pylori, Hp)感染与慢性胃炎、消化性溃疡和胃癌的关系日益受到国内外的关注,为深入开展Hp相关性研究工作,需建立一种简便易行的菌株传递方法. 我们对多年来开展的菌株传送方法进行简单总结,并将一种比较满意的传递方法介绍给大家.
, http://www.100md.com
1 材料和方法
1.1 材料 菌株:实验采用NCTC 11637,NCTC11639和CAPMN62三株Hp,其中NCTC 11637和NCTC11639为国际标准菌株,CAPMN62为国内分离株,实验前均经系统鉴定. 保存液配制:用蒸馏水配制200g/L的蔗糖溶液,经115℃,30min灭菌后,将蔗糖溶液与经灭活的小牛血清按1∶1比例混合即成为本实验所用菌株保存液,保存液平时可置普通冰箱冷冻备用.
1.2 方法 将三株实验用菌分别接种含100mL/L羊血的布氏琼脂平板后,置37℃混合气体(100mL/L CO2,50mL/L O2和850mL/L N2)环境培养. 取新鲜培养3d的Hp,用保存液(含100g/L蔗糖,500mL/L小牛血清)作成1013cfu/L的菌悬液,分别用满管和半管保存液量装于1.5mL灭菌小管中,经密封后放于37℃恒温箱中,于12,24,36,48,60,72,96,120,144和168h,按每种保存量各取出一管,接种双份平皿(100mL/L羊血布氏琼脂)后,于微需氧环境培养3d后观察细菌生长情况. 另外,将一满管1013cfu/L浓度的Hp用特快专递(EMS)自北京传送到上海(上海医科大学华山医院),收到后马上进行接种,并连续观察传递管中菌株在不同时间内的存活情况.
, http://www.100md.com
2 结果
在气温20℃~34℃条件下,经邮政特快专递(express mail service, EMS)传递的菌株存活良好,且在传递到达后可继续存活至55h. 在37℃保存液中不同条件下的存活情况见表1.
表1 Hp在37℃保存液中的存活能力
菌株号 | 保存液满度 | 不同时间(h)存活情况 | |||||||||
12 | 24 | 36 | 48 | 60 | 72 | 96 | 120 | 144 | 168 | ||
NCTC11637 | 满管 | + | + | + | + | + | + | + | + | - | - |
NCTC11637 | 半管 | + | + | + | + | + | + | - | - | - | - |
NCTC11639 | 满管 | + | + | + | + | + | + | + | + | - | - |
NCTC11639 | 半管 | + | + | + | + | + | + | - | - | - | - |
CAPM N62 | 满管 | + | + | + | + | + | + | + | + | + | - |
CAPM N62 | 半管 | + | + | + | + | + | + | + | - | - | - |
, 百拇医药
+:细菌存活; -:无活菌存在.
3 讨论
目前进行菌株传递的方法主要有冻干菌株传递、冷冻菌株传递和培养平皿(或培养小瓶)传递等几种方案[1-3]. 冻干菌株传递常被用来进行国际菌株传递,因冻干菌株可在室温条件下存活若干年,已被各国科研机构广泛采用,但菌株冻干(特别是Hp菌株的冻干)程序较繁琐,要求技术较高,要对国内大量用户采用冻干菌株供应存在一定困难,同时,因需冻干设备,此方案不适合各地向菌株库传送菌株. 鉴于以上情况,在近5a内,我们更多地采用了冷冻菌株传递方法,这种方法对菌株库的压力较小,已有几十家医院、大学和科研机构从菌株库成功获得菌株,但此方法一般需专人传递,而且在传递中要保持菌株一直处于冷冻状态,因而时有传递失败的情况发生.
, http://www.100md.com
我们在进行耗时较长的药敏实验中发现,Hp在布氏肉汤中能存活较长时间,但有研究资料表明,Hp在盐水、Stuart传送培养基、含200mL/L甘油的布氏汤、含170mL/L甘油的脱脂牛奶中,室温6h后活菌数即明显下降. 一般认为在最适保存液中,4℃至室温环境只能存活5h~48h[4-8]. 我们在实验中对Hp在以小牛血清蔗糖为主的保存液中的存活能力进行了观察,发现至少3d的存活能力已能利用遍布各地特快专递系统(EMS)进行有效传递;对此,我们在北京-上海间进行的实地传递实验也获得了成功.
本实验表明,使用液体保护液,在常温下通过EMS进行国内Hp的菌株传递能满足我国各地菌株传递的需要,此方法简单、实用,可被全国Hp菌株库及各临床、科研单位采用.
4 参考文献
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J Clin Microbiol,1992;30(6):1453-145, 百拇医药(张建中1 蒋秀高1 陈晶晶1 孙兆军1 盛涛1 李志芳2)