香港海鸥菌不同保存方法效果比较
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摘要
目的比较不同方法对香港海鸥菌的短期和长期保存效果,以期建立该菌的简单、实用和经济的短期和长期保存方法。方法采用半固体石蜡封存法,磁珠菌种保存管、10%甘油、15%甘油肉汤和30%甘油肉汤等5种方法保存香港海鸥菌,其中,半固体石蜡封存法的保存温度为室温和4℃,其他方法均在-20℃和-80℃进行菌种保存。定期进行菌种复苏,采用活菌菌落计数比较不同方法的保存效果,观察期为12个月。结果半固体石蜡封存法在室温环境下保存的活菌数在6个月内呈缓慢下降趋势(活菌数的对数值下降幅度<1),但6个月以后活菌数急速下降且菌落变小,至第9个月时为0 CFU/mL;而4℃保存的第1个月活菌数下降幅度小,但到第3个月时活菌数为0 CFU/mL;在12个月内,磁株及3种不同甘油浓度保存的活菌数在-80℃下保持相对稳定,但-20℃下保存时间短、效果差。结论室温条件下的半固体石蜡封存法适合短期保存香港海鸥菌,而-80℃低温条件下的磁株或甘油适合长期保存。
Objective To compare different methods for preserving Laribacter hongkongensis(LH) strains and to develop a simple and practicable preservation protocol for short-and long-term storage and reactivation LH strain. Methods The five methods for LH strain storage included:(i) semisolid with paraffin storage,(ii) Microbank vial,(iii)10% glycerol,(iv) 15% glycerol/85% Luria-Bertani(LB) broth culture,and(v) 30% glycerol/70% LB broth culture. The LH strains were put at 4 ℃ and room temperature when using methods i,while the stains were disposed at conventional freezing temperatures of-20 ℃ and-80 ℃ when using other methods,respectively. All storage techniques were evaluated periodically by counting with colony-forming unit over 12 months. Results For semisolid with paraffin storage,the viable strains were reduced gently up to 6 months at room temperature,then reduced rapidly and LH colonies became pinpoint size,contrasting with no colony growth on Mac Conkey agar at 3-month at 4 ℃. Preservation of LH strains in Microbank vial,10% glycerol,15% glycerol broth,and 30% glycerol broth stored at-80 ℃ were proved to be stable for up to 12 months. How ever,recovery of the strains from these methods stored at-20 ℃ showed poor growth after one month. Conclusion Semisolid with paraffin storage at room temperature is an effective way for short-term preservation of LH strain. Preservation in glycerol or Microbank vial at-80 ℃ is recommended for prolonged periods of LH strain storage.
Objective To compare different methods for preserving Laribacter hongkongensis(LH) strains and to develop a simple and practicable preservation protocol for short-and long-term storage and reactivation LH strain. Methods The five methods for LH strain storage included:(i) semisolid with paraffin storage,(ii) Microbank vial,(iii)10% glycerol,(iv) 15% glycerol/85% Luria-Bertani(LB) broth culture,and(v) 30% glycerol/70% LB broth culture. The LH strains were put at 4 ℃ and room temperature when using methods i,while the stains were disposed at conventional freezing temperatures of-20 ℃ and-80 ℃ when using other methods,respectively. All storage techniques were evaluated periodically by counting with colony-forming unit over 12 months. Results For semisolid with paraffin storage,the viable strains were reduced gently up to 6 months at room temperature,then reduced rapidly and LH colonies became pinpoint size,contrasting with no colony growth on Mac Conkey agar at 3-month at 4 ℃. Preservation of LH strains in Microbank vial,10% glycerol,15% glycerol broth,and 30% glycerol broth stored at-80 ℃ were proved to be stable for up to 12 months. How ever,recovery of the strains from these methods stored at-20 ℃ showed poor growth after one month. Conclusion Semisolid with paraffin storage at room temperature is an effective way for short-term preservation of LH strain. Preservation in glycerol or Microbank vial at-80 ℃ is recommended for prolonged periods of LH strain storage.
引文
[1]Yuen KY,Woo PC,Teng JL,et al.Laribacter hongkongensis gen.nov.,sp.nov.,a novel gram-negative bacterium isolated from a cirrhotic patient with bacteremia and empyema[J].J Clin M icrobiol,2001,39(12):4227-4232.
[2]Woo PC,Poon RW,Foo CH,et al.First report of Laribacter hongkongensis peritonitis in continuous ambulatory peritoneal dialysis[J].Perit Dial Int,2016,36(1):105-107.
[3]张爱梅,郭大城,王建丽,等.国内菌种保藏材料及保藏方法研究现状[J].河南预防医学杂志,2011,22(6):405-407.
[4]Woo PC,Lau SK.Tse H.et al.The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats[J].PLo S Genet,2009,5(3):e1000416.
[5]陈燕飞.两种细菌学检查方法的比较[J].中国农学通报,2010,26(17):129-131.
[6]Woo PC,Lau SK,Teng JL,et al.Association of Laribacter hongkongensis in community-acquired gastroenteritis with travel and eating fish:a multicentre case-control study[J].The Lancet,2004,363(9425):1941-1947.
[7]Lau SK,Woo PC,Hui WT,et al.Use of cefoperazone Mac Conkey agar for selective isolation of Laribacter hongkongensis[J].J Clin M icrobiol,2003,41(10):4839-4841.
[8]董昧.微生物菌种保藏方法[J].煤炭与化工,2009,32(7):34-35.
[9]许丽娟,刘红,魏小武.微生物菌种的保藏方法[J].现代农业科技,2008,16:99.
[10]栾军,祝长青,蒋原,等.空肠弯曲菌保存方法的比较[J].口岸卫生控制,2007,12(4):15-17.
[2]Woo PC,Poon RW,Foo CH,et al.First report of Laribacter hongkongensis peritonitis in continuous ambulatory peritoneal dialysis[J].Perit Dial Int,2016,36(1):105-107.
[3]张爱梅,郭大城,王建丽,等.国内菌种保藏材料及保藏方法研究现状[J].河南预防医学杂志,2011,22(6):405-407.
[4]Woo PC,Lau SK.Tse H.et al.The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats[J].PLo S Genet,2009,5(3):e1000416.
[5]陈燕飞.两种细菌学检查方法的比较[J].中国农学通报,2010,26(17):129-131.
[6]Woo PC,Lau SK,Teng JL,et al.Association of Laribacter hongkongensis in community-acquired gastroenteritis with travel and eating fish:a multicentre case-control study[J].The Lancet,2004,363(9425):1941-1947.
[7]Lau SK,Woo PC,Hui WT,et al.Use of cefoperazone Mac Conkey agar for selective isolation of Laribacter hongkongensis[J].J Clin M icrobiol,2003,41(10):4839-4841.
[8]董昧.微生物菌种保藏方法[J].煤炭与化工,2009,32(7):34-35.
[9]许丽娟,刘红,魏小武.微生物菌种的保藏方法[J].现代农业科技,2008,16:99.
[10]栾军,祝长青,蒋原,等.空肠弯曲菌保存方法的比较[J].口岸卫生控制,2007,12(4):15-17.