纳米细菌的分离培养和保存方法探讨

Method for isolation,culture and conservation of nanobacteria

目的以钙化的胎盘组织为例,寻求在钙化组织中分离纳米细菌的最佳方法,以便更多疑似与纳米细菌感染有关的疾病得以准确分离出纳米细菌菌株,并探讨培养和保存纳米细菌的最适宜的条件。方法 (1)胎盘钙化组织标本分别用盐酸脱矿与超声振荡脱矿的方法分离纳米细菌,计算其分离阳性率。(2)分离出的纳米细菌分别用细胞培养箱与细菌培养箱培养1个月,利用分光光度计记录两种条件下纳米细菌浓度的变化,并描绘生长曲线。(3)分别用4、-20与-80℃冰箱保存钙化组织和纳米细菌,记录纳米细菌分离和复苏的生长状况并绘制生长曲线。结果 (1)钙化组织用盐酸脱矿更易分离得到纳米细菌。(2)细胞与细菌培养环境下纳米细菌的生长速度并无明显差别。(3)4℃保存钙化组织和纳米细菌对于其分离和复苏都要优于-20℃和-80℃。结论对钙化组织进行盐酸脱矿可以更好的分离出纳米细菌,并且可以在细菌培养箱内培养纳米细菌,新鲜钙化组织标本和纳米细菌可以短时间保存在4℃。

Objective To find an optimal method for isolating nanobacteria in calcified tissues and the most suit-able condition to culture and observe nanobacteria. Method Calcified placental tissue were used in this research in a hope that the method could be used to other calcified tissues as well. （ 1 ） Calcified placental tissues were demineralized separately with hydrochloric acid and ultrasonic vibration to isolate nanobacteria from the samples; the positive rates of isolation were calculated and compared. （2）Nanobacteria were cultured separately in cells and bacteria culture conditions and concentration was recorded with spectophoto fluorometer. （3）Fresh calcified tissues and cultured nanobacteria were preserved in different low temperature conditions, recovery and concentration was recorded. Result （ 1 ） Nanobacteria were obtained more easily in HC1 demineralized group. （2）There was no obvious difference between the growth rate of Nanobacteria in bacteria culture conditions and in cell culture conditions. （3）Fresh samples and nanobacteria could be preserved in 4℃ condition for several weeks. Conclusion Nanobacteria can be isolated with HCl, cultured in bacteria culture conditions and preserved in 4 ℃ for a short time.