胆汁纳米细菌培养及鉴定

Bile nanobacteria cultivation and identification

①目的探讨胆汁中纳米细菌的培养、鉴定方法及微生物学特性。②方法对6例胆结石患者胆汁标本进行纳米细菌培养,观察其生长情况。采用透射电镜、间接免疫荧光染色、Von KOSSA钙染色进行纳米细菌鉴定并采用扫描电镜对其进行能谱分析。③结果 6例胆结石患者胆汁标本纳米细菌培养均为阳性;透射电镜下观察,纳米细菌约为80~350nm,呈椭球形或短棒状,聚集成簇状,其表面被覆细菌被膜;经Von KOSSA法钙染色,其形成的矿化外壳呈黑色;间接免疫荧光染色显示纳米细菌被荧光标记抗体结合,呈绿色荧光颗粒。扫描电镜能谱分析（EDX）显示,纳米细菌外壳含有钙、磷、镁等元素,其钙/磷比值为1.62;④结论胆囊结石患者胆汁中存在纳米细菌感染。纳米细菌体积微小,生长缓慢,能在生理条件下于其菌体表面产生矿化外壳。纳米细菌单克隆抗体联合透射电镜可鉴定纳米细菌,钙染色对纳米细菌的鉴定有着重要的参考价值。

Objective To explore the culture and identification of nanobacteria in bile and biological characteristic.Methods Six gallbladder bile samples were obtained from gallstone patients and then cultured for nanobacteria under cell culture conditions.Nanobacteria were identified by transmission electron microscopy（TEM）,and indirect immunofluorescence staining（IIFS） and Von KOSSA calcific staining.The scanning electron microscope was used for energy dispersive X-ray microanalysis of the cultured nanobacteria.Results The results of culture for nanobacteria were positive in all of 6 bile samples.Observing with transmission electron microscopy,nanobacteria revealed 80～350 nm particles.They appeared bacilli or coccoid in shape,predominantly as clusters by attaching to each other with biofilms-like material.Calcified nanobacteria（in biofilms）could be stained as black dots by Von KOSSA method.The result of indirect immunofluorescence staining showed that nanobacteria could be binded with fluorescently-labeled antibody,and produce green fluorescence.Elements of calcium,phosphor,magnesium,could be detected by EDX microanalysis,and the calcium-phosphate ratio of nanobacteria was 1.62,which was similar to that of hydroxyapatite.Conclusion Nanobacteria do exist in gallstone patients.The size of nanobacteria is super small.They grow slowly under mammalian cell culture conditions.They can produce calcified biofilms which are mainly composed of hydroxyapatite to escape damages.IIFS with monoclonal antibody of nanobacteria associated with TEM is useful in identifying nanobacteria.Calcific staining is of great value to identification of nanobacteria.