摘要
目的探讨细胞培养中黑点状污染物的种类及防治措施。方法倒置显微镜观察黑点状污染物的生长状态;革兰染色、金胺O染色以及电子显微镜术分析黑点状物质的形态学特征;16S rDNA基因测序鉴定细菌种类;药敏实验筛选污染物敏感的抗生素;采用RAW264.7细胞培养上清液对冻存的SH-SY5Y细胞进行复苏。结果倒置显微镜实时观察显示黑点状物质呈静止和运动两种生长状态;革兰染色呈阴性,金胺O染色呈阳性;电子显微镜负染技术揭示该污染物是一种短杆状细菌,游动期带有极性鞭毛;16S rDNA基因测序表明该污染物是一种名为phenylobacterium zucineum HLK1的苯基杆菌。药敏实验筛选出头孢曲松、羧苄西林和亚胺培南等抗生素对该细菌具有抑制作用,但是细胞培养实验表明上述抗生素无法清除SH-SY5Y细胞内的细菌。被污染细胞不能长期冷冻保存,但是采用RAW264.7细胞培养上清液进行复苏可显著提高细胞的存活效率。结论培养体系内的黑点状污染物是一种能够侵入细胞内部、生存能力极强的寡营养细菌,常规抗生素处理很难将它们从培养体系内清除。RAW264.7细胞培养上清液可能含有某种对抗p.z.HLK1的活性物质,采用RAW264.7细胞培养上清进行细胞复苏可以显著提高细胞的存活效率。
Objective To explore the type and control measure of black dot-like contaminants in cell cultures.Methods The growth state of bacteria was investigated under an inverted microscope;Their morphological characteristics were analyzed by Gram and auramine O staining as well as electron microscopy;16S rDNA gene sequencing was used to analyze bacterial species;Drug sensitivity test was used to screen antibiotics against the bacteria;Cryopreserved SH-SY5Y cells were resuscitated by cell culture supernatant of RAW264 cells.Results Inverted microscopic real-time observations showed that black dot-like substances had two growth states:static and moving.They were negative for Gram staining while positive for auramine O staining.Electron microscopy revealed that they were short rod-shaped bacteria with a polar flagellum during moving phase.16S rDNA gene sequencing showed that these bacteria werephenylobacterium zucineum HLK1.Ceftriaxone,carboxycillin and imipenem were screened by drug sensitivity test to have inhibitory effects on the bacteria,but cell culture experiments showed that they could not remove the bacteria from SH-SY5Y cells.Contaminated cells could not be cryopreserved for a long time,but resuscitation with RAW264.7 cell culture supernatant significantly improved the survival rate of cells.Conclusion The black dot-like contaminants in cell cultures are a special type of oligotrophic bacterium with strong viability that can invade the cells and cannot be cleared with antibiotic treatment.RAW264.7 cell culture supernatant seems contain some substances against bacteria,and resuscitating frozen cells with RAW264.7 cell culture supernatant may significantly improve the survival rate of cells.
作者
谭百宏
王艳玲
郑敬彤
于军
刘利
李艳超
Tan Baihong;Wang Yanling;Zheng Jingtong;Yu Jun;Liu Li;Li Yanchao(Laboratory Teaching Center of Basic Medicine,Norman Bethune Health Science Center of Jilin University,Changchun 130021;Dept of Pathogenic Biology,Norman Bethune Health Science Center of Jilin University,Changchun 130021;Dept of Histology and Embryology,Norman Bethune Health Science Center of Jilin University,Changchun 130021)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第1期126-132,共7页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81871014)。
