海鞘血细胞培养和细菌污染控制

Ascidian hemocytes culture and the prevention of bactirail contamination

以海鞘(Ciona savignyi)为研究对象,考察了海鞘血细胞原代培养方法及培养中细菌污染的鉴定和控制。血细胞在培养1 h后贴壁,细胞基本以圆形细胞为主,变形细胞一般呈不规则形。变形细胞存活时间较短,而圆形细胞存活时间较长。对于培养过程中的细菌污染,通过细菌分离、培养和纯培养发现两类菌株检出率较高,均为革兰氏阴性菌。经PCR扩增16SrDNA基因序列片断,结果显示这两类菌株分别属于弧菌属(Vibrio)和施万氏菌属(Shewanella)。药敏试验结果显示,弧菌属对氯霉素和环丙沙星等具有较强的敏感性;对施万氏菌属较敏感的药物依次为亚胺培南和氯霉素等。最后比较了几种抗生素组合控制血细胞培养中细菌污染的使用效果,其中氯霉素、亚胺培南加双抗的组合有较好的抑菌效果并对培养细胞的贴壁和生长没有影响。

In this study, ascidian hemocytes were cultured in vitro and the identification and prevention of bacterial contamination were investigated. After 1 h incubation, hemocytes spread and formed a flat sheet consisting of nine types of cells. Most cultured cells were round and several cells were amebocytes. Round cell types could be maintained for 2 weeks at least in vitro culture, whereas in vitro could survived for very short time. Two bacterial strains contaminated in cell culture were isolated from the bacterial contamination samples by culture and purity culture. Two bacterial strains were gram-negative bacterium. The detected bacteria were isolated and identified as the members of by 16S rDNA sequencing. Drug sensitivity test detected that Vibrio showed resistance to chloromycetin and andciprofloxacin, Shewanella showed resistance to imipenem and chloromycetin. In order to control the microbial contamination in cell culture, three antibiotics mixtures were compared. It is shown that gentanycin was efective to prevent bacterial contamination.