摘要
为体外构建水霉菌生物膜(BF)并对其形成过程与结构特征进行分析,本研究利用寄生水霉(Saprolegnia parasitica)ATCC200013在玻片和聚乙烯网片上构建水霉菌BF,筛选适宜水霉菌粘附的基质材料,并在BF形成过程中,采用CCK-8法(Cell Counting Kit-8)定量检测BF的活力变化,利用Calcofluor White M2R染色后,荧光显微镜观察细胞外基质;扫描电镜观察水霉菌BF的形态结构;SYTO 9/PI染色后,激光共聚焦显微镜观察水霉菌BF的活力及结构特征。结果显示,水霉菌能够在聚乙烯网片上形成较好的BF,并且BF能够与聚乙烯网片紧密粘附,完全覆盖聚乙烯网片,而与盖玻片粘附不紧密。水霉菌BF OD450nm值在培养前24 h升高较快,48 h后趋于稳定。水霉菌在第6 h即开始有散在的孢子黏附于聚乙烯网片上;12 h^24 h形成单细胞层;36 h^60 h为中间形成阶段,至72 h菌丝缠绕形成多层的、弥散着大量细胞外基质的立体结构,同时死亡菌随着培养时间延长而增多。本研究体外构建了水霉菌BF并了解其结构特征,为水霉菌BF的进一步研究奠定了基础。
To establish an in vitro model and investigate the structural characteristics of Saprolegnia biofilm, the biofilm of Saprolegnia parasitica ATCC200013 was developed on glass slide and polyethylene netting. The activity of biofilm was measured and quantitatively analyzed by CCK-8 （Cell Counting Kit-8） to detect the process. Morphological characteristics of S.parasitica was observed under fluorescence microscope stained by Calcofluor White M2R, scanning electron microscope, and confocal laser scanning microscopy stained by SYTO 9/PI. The results showed that biofilm was able to develop more efficiently on polyethylene netting than that on glass slide. S.parasitica biofilm adhered to polyethylene netting tightly and covered it completely, while the biofilm on glass slide was easy to fall off. The OD45~ value of S.parasitica biofllm developed quickly before 24 hrs and stabilized after 48 hrs. S.parasitica spore adhered to materials occurred at 6 hrs, hyphae intertwined to form single layer structure from 12 to 24 hrs and middle stage forming was from 36 to 60 hrs. Then the complex three dimensional structure was set up at 72 hrs. In the maturation, more and more extracellular matrix appeared around hyphae and dead cells increased with the incubation time. In conclusion, the successful establishment of S.parasitica biofilm in vitro and investigation of the biofilm structural characteristics provided the bases for fiu＇ther related researches.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2015年第9期669-673,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家863计划项目(2011AA10A216)
上海市科技兴农重点攻关项目[沪农科攻字(2011)第4-8号]
国家水产种质资源平台运行服务项目
上海高校知识服务平台资助
国家自然科学基金项目(31172430)
Shanghai Universities First-class Disciplines Project of Fisheries and Shanghai University Knowledge Service Platform(ZF1206)
公益性行业(农业)科研专项经费"渔药使用风险评估及其控制技术研究与示范"(201203085)
关键词
水霉菌
生物膜
体外模型
结构特征
Saprolegnia
biofilm
in vitro model
structural characteristic
