利用流式细胞仪计数微型浮游生物的方法  

Application of flow cytometry to enumerate small plankton

作　　者：李胜男[1,2] 王秀娟[1,2] 周建[1,2] 孔繁翔[1] 史小丽[1] 

机构地区：[1]中国科学院南京地理与湖泊研究所湖泊与环境国家重点实验室,南京210008 [2]中国科学院大学,北京100049

出　　处：《湖泊科学》2015年第5期757-766,共10页Journal of Lake Sciences

基　　金：国家自然科学基金项目(31270507;31070420)资助

摘　　要：微型浮游生物(细胞粒径<20μm)在水生生态系统的物质循环和能量流动中起着重要的作用,对其丰度的准确测定是进一步研究微型浮游生物在不同水生生态系统中作用的重要基础.相对于传统的显微镜检测技术,流式细胞术不仅具有分析速度快、灵敏度和准确度高等优点,而且可以同时测量单个细胞的多个生理参数.不同类型微型浮游生物流式细胞术的应用原理是不同的.对于自养型浮游藻类,主要根据藻体内色素的自发荧光对其进行分辨和计数;而对于异养型细菌、原生动物及浮游病毒等,还需借助外源荧光染料对细胞核酸染色后再进行分析.目前流式细胞术已成为浮游藻类和异养细菌丰度检测的常规方法,但是,由于原生动物具有更大的细胞体积且在自然水体中丰度较低;而浮游病毒粒径又太小,甚至低于光源激发波长,因此流式细胞术应用一直受到限制,直到近10年来才有相关报道.本文对运用流式细胞术计数浮游藻类、浮游细菌、原生动物和浮游病毒的具体原理、方法和进展进行综述,并对流式细胞仪在未来水生微生物学领域的应用进行展望.The small plankton（ cell size less than 20 μm） plays an important role in the circulation of materials and energy flow of the aquatic system. Accurate enumeration of these organisms is the basis for understanding their ecological role in various water bodies. In comparison with microscopy observation,flow cytometry analysis is much more advantageous in terms of speed,sensitivity and accuracy. Moreover,multiple parameters of a single cell could be measured simultaneously using flow cytometry. The protocol to enumerate plankton depends on the type of plankton. Discrimination of several major groups of phytoplankton is mainly based on the differences in the fluorescence properties of their photosynthetic pigments. While for the heterotrophic bacteria,protozoan and viruses,a combination of exogenous fluorochromes staining on cell components（ mainly nucleic acids） is required to better characterize different cell groups. Now flow cytometry has become a routine methodology for detecting density of the autotrophic phytoplankton and heterotrophic bacterioplankton. However,it has been only used in quantification of protozoan and viruses in the recent 10 years,for those applications which are much more difficult and complicated for the larger cell size and less abundant densities of protozoan and much smaller cell size（ even smaller than the wavelength of the laser light used） of viruses compared to bacterioplankton and small phytoplankton. The different principles and protocols used to discriminate autotrophic phytoplankton,heterotrophic bacteria,protozoan and viruses through flow cytometry were reviewed in detail,and future applications of flow cytometry in aquatic microbiology were also prospected.

关 键 词：流式细胞仪 浮游植物 异养细菌 原生动物 病毒 色素 荧光染料 

分 类 号：Q-33[生物学]