| 摘要: |
| 病毒在海洋生态系统的物质循环和能流中起着重要作用，对其进行有效定量是研究其数量变动和生态功能的前提。现行的海洋沉积物病毒定量主要采用荧光染色计数法，但对技术流程中一些处理方法的定量效能尚待验证。本研究利用不同类型海洋沉积物对荧光计数法进行了效能评估和条件优化。结果表明：将沉积物采样后染色封片直接计数或-20℃保存为最佳，保存3个月后未见丰度降低。而不同类型的沉积物经液氮闪冻后于-80℃冷冻保存后的定量效能不同：沙质沉积物保存1个月后病毒丰度下降1个数量级；泥沙质沉积物保存1个月后丰度降低了约27%，2个月后则下降了60%。对焦磷酸钠最优终浓度实验结果显示，使用3mmol/L终浓度获得的病毒计数定量效能优于目前普遍使用的5mmol/L终浓度。水浴超声分离在常温下的定量效果更优，在水浴中添加冰块导致获取的沙质沉积物中的病毒丰度下降约37%。研究发现：处理中用稀释替代离心不仅可降低沉积物颗粒的干扰，而且定量效能更高；离心处理获取的沙质和泥沙质沉积物中的病毒丰度较稀释处理低约40%。对海洋沉积物中原核生物的定量效能与病毒有相似的结果。据此，本文提出了改进的沉积物病毒荧光计数法的操作流程。 |
| 关键词: 海洋沉积物 病毒 原核生物 定量效能 荧光计数法 |
| DOI：10.11693/hyhz20161100239 |
| 分类号:Q938 |
| 基金项目:中国科学院战略性先导科技专项项目三“深海海洋环境与生态系统”，XDA11030201号；973项目“超深渊生物群落及其与关键环境要素的相互作用机制研究”，2015CB755902号。 |
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| OPTIMIZATION OF VIRAL ENUMERATION IN EPIFLUORESCENCE MICROSCOPY FOR MARINE SEDIMENTS |
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WEI Miao1,2, XU Kui-Dong1,2
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1.Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.University of Chinese Academy of Sciences, Beijing 100049, China
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| Abstract: |
| Viruses play key roles in material cycles and energy flows of marine ecosystems. Effective enumeration of viruses is critical for understanding their ecological roles and dynamics. Epifluorescence microscopy has been used widely to enumerate viruses in marine sediments, but the efficiency in some procedures remains unknown. We evaluated and optimized the procedures of viral enumeration based on three types of marine sediments using epifluorescence microscopy. The results suggest that the preparation of dye-stained slides immediately after sampling and storage at -20℃ yielded the greatest counting efficiency, and no decrease was observed after three months. By contrast, the counting efficiency of snap freezing in liquid nitrogen and storage at -80℃ varied with different types of sediments:declined an order of magnitude in sandy sediment after one month; and decreased by about 27% after one month, and about 60% after two months in muddy-sand sediment. The results show that for the optimal performance, the final concentration of sodium pyrophosphate shall be set at 3mmol/L rather than at 5mmol/L that commonly used at present, sonication shall be placed in water at ambient temperature rather than a mixture of ice-water, with which a loss of 37% might be reached for enumeration of viruses in the sandy sediment; and dilution instead of centrifugation resulted in an increase of the viral counts by about 40% for the sandy and muddy-sand sediments. Similar efficiencies were reached for prokaryotes in all tests. Therefore, this protocol of viral counting for marine sediments in epifluorescence microscopy is improved and thus shall be recommended. |
| Key words: marine sediment virus prokaryote quantitative efficiency epifluorescence microscopy |
