菲律宾蛤仔血细胞透射电镜样品制备的初步研究

Preliminary Study on Sample Preparation of Ruditapes philippinarum Haemocyte for Transmission Electron Microscopy

透射电镜样品制备的好坏直接影响着透射电镜的观察和电镜图片的质量。该论文针对贝类血细胞普遍存在易凝集、密度低、类型不单一的现象,选取菲律宾蛤仔血细胞为研究对象,从取样方法、琼脂预包埋方式和锇酸固定时间3个方面比较和筛选适合其透射电镜的样品制备方法。实验结果显示:用预先抽取固定液的针管抽取血淋巴能有效防止血细胞凝集;离心式的琼脂预包埋方法能使血细胞集中,防止血细胞丢失;3000r/min^10 000r/min的转速均能达到较好的制样效果;锇酸固定10min,细胞质基质丢失严重,线粒体、高尔基体、粗面内质网等超微结构模糊。随着锇酸固定时间的增加,血细胞超微结构的保存逐渐转好,锇酸固定2h得到的血细胞透射电镜样品制备效果最佳。这些研究结果为菲律宾蛤仔和其它贝类血细胞超微结构及其相关的免疫学、病理学的研究提供基本技术参考。

The sample preparation directly affects the transmission electron microscopy(TEM)observation and the quality of the electron microscope pictures.Due to the bivalve haemocyte has the characteristics of agglutination,low density and morphological diversity,in order to find the proper haemocyte preparation of bivalve for TEM we compared the effects of sampling method,agar pre-embedded mode and osmium tetroxide fixed time on the haemocyte sample preparation of Ruditapes philippinarumin this paper.The results showed that using anaxenic syringein which contained fixative could effectively prevent haemocytes agglutination.In the agar pre-embedded procedure,it was better to centrifuge the haemocytes after blending the haemocyte into agar and 3000r/min^10000r/min rotate speed could achieve good effect of sample preparation.It had significantly effects of osmium tetroxide fixed time on the Ruditapes philippinarum haemocyte ultrastructure,in osmium tetroxide fixed 10 min treatment the cytoplasmic matrix lost seriously,the organelles ultrastructures were not well preserved,with the fixed time increasing,the haemocyte ultrastructures got better,and the osmium tetroxide fixed 2h groups got the best effect of sample preparation for haemocyte.These results provided basic technical reference for studing the haemocyte ultrastructure,related immunology and pathology of R.philippinarumand and other bivalve.