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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。
TEM sample preparation directly affects the quality of transmission electron microscopy and electron microscopy images. This paper aims at the universality of shellfish blood cells that are easy to agglutinate, the density is low and the type is not uniform. The blood cells of Clarias clarkii were selected as the research object. The methods of sampling, agar pre-embedding and osmium tetroxide fixation were compared and screened Its transmission electron microscopy sample preparation method. The experimental results show that the extraction of hemolymph with a pre-drawn needle can effectively prevent hemagglutination. Centrifugal agar pre-embedding method can concentrate blood cells and prevent the loss of blood cells. The speed of 3000r / min ~ 10 000r / min can reach Good sample preparation effect; osmium acid fixation 10min, cytoplasmic matrix lost serious, mitochondria, Golgi apparatus, rough endoplasmic reticulum and other ultrastructure fuzzy. With the increase of osmium tetroxide fixation time, the preservation of blood cell ultrastructure was gradually improved, and the best result was obtained by transmission electron microscopy (TEM). These findings provide the basic technical reference for the research on ultrastructure of clam and other shellfish blood cells in Philippine and their related immunology and pathology.