菲律宾蛤仔血细胞透射电镜样品制备固定液的筛选

Screening of Specimen Preparation and Fixatives of Manila Clam Ruditapes philippinarum Haemocytes for Transmission Electron Microscopy

对样品的固定是透射电镜样品制备的关键环节,通过在透射电镜下观察和比较不同固定液固定菲律宾蛤仔血细胞后血细胞超微结构的变化,初步筛选出适合菲律宾蛤仔血细胞透射电镜制样的固定液。试验结果表明,固定液可直接影响到透射电镜制样品质的高低,2.5%、5%戊二醛低渗固定液引起了血细胞细胞器的肿胀,其中线粒体肿胀尤为严重,肿胀程度2.5%戊二醛组大于5%戊二醛组;等渗固定液固定的血细胞细胞器不肿胀,但等渗5%戊二醛与4%多聚甲醛-2.5%戊二醛固定液对血细胞的精细结构保存不佳,而2%多聚甲醛-2.5%戊二醛固定液对血细胞超微结构保存较好,表现为细胞外膜结构清晰,细胞质基质丰富且分布均匀,细胞内线粒体、内质网、高尔基体、细胞核等细胞器固定效果较好。试验得出,低渗固定液会导致细胞肿胀,线粒体对渗透压的要求较其他细胞器更加敏感;提高固定液含量会减轻因低渗引起的细胞肿胀程度,但高含量固定液会损坏细胞内精细结构,故适合的样品固定液是细胞精细结构较好保存的基础。

Fixation is the key step of the specimen preparation for transmission electron microscopy.The aim of this paper is to investigate and compare the influence of 5different fixatives on the ultrastructure alterations of haemocytes in Manila clam Ruditapes philippinarum using transmission electron microscope in order to find the proper fixative for Manila clam.The results showed that fixative affected the haemocyte ultrastructure directly.The haemocytes were swelled in 2.5% and 5% glutaraldehyde groups,the higher in 2.5% glutaraldehyde groups than in 5% glutaraldehyde fixative treatments,and higher swollen in the haemocytes mitochondria than in the other organelles.No organelles swelling was found in isotonic fixative treatments,while the inner structures of the haemocytes were not preserved very well in 5% glutaraldehyde fixative with 7% saccharose groups and 4% paraformaldehyde-2.5% glutaraldehyde fixative treatments,especially the endomembrane system.In 2% paraformaldehyde-2.5% glutaraldehyde fixative treatments,the haemocytes had better ultrastructure than in other groups,with clear outer membrane structure of haemocytes.The cytoplasmic matrix was rich and evenly distributed,as well the ultrastructure of the organelles such as mitochondria,endoplasmic reticulum,Golgi apparatus and the nucleus were better preserved.From this study we can concluded that the hypotonic fixative could cause haemocytes swelling and that the mitochondria are more sensitive to the fixative osmotic pressure than other organelles.Raised fixative concentration reduces the cell swelling degree,but the high fixative concentration is not benefit to the haemocytes ultrastructure.Thus the suitable fixative is the foundation of the cell ultrastructure preservation.