Seawater fish ovum electron-microscope scanning sample preparing method

Abstract

The disclosed preparation method for ovum of sea fish as TEM sample comprises: fixing fresh fish ovum by glutaral solution diluted by phosphate buffer, cleaning, dewatering the ovum by 20-70% alcohol with concentration gradient increased by 10% and 75-100% alcohol with concentration gradient increased by 5% as two stages, then dewatering in pure acetone with CaCl2 as agent; then, drying at critic point, and spraying gold for the sample. This invention has ovum completion rate as 93.8-96.7% with low cost and convenient operation.

Description

technical field [0001] The invention relates to a preparation technology of seawater fish egg samples for electron microscope scanning observation. Background technique [0002] Fish fertilization includes two types of in vitro fertilization and internal fertilization. The fertilization process is that the sperm pass through the membrane hole of the egg and fuse with the egg nucleus to complete the combination of the gametes of the two sexes. Since the sperm of teleosts generally do not have acrosomes, the combination of mammalian sperm and eggs is due to the fact that fertilin and antifertilin recognize and interact with each other and produce sperm acrosomes in different fertilization methods. There is a sunken, usually funnel-shaped egg membrane pore on the shell membrane, which provides the only channel for the combination of sperm and eggs in teleosts, making the fertilization process have remarkable specificity (Wang Ruixia, 1984; Kuchnow, 1987; Kobayashi, 1987). How ...

AI Technical Summary

Problems solved by technology

Because fish are yolk eggs, the egg structure is special, the protoplasm and yolk are distributed in polarity, and the egg is surrounded by a very thin egg membrane with dense micropore distribution. In the fixative solution, due to the reaction of osmic acid molecules and aldehydes And generate precipitation, or cause lipoprotein complex dissolving because of too long fixation time, easily make ovum become brittle, break easily, affect the production quality of ovum sample, meanwhile, in fixative, because osmotic pressure effect ovum absorbs a large amount of water and To maintain a spherical shape, use the traditional ethanol dehydration method. Due to the high concentration of ethanol, the egg loses water too quickly, which will lead to an imbalance of pressure inside and outside the egg, and changes in surface tension, causing the egg membrane and yolk sac to collapse and deform, and dry at the critical point. Usually, the biological tissue samples dehydrated by ethanol and acetone are replaced with isoamyl acetate. This step will lead to large irregular shrinkage and collapse of the dehydrated fish eggs, and drastic changes in morphology, which will seriously affect the quality of the samples. This leads to difficulties in electron microscope observation and detection errors. Therefore, the traditional method of making electron microscope samples of animal and plant tissues or organs is not suitable for the preparation of fish eggs. Improvements are needed to establish a system that can maintain a high degree of integrity and good morphological and structural characteristics. Electron microscope observation samples of seawater fish eggs