Preliminary Establishment About Sampling-Culturing Of The Freshwater Cell Culture And The Key Points Of The Technology Research

According to the principle that the tissue cells of infected fish are the sensitive cells of the particular pathogen, the cell culture method of Sampling-Culturing was employed. It starts with sampling and culturing the tissue cells from the same kind of health fish.The subject started from overall arrangement of laboratory. Taking the kidney of grass carp, the snout and kidney of catfish as the experimental objects, and at the same time, purchasing the CO cells-line served as control group of the cell culture. The results showed that:After digested by trypsinization, CO cells could attach the bottom and subcultured. The cells were in good condition, and has been successfully subcultured to16th generations. The two kinds of kidney tissue from grass carp and catfish were digested by trypsinization while the snout and kidney from catfish were in the method of explant culture, obtained a lot of single and dispersed primary cells, staining by trypan blue, we could observe the cells with a convert microscope in shape, and count the survival rate of cells, the result survival rate was more than92%. But the cells which obtained by trypsin digestion failed to attach the bottom though we operated six measures to choose and optimize the culture conditions. The cells from explant culture can attach the bottom, because of the migration cells which were nearby the tissue were too crowded to grow, and displayed the contact inhibition, the following phenomena were observed:cell contact becomes lossing much more cavitation and granules appear in cell plasma, and finally cracked to pieces. Aftert washed and blown, the separate cells were inoculated, but the cells couldn’t attach the bottom any more, influenced the subculture.After optimization, the cells still couldn’t attach the bottom, further analysis were needed. We can see that the’sampling-culturing’is feasible, explant culture is better than trypsinization. Although we have tried our best, a key technological failed to be broke through, we still need a further study.