RESUMO
Lethal-challenge experiments are provided by German law till now for testing fish vaccines. Finding an in vitro-assay which could replace this experiments was subject of our work. Two questions must have been answered to develop the lymphocyte blastogenesis assay: Do lymphocytes of immunised fish react the same way as lymphocytes of naive fish, being incubated with antigen and is there a coherence between the lymphocyte's in-vitro-response and the immune status of challenged fish. Methods to take blood samples from anaesthetised fish and to obtain lymphocytes by centrifugation on gradients were worked out and the lymphocyte's demands on culture requirements were evaluated thoroughly because the ability to proliferate depends on many exogenous parameters. Proliferation was represented and measured by a tetrazolium based colorimetric assay (MTT).
