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Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Transfer floating cells to new flasks.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.03% (w/v) EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. 
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 23°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Medium Renewal: At the time of subcultivation

Wolf K, et al. Lymphocystis virus: isolation and propagation in centrarchid fish cell lines. Science 151: 1004-1005, 1966. PubMed: 4955835

Zwillenberg LO, Wolf K. Ultrastructure of lymphocystis virus. J. Virol. 2: 393-399, 1968. PubMed: 4986903

Wolf K, et al. Tadpole edema virus: a viscerotropic pathogen for anuran amphibians. J. Infect. Dis. 118: 253-262, 1968. PubMed: 5674065