Division I.5.1 Cell cultures and media

Bluegill fry cell line -2 (BF-2) or Rainbow trout gonad cell line - 2 (RTG-2) and either Epithelioma papulosum cyprini (EPC) or Fathead minnow (FHM) cells shall be grown at 20 to 30 °C in suitable medium, namely Eagle's Minimum essential medium (MEM) or modifications thereof, with a supplement of 10 % foetal bovine serum and antibiotics in standard concentrations.

When the cells are cultivated in closed vials, the medium shall be buffered with bicarbonate. The medium used for cultivation of cells in open units may be buffered with tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) (23 mM) and sodium bicarbonate (6 mM). The pH must be 7,6 ± 0,2.

The cell cultures to be used for inoculation with fish tissue material shall be young, normally 1 day old cell culture monolayers where possible; however, a range between 4 to 48 hours old may be accepted. The cells must be actively growing at inoculation.