Division I.5.2 Inoculation of cell cultures

Antibiotic-treated organ suspension shall be inoculated into cell cultures in two dilutions, namely the primary dilution and, in addition, a 1:10 dilution thereof, resulting in final dilutions of tissue material in cell culture medium of 1:100 and 1:1 000, respectively, in order to prevent homologous interference. At least two cell lines shall be inoculated as referred to in point I.5.1. The ratio between inoculum size and volume of cell culture medium shall be about 1:10.

For each dilution and each cell line, a minimum of about 2 cm² cell area, corresponding to one well in a 24-well cell culture tray, shall be utilised. Cell culture trays shall be used where possible.