Division I.5.5 Subcultivation

If no CPE has developed after the primary incubation for 7 to 10 days, subcultivation shall be performed to fresh cell cultures utilising a cell area similar to that of the primary culture.

Aliquots of medium (supernatant) from all cultures or wells constituting the primary culture shall be pooled according to cell line 7 to 10 days after inoculation. The pools shall then be inoculated into homologous cell cultures undiluted and diluted 1:10 (resulting in final dilutions of 1:10 and 1:100, respectively, of the supernatant) as described in point I.5.2. Alternatively, aliquots of 10 % of the medium constituting the primary culture shall be inoculated directly into a well with fresh cell culture (namely, well to well subcultivation). The inoculation may be preceded by pre-incubation of the dilutions with the antiserum to IPN virus at appropriate dilution as described in point I.4.3.

The inoculated cultures shall then be incubated for 7 to 10 days at 15 °C and inspected in accordance with point I.5.4.

If toxic CPE occurs within the first 3 days of incubation, subcultivation shall be performed at that stage, but the cells shall then be incubated for 7 days and sub cultivated again with further 7 days incubation. When toxic CPE develops after 3 days, the cells shall be passed once and incubated to achieve the total of 14 days from the primary inoculation. There must be no evidence of toxicity in the final 7 days of incubation.

If bacterial contamination occurs despite treatment with antibiotics, subcultivation shall be preceded by centrifugation at 2 000 to 4 000 × g for 15 to 30 minutes at 2 to 5 °C, or filtration of the supernatant through a 0,45 μm filter or both (low protein-binding membrane). In addition to this, subcultivation shall follow the same procedures as described for toxic CPE in the fourth paragraph of this point.

If no CPE occurs, the test may be declared negative.