{
  "url": "https://openlegislation.uk/eu-decision-2015-1554/annex-II/part-3/division-II/division-II.2/division-II.2.3",
  "json_url": "https://openlegislation.uk/eu-decision-2015-1554/annex-II/part-3/division-II/division-II.2/division-II.2.3.json",
  "legislation": "eudn/2015/1554",
  "legislation_title": "Commission Implementing Decision (EU) 2015/1554 of 11 September 2015 laying down rules for the application of Directive 2006/88/EC as regards requirements for surveillance and diagnostic methods (notified under document C(2015) 6188) (Text with EEA relevance)",
  "legislation_url": "https://openlegislation.uk/eu-decision-2015-1554",
  "path": "annex/II/part/3/division/II/division/II.2/division/II.2.3",
  "kind": "division",
  "number": "II.2.3",
  "heading": "Subcultivation",
  "text": "Subcultivation shall be carried out between days 13 to 15. Culture supernatant shall be added to wells containing fresh actively growing cells in appropriate dilution (1/10) in multi-well plates and incubated at 14 ± 2 °C for up to 18 days. Using a microscope, cell cultures shall be examined for CPE twice, between days five to seven and days 14 to 18 following inoculation. If any pool shows CPE, virus identification procedures shall be initiated immediately in accordance with point II.2.4. If no CPE is observed by days 14 to 18, a haemadsorption or an RT-PCR test shall be performed.\nIf cytotoxicity occurs within the first 7 days of incubation, subcultivation shall be performed at that stage, and the cells shall be incubated for 14 to 18 days and sub cultivated again with a further period of 14 to 18 days incubation. If cytotoxicity occurs after 7 days, subcultivation shall be performed once and the cells shall be incubated to achieve the total period of 28 to 36 days incubation from the primary inoculation.\nIf bacterial contamination occurs in the primary culture, the test shall be set up again using the tissue homogenate stored at – 80 °C. Prior to inoculation, the tissue homogenate shall be centrifuged at 4 000 × g for 15 to 30 minutes at 0 to 6 °C and the supernatant shall be filtered at 0,22 μm. If bacterial contamination occurs during the subcultivation step the supernatant shall be filtered at 0,22 μm, inoculated onto fresh cells and incubated for a further 14 to 18 days.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudn/2015/1554/annex/II/part/3/division/II/division/II.2/division/II.2.3",
    "retrieved_at": "2026-11-01T15:14:14.330569+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}