{
  "url": "https://openlegislation.uk/eu-directive-1993-85/annex-I/appendix-5",
  "json_url": "https://openlegislation.uk/eu-directive-1993-85/annex-I/appendix-5.json",
  "legislation": "eudr/1993/85",
  "legislation_title": "Council Directive 93/85/EEC of 4 October 1993 on the control of potato ring rot",
  "legislation_url": "https://openlegislation.uk/eu-directive-1993-85",
  "path": "annex/I/appendix/5",
  "kind": "appendix",
  "number": "5",
  "heading": "Media for isolation and culture of C. m. subsp. sepedonicus",
  "text": "(a)\nGeneral growth media\nNutrient agar (NA)\nNutrient Agar (Difco)\n23,0 g\nDistilled water\n1,0 L\nDissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.\nNutrient dextrose agar (NDA)\nDifco bacto nutrient agar containing 1 % D(+) glucose (monohydrate). Sterilize by autoclaving at 115 °C for 20 minutes.\nYeast peptone glucose agar (YPGA)\nYeast Extract (Difco)\n5,0 g\nBacto-Peptone (Difco)\n5,0 g\nD(+) Glucose (monohydrate)\n10,0 g\nBacto-Agar (Difco)\n15,0 g\nDistilled water\n1,0 L\nDissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.\nYeast extract mineral salts medium (YGM)\nBacto-Yeast-Extract (Difco)\n2,0 g\nD(+) Glucose (monohydrate)\n2,5 g\nK 2 HPO 4\n0,25 g\nKH 2 PO 4\n0,25 g\nMgSO 4 .7H 2 O\n0,1 g\nMnSO 4 .H 2 O\n0,015 g\nNaCl\n0,05 g\nFeSO 4 .7H 2 O\n0,005 g\nBacto-Agar (Difco)\n18 g\nDistilled water\n1,0 L\nDissolve ingredients and sterilize 0,5 litre volumes of medium by autoclaving at 115 °C for 20 minutes.\n(b)\nValidated selective growth media\nMTNA medium\nUnless otherwise stated all media components are from BDH.\nYeast extract (Difco)\n2,0 g\nMannitol\n2,5 g\nK 2 HPO 4\n0,25 g\nKH 2 PO 4\n0,25 g\nNaCl\n0,05 g\nMgSO 4 .7H 2 O\n0,1 g\nMnSO 4 .H 2 O\n0,015 g\nFeSO 4 .7H 2 O\n0,005 g\nAgar (Oxoid no. 1)\n16,0 g\nDistilled water\n1,0 L\nDissolve ingredients, adjust pH to 7,2. After autoclaving (at 121 °C for 15 minutes) and cooling down to 50 °C, add the antibiotics: trimethoprim 0,06 g, nalidixic acid 0,002 g, amphotericin B 0,01 g.\nStock antibiotic solutions: trimethoprim (Sigma) and nalidixic acid (Sigma) (both at 5 mg/ml), in 96 % methanol, amphotericin B (Sigma) (1 mg/ml) in dimethyl sulfoxide. Stock solutions are filter-sterilized.\nNote:\nDurability of basal medium is three months. After antibiotics are added durability is one month when stored refrigerated.\nNCP-88 medium\nNutrient agar (Difco)\n23 g\nYeast extract (Difco)\n2 g\nD-mannitol\n5 g\nK 2 HPO 4\n2 g\nKH 2 PO 4\n0,5 g\nMgSO 4 .7H 2 O\n0,25 g\nDistilled water\n1,0 L\nDissolve ingredients, adjust pH to 7,2. After autoclaving and cooling down to 50 °C, add the following antibiotics: Polymyxin B sulphate (Sigma) 0,003 g, nalidixic acid (Sigma) 0,008 g, Cycloheximide (Sigma) 0,2 g.\nDissolve antibiotics in stock solutions as follows: nalidixic acid in 0,01 M NaOH, cycloheximide in 50 % ethanol, polymyxin B sulphate in distilled water. Stock solutions are filter-sterilized.\nNote:\nDurability of basal medium is three months. After antibiotics are added durability is one month when stored refrigerated.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "F",
      "label": "F1",
      "text": "Substituted by Commission Directive 2006/56/EC of 12 June 2006 amending the Annexes to Council Directive 93/85/EEC on the control of potato ring rot."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/1993/85/annex/I/appendix/5",
    "retrieved_at": "2026-11-01T15:23:17.858184+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."
}