{
  "url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-4",
  "json_url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-4.json",
  "legislation": "eudr/1998/57",
  "legislation_title": "Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.",
  "legislation_url": "https://openlegislation.uk/eu-directive-1998-57",
  "path": "annex/II/appendix/4",
  "kind": "appendix",
  "number": "4",
  "heading": "Buffers for test procedures",
  "text": "General: Unopened sterilised buffers can be stored for up to one year\n1.\nBuffers for extraction procedure\n1.1.\nExtraction buffer (50 mM phosphate buffer, pH 7,0)\nThis buffer is used for extraction of the bacterium from plant tissues by homogenisation or shaking.\nNa 2 HPO 4 (anhydrous)\n4,26 g\nKH 2 PO 4\n2,72 g\nDistilled water\n1.00 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.\nAdditional components may be useful as follows:\nFor use with homogenisation extraction method\nPurpose\nQuantity (per l)\nLubrol flakes\nDeflocculant\n0,5 g\nDC silicone antifoam\nAnti-foam agent\n1,0 ml\nTetrasodium pyrophosphate\nAnti-oxidant\n1,0 g\nPolyvinylpyrrolidone-40000 (PVP-40)\nBinding of PCR inhibitors\n50 g\n1.2.\nPellet buffer (10 mM phosphate buffer, pH 7,2)\nThis buffer is used for resuspension and dilution of potato tuber heel-end core extracts following concentration to a pellet by centrifugation.\nNa 2 HPO 4 .12H 2 O\n2,7 g\nNaH 2 PO 4 .2H 2 O\n0,4 g\nDistilled water\n1,0 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.\n2.\nBuffers for the IF test\n2.1.\nIF-Buffer (10 mM phosphate buffered saline (PBS), pH 7.2)\nThis buffer is used for dilution of antibodies\nNa 2 HPO 4 .12H 2 O\n2,7 g\nNaH 2 PO 4 .2H 2 O\n0,4 g\nNaCL\n8,0 g\nDistilled water\n1,0 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.\n2.2.\nIF-buffer-Tween\nThis buffer is used to wash slides.\nAdd 0,1 % Tween 20 to the IF buffer.\n2.3.\nPhosphate buffered glycerol, pH 7,6\nThis buffer is used as a mountant fluid on the windows of IF slides to enhance fluorescence.\nNa 2 HPO 4 .12H 2 O\n3,2 g\nNaH 2 PO 4 .2H 2 O\n0,15 g\nGlycerol\n50 ml\nDistilled water\n100 ml\nAnti-fading mountant solutions are commercially available e.g. Vectashield ® (Vector Laboratories) or Citifluor ® (Leica).\n3.\nBuffers for the Indirect ELISA test\n3.1.\nDouble strength coating buffer, pH 9,6.\nNa 2 CO 3\n6,36 g\nNaHCO 3\n11,72 g\nDistilled water\n1,00 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.\nSodium sulphite (0.2 %) may be added as antioxidant if required to prevent build up of oxidised aromatic compounds.\n3.2.\n10X Phosphate buffered saline (PBS), pH 7,4\nNaCL\n80,0 g\nKH 2 PO 4\n2,0 g\nNa 2 HPO 4 .12H 2 O\n29,0 g\nKCl\n2,0 g\nDistilled water\n1,0 L\n3.3.\nPBS-Tween\n10X PBS\n100 ml\n10 % Tween 20\n5 ml\nDistilled water\n895 ml\n3.4.\nBlocking (antibody) buffer (must be freshly prepared).\n10X PBS\n10,0 ml\nPolyvinylpyrrolidone-44000 (PVP-44)\n2,0 g\n10 % Tween 20\n0,5 ml\nMilk powder\n0,5 g\nDistilled water\nmake up to 100 ml\n3.5.\nAlkaline phosphatase substrate solution, pH 9,8\nDiethanolamine\n97 ml\nDistilled water\n800 ml\nMix and adjust to pH 9,8 with concentrated HCl.\nMake up to 1 L with distilled water.\nAdd 0,2 g MgCl 2 .\nDissolve 2 phosphatase substrate 5 mg tablets (Sigma) per 15 ml of solution.\n4.\nBuffers for DASI ELISA test\n4.1.\nCoating buffer, pH 9,6\nNa 2 CO 3\n1,59 g\nNaHCO 3\n2,93 g\nDistilled water\n1 000 ml\nDissolve ingredients and check pH 9,6\n4.2.\n10X Phosphate saline buffer(PBS) pH 7,2 to 7,4\nNaCl\n80,0 g\nNaH 2 PO 4 .2 H 2 O\n4,0 g\nNa 2 HPO 4 .12H 2 O\n27,0 g\nDistilled water\n1 000 ml\n4.3.\nPBS-Tween\n10X PBS\n50 ml\n10 % Tween 20\n5 ml\nDistilled water\n950 ml\n4.4.\nSubstrate buffer, pH 9,8\nDiethanolamine\n100 ml\nDistilled water\n900 ml\nMix and adjust to pH 9,8 with concentrated HCl.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "F",
      "label": "F1",
      "text": "Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.."
    },
    {
      "type": "footnote",
      "label": "f1",
      "text": "For use with homogenisation extraction method"
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/1998/57/annex/II/appendix/4",
    "retrieved_at": "2026-11-01T15:23:06.931886+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."
}