Council Directive 93/85/EEC of 4 October 1993 on the control of potato ring rot (c. 85)

[F1 ANNEX I U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF THE RING ROT BACTERIUM, CLAVIBACTER MICHIGANENSIS (Smith) Davis et al. ssp. SEPEDONICUS (Spieckermann et Kotthoff) Davis et al. SCOPE OF THE TEST SCHEME

Textual Amendments

F1 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.

5. FISH TEST U.K.

5.2. Pre-hybridisation and hybridisation U.K.

5.2.1. Prepare a lysozyme solution containing 10 mg lysozyme (Sigma L–6876) in 10 ml buffer (100 mM Tris-HCl, 50 mM EDTA, pH8.0). This solution can be stored but it should only be freeze-thawed once. Cover each sample well with approximately 50 µl of lysozyme solution and incubate for 10 minutes at room temperature. Then dip the slides in demineralised water, once only and dry with filter paper. U.K.

Alternatively, instead of lysozyme add 50 µl of 40 to 400µg ml ^–1 proteinase K in buffer (20 mM Tris-HCl, 2 mM CaCl ₂ , pH 7,4) to each well and incubate at 37 °C for 30 minutes.

5.2.2. Dehydrate the cells in a graded ethanol series of 50 %, 80 % and 96 % for one minute each. Air dry the slides in a slide-holder. U.K.

5.2.3. Prepare a moist incubation chamber by covering the bottom of an air-tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7). Pre-incubate the box in the hybridisation oven at 55 °C for at least 10 minutes. U.K.

5.2.4. Prepare the hybridisation solution (Appendix 7) allowing 45 µl per slide, and preincubate for five minutes at 55 °C. U.K.

5.2.5. Place slides on a hot plate at 45 °C and apply 10 µl of hybridization solution to each of the four wells on the slide(s). U.K.

5.2.6. Apply two coverslips (24 × 24 mm) to each slide without trapping air. Place the slides in the pre-warmed moist chamber and hybridise overnight in the oven at 55 °C in the dark. U.K.

5.2.7. Prepare three beakers containing 1 l of Ultra pure water (UPW), 1 l of 1x hybmix (334 ml 3x hybmix and 666 ml UPW) and 1 l of 1/2x hybmix (167 ml 3x hybmix and 833 ml UPW). Pre-incubate each in a waterbath at 55 °C. U.K.

5.2.8. Remove the coverslips from the slides and place the slides in a slide holder. U.K.

5.2.9. Wash away excess probe by incubation for 15 mins. in the beaker with 1x hybmix at 55 °C. U.K.

5.2.10. Transfer the slide holder to 1/2 hybmix washing solution and incubate for a further 15 mins. U.K.

5.2.11. Dip the slides briefly in UPW and place them on filter paper. Remove excess moisture by covering the surface gently with filter paper. Pipette 5 to 10 μl of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA or equivalent) on each window and apply a large coverslip (24 × 60 mm) over the whole slide.] U.K.