Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. (c. 57)

[F1 ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.

Textual Amendments

F1 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..

Appendix 4 Buffers for test procedures

3. Buffers for the Indirect ELISA test U.K.

3.1. Double strength coating buffer, pH 9,6. U.K.

Na 2 CO 3	6,36 g
NaHCO 3	11,72 g
Distilled water	1,00 l

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

Sodium sulphite (0.2 %) may be added as antioxidant if required to prevent build up of oxidised aromatic compounds.

3.2. 10X Phosphate buffered saline (PBS), pH 7,4 U.K.

NaCL	80,0 g
KH 2 PO 4	2,0 g
Na 2 HPO 4 .12H 2 O	29,0 g
KCl	2,0 g
Distilled water	1,0 L

3.3. PBS-Tween U.K.

10X PBS	100 ml
10 % Tween 20	5 ml
Distilled water	895 ml

3.4. Blocking (antibody) buffer (must be freshly prepared). U.K.

10X PBS	10,0 ml
Polyvinylpyrrolidone-44000 (PVP-44)	2,0 g
10 % Tween 20	0,5 ml
Milk powder	0,5 g
Distilled water	make up to 100 ml

3.5. Alkaline phosphatase substrate solution, pH 9,8 U.K.

Diethanolamine	97 ml
Distilled water	800 ml

Mix and adjust to pH 9,8 with concentrated HCl.

Make up to 1 L with distilled water.

Add 0,2 g MgCl ₂ .

Dissolve 2 phosphatase substrate 5 mg tablets (Sigma) per 15 ml of solution.]