# Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al., Appendix 4: Buffers for test procedures

Extent: E+W+S+N.I.

General: Unopened sterilised buffers can be stored for up to one year
1.
Buffers for extraction procedure
1.1.
Extraction buffer (50 mM phosphate buffer, pH 7,0)
This buffer is used for extraction of the bacterium from plant tissues by homogenisation or shaking.
Na 2 HPO 4 (anhydrous)
4,26 g
KH 2 PO 4
2,72 g
Distilled water
1.00 l
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.
Additional components may be useful as follows:
For use with homogenisation extraction method
Purpose
Quantity (per l)
Lubrol flakes
Deflocculant
0,5 g
DC silicone antifoam
Anti-foam agent
1,0 ml
Tetrasodium pyrophosphate
Anti-oxidant
1,0 g
Polyvinylpyrrolidone-40000 (PVP-40)
Binding of PCR inhibitors
50 g
1.2.
Pellet buffer (10 mM phosphate buffer, pH 7,2)
This buffer is used for resuspension and dilution of potato tuber heel-end core extracts following concentration to a pellet by centrifugation.
Na 2 HPO 4 .12H 2 O
2,7 g
NaH 2 PO 4 .2H 2 O
0,4 g
Distilled water
1,0 l
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.
2.
Buffers for the IF test
2.1.
IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7.2)
This buffer is used for dilution of antibodies
Na 2 HPO 4 .12H 2 O
2,7 g
NaH 2 PO 4 .2H 2 O
0,4 g
NaCL
8,0 g
Distilled water
1,0 l
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.
2.2.
IF-buffer-Tween
This buffer is used to wash slides.
Add 0,1 % Tween 20 to the IF buffer.
2.3.
Phosphate buffered glycerol, pH 7,6
This buffer is used as a mountant fluid on the windows of IF slides to enhance fluorescence.
Na 2 HPO 4 .12H 2 O
3,2 g
NaH 2 PO 4 .2H 2 O
0,15 g
Glycerol
50 ml
Distilled water
100 ml
Anti-fading mountant solutions are commercially available e.g. Vectashield ® (Vector Laboratories) or Citifluor ® (Leica).
3.
Buffers for the Indirect ELISA test
3.1.
Double strength coating buffer, pH 9,6.
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
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
10X PBS
100 ml
10 % Tween 20
5 ml
Distilled water
895 ml
3.4.
Blocking (antibody) buffer (must be freshly prepared).
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
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.
4.
Buffers for DASI ELISA test
4.1.
Coating buffer, pH 9,6
Na 2 CO 3
1,59 g
NaHCO 3
2,93 g
Distilled water
1 000 ml
Dissolve ingredients and check pH 9,6
4.2.
10X Phosphate saline buffer(PBS) pH 7,2 to 7,4
NaCl
80,0 g
NaH 2 PO 4 .2 H 2 O
4,0 g
Na 2 HPO 4 .12H 2 O
27,0 g
Distilled water
1 000 ml
4.3.
PBS-Tween
10X PBS
50 ml
10 % Tween 20
5 ml
Distilled water
950 ml
4.4.
Substrate buffer, pH 9,8
Diethanolamine
100 ml
Distilled water
900 ml
Mix and adjust to pH 9,8 with concentrated HCl.

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Source: https://www.legislation.gov.uk/eudr/1998/57/annex/II/appendix/4 (the official version)
Page: https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-4
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.