F1ANNEX IITEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
(b)Validated selective growth media
SMSA medium (Englebrecht, 1994 as modified by Elphinstone et al., 1996)
Basal medium | |
Casamino acids (Difco) | 1,0 g |
Bacto-Peptone (Difco) | 10,0 g |
Glycerol | 5,0 ml |
Bacto-Agar (Difco); see Note 2. | 15,0 g |
Distilled water | 1,0 L |
Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.
Cool to 50 °C and add filter-sterilised aqueous stock solutions of the following ingredients to obtain the specified final concentrations:
Crystal Violet (Sigma) | 5 mg per l |
Polymixin-B-Sulphate (Sigma P-1004) | 600 000 U (approximately 100 mg) per l |
Bacitracin (Sigma B-0125) | 1 250 U (approximately 25 mg) per l |
Chloramphenicol (Sigma C-3175) | 5 mg per l |
Penicillin-G (Sigma P-3032) | 825 U (approximately 0,5 mg) per l |
2,3,5-triphenyl tetrazolium chloride (Sigma) | 50 mg per l |
Note:
1.
Use of reagents other than those specified above may affect growth of R. solanacearum.
2.
Oxoid Agar #1 can be used in place of Bacto-Agar (Difco). In this case growth of R. solanacearum will be slower, although growth of competing saprophytes may also be reduced. Typical colonies of R. solanacearum may take 1 to 2 days longer to form and the red colouration may be lighter and more diffuse than on Bacto-Agar.
3.
Increasing bacitracin concentration to 2 500 U per l may reduce populations of competing bacteria without affecting growth of Ralstonia solanacearum.
Store media and stock solutions of antibiotics at 4 °C in the dark and use within one month.
Plates should be free from surface condensation before use.
Avoid excess drying of plates.
Quality control should be performed after preparation of each new batch of medium by plating a suspension of a reference culture of R. solanacearum (see Appendix 3) and observing formation of typical colonies after incubation at 28 °C for two to five days.
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..