# 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, Appendix 5: Media for isolation and culture of C. m. subsp. sepedonicus

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

(a)
General growth media
Nutrient agar (NA)
Nutrient Agar (Difco)
23,0 g
Distilled water
1,0 L
Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.
Nutrient dextrose agar (NDA)
Difco bacto nutrient agar containing 1 % D(+) glucose (monohydrate). Sterilize by autoclaving at 115 °C for 20 minutes.
Yeast peptone glucose agar (YPGA)
Yeast Extract (Difco)
5,0 g
Bacto-Peptone (Difco)
5,0 g
D(+) Glucose (monohydrate)
10,0 g
Bacto-Agar (Difco)
15,0 g
Distilled water
1,0 L
Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.
Yeast extract mineral salts medium (YGM)
Bacto-Yeast-Extract (Difco)
2,0 g
D(+) Glucose (monohydrate)
2,5 g
K2HPO4
0,25 g
KH2PO4
0,25 g
MgSO4.7H2O
0,1 g
MnSO4.H2O
0,015 g
NaCl
0,05 g
FeSO4.7H2O
0,005 g
Bacto-Agar (Difco)
18 g
Distilled water
1,0 L
Dissolve ingredients and sterilize 0,5 litre volumes of medium by autoclaving at 115 °C for 20 minutes.
(b)
Validated selective growth media
MTNA medium
Unless otherwise stated all media components are from BDH.
Yeast extract (Difco)
2,0 g
Mannitol
2,5 g
K2HPO4
0,25 g
KH2PO4
0,25 g
NaCl
0,05 g
MgSO4.7H2O
0,1 g
MnSO4.H2O
0,015 g
FeSO4.7H2O
0,005 g
Agar (Oxoid no. 1)
16,0 g
Distilled water
1,0 L
Dissolve ingredients, adjust pH to 7,2. After autoclaving (at 121 °C for 15 minutes) and cooling down to 50 °C, add the antibiotics: trimethoprim 0,06 g, nalidixic acid 0,002 g, amphotericin B 0,01 g.
Stock antibiotic solutions: trimethoprim (Sigma) and nalidixic acid (Sigma) (both at 5 mg/ml), in 96 % methanol, amphotericin B (Sigma) (1 mg/ml) in dimethyl sulfoxide. Stock solutions are filter-sterilized.
Note:
Durability of basal medium is three months. After antibiotics are added durability is one month when stored refrigerated.
NCP-88 medium
Nutrient agar (Difco)
23 g
Yeast extract (Difco)
2 g
D-mannitol
5 g
K2HPO4
2 g
KH2PO4
0,5 g
MgSO4.7H2O
0,25 g
Distilled water
1,0 L
Dissolve ingredients, adjust pH to 7,2. After autoclaving and cooling down to 50 °C, add the following antibiotics: Polymyxin B sulphate (Sigma) 0,003 g, nalidixic acid (Sigma) 0,008 g, Cycloheximide (Sigma) 0,2 g.
Dissolve antibiotics in stock solutions as follows: nalidixic acid in 0,01 M NaOH, cycloheximide in 50 % ethanol, polymyxin B sulphate in distilled water. Stock solutions are filter-sterilized.
Note:
Durability of basal medium is three months. After antibiotics are added durability is one month when stored refrigerated.

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Source: https://www.legislation.gov.uk/eudr/2006/56/annex/I/appendix/5 (the official version)
Page: https://openlegislation.uk/eu-directive-2006-56/annex-I/appendix-5
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.