F1ANNEX ITEST 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

Annotations:
Amendments (Textual)
Appendix 5Media for isolation and culture of C. m. subsp. sepedonicus

(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.