Appendix 5 Media for isolation and culture of C. m. subsp. sepedonicusF1
United Kingdom
(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 |
| K 2 HPO 4 | 0,25 g |
| KH 2 PO 4 | 0,25 g |
| MgSO 4 .7H 2 O | 0,1 g |
| MnSO 4 .H 2 O | 0,015 g |
| NaCl | 0,05 g |
| FeSO 4 .7H 2 O | 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 |
| K 2 HPO 4 | 0,25 g |
| KH 2 PO 4 | 0,25 g |
| NaCl | 0,05 g |
| MgSO 4 .7H 2 O | 0,1 g |
| MnSO 4 .H 2 O | 0,015 g |
| FeSO 4 .7H 2 O | 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 |
| K 2 HPO 4 | 2 g |
| KH 2 PO 4 | 0,5 g |
| MgSO 4 .7H 2 O | 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.