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

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.

Difco bacto nutrient agar containing 1 % D(+) glucose (monohydrate). Sterilize by autoclaving at 115 °C for 20 minutes.

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.

Dissolve ingredients and sterilize 0,5 litre volumes of medium by autoclaving at 115 °C for 20 minutes.

Unless otherwise stated all media components are from BDH.

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.

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.