# 474: MEDIUM WITH POLYHYDROXYBUTYRIC ACID AS CARBON SOURCE ## Main sol. 474 1. Prepare a suspension of 3.0 g poly-ß-hydroxybutyric acid (PHB) in 100 ml distilled water, stir over night and sonicate until a white homogenous suspension is obtained. Autoclave for 5 minutes at 121°C. Prepare double layer agar plates. First layer: medium 457 with 16.0 g agar per l. 1000 ml **Main sol. 457** 16 g/l Agar 2. Second layer: medium 457 with agar, supplemented with 6.6 ml sterile PHB-suspension per 100 ml. 6.6 ml **Poly-ß-hydroxybutyric acid solution 3%(w/v)** 100 ml **Main sol. 457** ## Poly-ß-hydroxybutyric acid solution 3%(w/v) 3 g poly-ß-hydroxybutyric acid 100 ml Distilled water ## Main sol. 457 2.44 g Na2HPO4 1.52 g KH2PO4 0.5 g (NH4)2SO4 0.2 g MgSO4 x 7 H2O 0.05 g CaCl2 x 2 H2O 10 ml **Trace element solution SL-4** 1000 ml Distilled water 1. Adjust pH to 6.9. 2. Prepare a separate solution of the phosphates and autoclave separately. Combine the two solutions after cooling. 3. Rehydrate and cultivate lyophilized cells in complex medium (e.g. medium 1, 220 or 535). After this reactivation, cultivate in mineral medium 457 with the appropriate carbon source. ## Trace element solution SL-4 0.5 g EDTA 0.2 g FeSO4 x 7 H2O 100 ml **Trace element solution SL-6** 900 ml Distilled water ## Trace element solution SL-6 0.1 g ZnSO4 x 7 H2O 0.03 g MnCl2 x 4 H2O 0.3 g H3BO3 0.2 g CoCl2 x 6 H2O 0.01 g CuCl2 x 2 H2O 0.02 g NiCl2 x 6 H2O 0.03 g Na2MoO4 x 2 H2O 1000 ml Distilled water ## Main sol. 1 5 g Peptone 3 g Meat extract 15 g Agar, for solid medium 1000 ml Distilled water 1. Adjust pH to 7.0. 2. For Bacillus strains the addition of 10.0 mg MnSO4 x H2O is recommended for sporulation. ## Main sol. 220 15 g Casein peptone 5 g Soy peptone 5 g NaCl 15 g Agar 1000 ml Distilled water 1. Adjust pH to 7.3. Medium is identical with Tryptone Soya Agar (Oxoid CM 131). ## Main sol. 535 30 g Trypticase soy broth (BBL 11768, Oxoid CM129 or Merck 5459) 20 g Agar 1000 ml Distilled water 1. pH 7.3 2. Autoclave at 121°C for 15 min.