# 1761: PFENNIG'S MEDIUM III ## Main sol. 1761 46 ml **Solution A** 2.6 ml **Solution B** 5 ml **Solution C** 45 ml **Solution D** 2.6 ml **Solution E** 0.1 ml **Solution F** 1 ml **Solution G** 1. Adjust pH to 6.0. 2. Mix solution D, C and E in the cotton-stoppered Erlenmeyer flask. Bubble with CO2 in an ice bath under sterile conditions (5-10 min). 3. Fill 50 ml of the mixed solution (C,D,E) into each 100 ml bottle of solution A (for a total volume of approx. 96 ml). 4. Before using, add 2,6 ml solution B (Na2S x 9 H2O), 0.1 ml solution F (Vitamin B12) and 1 ml of solution G (2,5% Ammonium-acetate and Magnesium-acetate) to each 100ml bottle. 5. Adjust the pH with filter-sterilised 1M Na2CO3 to 7.1-7.3. 6. Fill in sterile, N2 gassed screw-cap tubes under N2 gas. 7. For the growth of DSM 11081 feed the culture with neutralized sulfide feeding solution regularly (approx. every 3 days). 0,5-1 ml/ 100 mL ## Solution A 0.25 g CaCl2 x 2 H2O 460 ml Distilled water 1. Fill 10x 46ml in 100 ml screw-cap bottles. Bubble with N2/CO2 and autoclave at 121°C for 15 min. ## Solution B 2 g Na2S x 9 H2O 135 ml Distilled water 1. Prepare in a screw-cap bottle, bubble with N2 to replace air, close tightly and autoclave. ## Solution C 1.5 g NaHCO3 50 ml H2O 1. Bubble with CO2 and filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle. ## Solution D 0.5 ml Resazurin (0,1%) 450 ml Distilled water 1. Autoclave in a cotton-stoppered Erlenmeyer flask with an outlet tube for medium, connected to a glass outlet at the bottom of the vessel and has, at the other end, a silicon rubber tube with a pinch cock and a bell for aseptic dispensing of the medium into bottles. Cool to room temperature under an atmosphere of N2/CO2 in an ice bath (10-15 min). ## Solution E 0.4 g Ammonium chloride 0.5 g MgSO4 x 7 H2O 0.34 g KCl 0.34 g KH2PO4 1 ml **Trace element solution SL-12 B** 25 ml Distilled water 1. Filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle. ## Solution F 2 mg Vitamin B12 100 ml Distilled water 1. Filter sterilized ## Solution G 2.5 g Ammonium acetate 2.5 g Magnesium acetate 100 ml Distilled water 1. Autoclave at 121°C for 15 min. ## Trace element solution SL-12 B 1000 ml Distilled water 3 g Na2-EDTA 1.1 g FeSO4 x 7 H2O 190 mg CoCl2 x 6 H2O 50 mg MnCl2 x 2 H2O 42 mg ZnCl2 24 mg NiCl2 x 6 H2O 18 mg Na2MoO4 x 2 H2O 300 mg H3BO3 2 mg CuCl2 x 2 H2O ## Neutralized sulfide solution 3% (w/v) 3 g Na2S x 9 H2O 100 ml Distilled water 1. The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 M H2SO4 drop-wise with a syringe without opening the bottle.