# 867: RHODOBACTER VELDKAMPII MEDIUM ## Main sol. 867 1000 ml **Main sol. 28** 0.25 % Yeast extract 0.3 % Na-acetate 2 mg Vitamin B12 10 ml **Standard vitamin solution** 1. supplement medium 28 with 0,25% Yeast-extract, 0,3% Na-acetate, 2mg/l Vitamin B12 and 10ml/l Standard vitamin solution (see below). ## Main sol. 28 460 ml **Solution A** 1. Aliquot Solution A into 100 mL screw-cap bottles, filled with 46 mL each. Bubble with N2/CO2 and autoclave at 121°C for 15 min (as decribed below). 2. Prepare the following solutions (resazurin, bicarbonate and Pfennig's heterotrophic salts) and sterilize as given below. 450 ml **Resazurin solution** 50 ml **Bicarbonate solution** 26 ml **Pfennig's heterotrophic salts solution** 3. Add bicarbonate solution and Pfennig's heterotrophic salts to the resazurin (complete volumina, i.e. 50 mL bicarbonate solution and 26 mL Pfennig's heterotrophic salts solution). Bubble with CO2 in an ice bath under sterile conditions. 4. Fill 50 ml of this mixture to each bottle of solution A (46 mL + 50 mL). 5. Before use, add 4 ml sulfide solution (1.5%) and 0.1 ml Vitamin B12 solution to each 100 mL bottle. 40 ml **Sulfide solution, 1.5%** 1 ml **Vitamin B12 solution** 6. Adjust the pH with filter-sterilised 1M Na2CO3 to 7.1-7.3. 7. If needed, aliquot into sterile, N2 gassed screw-cap tubes under N2 gas. 8. During the first 24 h, the iron of the medium precipitates in the form of black flocks. No other sediment should arise in the otherwise clear medium. 9. Feed the actively growing culture periodically with neutralized 3% solution of sodium sulfide (use 1 -3 mL/100 mL depending on strain and cultivation stage) to replenish sulfide and with other supplement solutions (see Ref. 3365). 10 ml **Neutralized sulfide solution 3% (w/v)** ## Solution A 0.25 g CaCl2 x 2 H2O 0.25 g Yeast extract 460 ml Distilled water 1. Aliquot Solution A into 100 mL screw-cap bottles, filled with 46 mL each. Bubble with N2/CO2 and autoclave at 121°C for 15 min. ## Sulfide solution, 1.5% 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. ## Bicarbonate solution 1.5 g NaHCO3 50 ml H2O 1. Bubble with CO2 and filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle. ## Resazurin solution 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. 2. Cool to room temperature under an atmosphere of N2/CO2 in an ice bath. ## Pfennig's heterotrophic salts solution 0.35 g Ammonium chloride 0.25 g Ammonium acetate 0.25 g Pyruvic acid sodium salt 0.25 g Dextrose 0.5 g MgSO4 x 7 H2O 0.35 g KCl 0.35 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. ## Vitamin B12 solution 0.01 g Vitamin B12 100 ml Distilled water 1. Filter sterilized ## Standard vitamin solution 10 mg Riboflavin 50 mg Thiamine-HCl x 2 H2O 50 mg Nicotinic acid 50 mg Pyridoxine hydrochloride 50 mg Calcium pantothenate 0.1 mg Biotin 0.2 mg Folic acid 1 mg Vitamin B12 100 ml Distilled water ## 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.