# 377: PYROCOCCUS MEDIUM ## Main sol. 377 13.85 g NaCl 3.5 g MgSO4 x 7 H2O 2.75 g MgCl2 x 6 H2O 0.33 g KCl 0.05 g NaBr 15 mg H3BO3 7 ml SrCl2 x 6 H2O (0.1% w/v) 10 mg (NH4)2SO4 5 ml Citric acid (0.1% w/v) 0.5 ml KI (0.01% w/v) 0.75 g CaCl2 x 2 H2O 0.5 g KH2PO4 2 ml NiCl2 x 6 H2O (0.1% w/v) 10 ml **Modified Wolin's mineral solution** 0.5 ml Sodium resazurin (0.1% w/v) 30 g Sulfur (powdered) 5 g Peptone (BD Bacto) 1 g Yeast extract (OXOID) 15 ml **Neutralized sulfide solution 3% (w/v)** 960 ml Distilled water 1. Dissolve ingredients except for sulfur, peptone, yeast extract, and sulfide, adjust the pH to 6.5 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Distribute medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials that already contain the appropriate amount of sulfur. Sterilize the medium by autoclaving at 110°C for 20 min. After sterilization, add peptone and yeast extract from sterile stock solutions prepared under 100% N2 gas atmosphere and sulfide from a neutralized stock solution. ## Modified Wolin's mineral solution 1.5 g Nitrilotriacetic acid 3 g MgSO4 x 7 H2O 0.5 g MnSO4 x H2O 1 g NaCl 0.1 g FeSO4 x 7 H2O 0.18 g CoSO4 x 7 H2O 0.1 g CaCl2 x 2 H2O 0.18 g ZnSO4 x 7 H2O 0.01 g CuSO4 x 5 H2O 0.02 g AlK(SO4)2 x 12 H2O 0.01 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.03 g NiCl2 x 6 H2O 0.3 mg Na2SeO3 x 5 H2O 0.4 mg Na2WO4 x 2 H2O 1000 ml Distilled water 1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. ## 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.