ATCC medium: 1340 MS medium for methanogens
Mineral Solution #1 (see below).....................15.0 ml
Mineral Solution #2 (see below).....................15.0 ml
20% Yeast Extract - Trypticase Solution (see below)..4.0 ml
20% Sodium formate...................................6.0 ml
25% Sodium acetate...................................4.0 ml
0.2% FeSO4 . 7H2O ....................................0.4 ml
0.1% Resazurin.......................................0.4 ml
Wolfe's Vitamin Solution (see below).................4.0 ml
Wolfe's Mineral Solution (see below).................4.0 ml
NaHCO3 ...............................................2.4 g
Agar (if needed).....................................8.0 g
Cysteine-Sulfide Reducing Agent (see below).........16.0 ml
Distilled water....................................340.0 ml
Bring to boil under 80% N2, 20% CO2. Wait until reduced. Stopper with butyl rubber. Dispense under same gas phase into anaerobic aluminum seal type culture tubes (Bellco #2048-18150).
Adjust final pH to 7.0. Autoclave at 121C for 15 min.
Mineral Solution #1: K2HPO4 ..............................6.0 g
Distilled water.....................1.0 L
Mineral Solution #2: KH2PO4 ..............................6.0 g
(NH4)2SO4............................6.0 g
NaCl................................12.0 g
MgSO4 . 7H2O ........................2.6 g
CaCl2 . 2H2O ........................0.16 g
Distilled water.....................1.0 L
20% Yeast Extract - Trypticase Solution:
Yeast extract.......................20.0 g
Trypticase Peptone (BD 211921)......20.0 g
Distilled water to..................100.0 ml
Cysteine-Sulfide Reducing Agent: N NaOH (4 g/100 ml).................................30.0 ml
L-Cysteine . HCl.....................................5.0 g
12.5% Na2S stock solution (freshly prepared) ........40.0 ml
Distilled water....................................330.0 ml
Add distilled water to 500 ml round-bottom flask. Add fresh NaOH; bring to boil under N2.
Remove gassing probe, add cysteine (swirling to dissolve), then Na2S solution.
Renew gassing for several minutes; seal with butyl rubber stoppers. Transfer to hood and dispense into 8.0 ml/18 mm Hungate tubes.