Medium data

Search for medium no. = [682].

Unless otherwise stated, sterilize media by autoclaving at 121°C for 15 min.

682 METHANOFOLLIS ETHANOLICUS MEDIUM

Solution A:

Yeast extract (BD-Difco)	`0.1`	g
KH₂PO₄	`0.14`	g
MgCl₂·6H₂O	`0.2`	g
CaCl₂·2H₂O	`0.15`	g
NH₄Cl	`0.54`	g
Trace vitamins solution (see below)	`2.0`	ml
Trace element solution (see below)	`1.0`	ml
NaHCO₃	`2.5`	g
Resazurin	`1.0`	mg
Distilled water	`900.0`	ml

Mix components, except NaHCO₃, and adjust pH to 6.5. Bring to a boil for 5-10 sec., then, cool down under a stream of N₂-CO₂ (80:20, v/v) and add NaHCO₃ to the solution. Dispense 0.9 volume of the medium into culture vessels (e.g., 18 ml of the medium in 50 ml serum bottle) under a stream of N₂-CO₂ (80:20, v/v) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight.
Solution B:

Ethanol	`0.46`	ml
Distilled water	`100.0`	ml

Adjust pH to 7.0 and filter-sterilize. Keep the solution under a N₂ atmosphere.
To complete the medium, add 0.1 volume of solution B to the culture vessels containing solution A. Prior to inoculation, aseptically and anaerobically add 0.01 volume of L-cysteine·HCl·H₂O and Na₂S·9H₂O solutions to the medium (3% solutions, autoclaved and stored under a N₂ atmosphere). Incubate with a 10% inoculum.
Trace vitamins solution:

Biotin	`4.9`	mg
Folic acid	`8.8`	mg
Pyridoxine·HCl	`4.1`	mg
Thiamine·HCl	`6.7`	mg
Riboflavin	`7.5`	mg
Nicotinic acid	`2.5`	mg
DL-Calcium pantothenate	`9.5`	mg
Vitamin B₁₂	`0.1`	mg
p-Aminobenzoic acid	`2.7`	mg
Lipoic acid	`4.1`	mg
Distilled water	`1.0`	L

Trace element solution:

FeCl₃·6H₂O	`1.27`	g
MnCl₂·4H₂O	`0.2`	g
CoCl₂·6H₂O	`0.13`	g
ZnCl₂	`0.14`	g
CuCl₂·2H₂O	`0.001`	g
AlCl₃	`0.01`	g
H₃BO₃	`0.006`	g
Na₂MoO₄·2H₂O	`0.02`	g
NiCl₂·6H₂O	`0.01`	g
Na₂SeO₃	`0.002`	g
Na₂WO₄·2H₂O	`0.003`	g
Distilled water	`1.0`	L