Medium data

Search for medium no. = [556].

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

556 METHYLOSOMA DIFFICILE MEDIUM

NaCl	`0.1`	g
MgCl₂·6H₂O	`0.04`	g
KCl	`0.05`	g
CaCl₂·2H₂O	`0.015`	g
Na₂SO₄	`0.016`	g
Distilled water	`993.0`	ml

Mix components and autoclave. After cooling, add the following solutions (autoclaved or filter-sterilized) to the medium:

2 M HEPES buffer (pH 7.0)	`5.0`	ml
1 M Phosphate buffer (pH 7.2, see below)	`0.15`	ml
0.09 M KNO₃ solution	`0.544`	ml
FeCl₂ solution (see Medium No. 187)	`1.0`	ml
Trace element solution (see Medium No. 187)	`1.0`	ml
Vitamin solution (filter-sterilized, see below)	`1.0`	ml

Distribute the culture medium into culture tubes (leave >80% volume as a gas phase) and seal with butyl rubber stoppers.
Finally, replace the gas phase with 20% methane and 5% CO₂ in air (by volume).
1 M Phosphate buffer:
Mix 28 ml of 1 M KH₂PO₄ and 78 ml of 1 M Na₂HPO₄ to prepare 100 ml of 1 M phosphate buffer.
Vitamin solution:

Vitamin B₁₂	`50.0`	mg
p-Aminobenzoic acid	`50.0`	mg
Biotin	`10.0`	mg
Nicotinic acid	`100.0`	mg
DL-Calcium pantothenate	`25.0`	mg
Pyridoxine·HCl	`250.0`	mg
Thiamine·HCl	`50.0`	mg
Distilled water	`1.0`	L