Medium data

Search for medium no. = [923].

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

923 PEAT MEDIUM 1 FOR METHANOREGURA BOONEI

Major metals (see below)	`10.0`	ml
Trace metal 1 solution (see below)	`1.0`	ml
Distilled water	`1.0`	L

Mix components thoroughly and adjust pH to 5.0 with HCl. Bring to a boil for 5 min, then, cool down under a stream of a N₂-CO₂ (80:20, v/v) gas mixture. Distribute the medium into Balch-type culture tubes (5 ml each) under the same gas mixture, seal with thick blue butyl rubber stoppers and autoclave.
To complete the medium, replace the gas phase with H₂-CO₂ (80:20, v/v) gas mixture, and add the following solutions to each of the culture tubes (containing 5 ml medium) from sterile anaerobic stocks (autoclaved or *filter-sterilized):

83 mM TiNTA solution* (see below)	`0.06`	ml
0.5 M HOMOPIPES solution* (pH 5.4)	`0.05`	ml
Vitamin solution* (see below)	`0.05`	ml
2% Yeast extract solution	`0.05`	ml
50 mM Coenzyme M solution	`0.05`	ml
20 mM Sodium acetate	`0.05`	ml

Check the final pH of the medium to be about 5.1. Pressurize inoculated tubes to 70 kPa H₂-CO₂ (80:20, v/v).
Major metals:

KCl	`0.15`	g
KH₂PO₄	`1.36`	g
NH₄Cl	`2.68`	g
Distilled water	`1.0`	L

Trace metal 1 solution:

CoCl₂·6H₂O	`24.0`	mg
ZnCl₂	`75.0`	mg
H₃BO₃	`19.0`	mg
NiCl₂·6H₂O	`24.0`	mg
Na₂MoO₄·2H₂O	`24.0`	mg
FeCl₂·4H₂O	`1.344`	g
MnSO₄·xH₂O	`26.0`	mg
MgSO₄·7H₂O	`1.556`	g
CaCl₂·2H₂O	`2.336`	g
CuSO₄·5H₂O	`9.0`	mg
AlK(SO₄)₂·12H₂O	`3.446`	g
Distilled water	`1.0`	L

83 mM TiNTA solution:

1 M Tris base (pH 8)	`7.2`	ml
0.5 M Nitrilotriacetic acid, disodium salt	`4.8`	ml
15% TiCl3 solution	`0.55`	ml

Vitamin solution:

Biotin	`20.0`	mg
Folic acid	`20.0`	mg
Pyridoxine·HCl	`100.0`	mg
Thiamine·HCl	`50.0`	mg
Riboflavin	`50.0`	mg
Nicotinic acid	`50.0`	mg
DL-Calcium pantothenate	`50.0`	mg
Vitamin B₁₂	`1.0`	mg
p-Aminobenzoic acid	`50.0`	mg
Lipoic acid	`50.0`	mg
Distilled water	`1.0`	L

Filter-sterilize and store under N₂ at 4C in the dark.