Medium data

Search for medium no. = [872].

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

872 METHANOBACTERIUM MEDIUM (IV)

KH₂PO₄	`0.5`	g
MgSO₄·7H₂O	`0.4`	g
NaCl	`5.0`	g
NH₄Cl	`0.4`	g
CaCl₂·2H₂O	`0.05`	g
FeSO₄·7H₂O	`0.002`	g
FeCl₂ solution (see Medium No. 187)	`1.0`	ml
Trace element solution (see Medium No. 187)	`1.0`	ml
Yeast extract	`1.0`	g
Sludge fluid (see below)	`50.0`	ml
Sodium acetate	`1.0`	g
Sodium formate	`2.0`	g
NaHCO₃	`4.0`	g
Fatty acid mixture (see Medium No. 266)	`20.0`	ml
Resazurin	`1.0`	mg
Distilled water	`930.0`	ml

Mix components except NaHCO₃ and bring to a boil for 5-10 sec. Cool down under a stream of H₂-CO₂ (80:20, v/v) and add NaHCO₃ to the medium. Dispense the medium into culture vessels under a stream of H₂-CO₂ (80:20, v/v), seal with butyl rubber stoppers and autoclave. Prior to inoculation, add per liter the following solutions (autoclaved and stored under a N₂ atmosphere):

5% Na₂S·9H₂O solution	`10.0`	ml
5% L-Cysteine·HCl·H₂O solution	`10.0`	ml

Pressurize inoculated vessels to 200 kPa with a H₂-CO₂ (80:20, v/v) gas mixture.
Sludge fluid:
Add 0.4% yeast extract to sludge from an anaerobic digester and gassing with a N₂ gas for a few minutes. Incubate it at 37C for 24 hr under a N₂ atmosphere. Then centrifuge the sludge at 13,000 x g and autoclave the resulting clear supernatant in screw-capped vessels under a N₂ atmosphere. The sludge fluid can be stored at room temperature in the dark.