Medium data

Search for medium no. = [1424].

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

1424 METHANOBREVIBACTER CUTICULARIS MEDIUM

Rumen fluid, clarified (see Medium No. 266)	`20.0`	ml
NaCl	`1.0`	g
KCl	`0.5`	g
MgCl₂·6H₂O	`0.4`	g
CaCl₂·2H₂O	`0.1`	g
NH₄Cl	`0.3`	g
KH₂PO₄	`0.2`	g
Na₂SO₄	`0.15`	g
Casamino acids (BD Difco)	`0.5`	g
Yeast extract (Oxoid)	`0.5`	g
FeCl₂ solution (see Medium No. 187)	`1.0`	ml
Trace element solution (see Medium No. 187)	`1.0`	ml
Selenite-tungstate solution (see Medium No. 431)	`1.0`	ml
Resazurin	`0.5`	mg
Distilled water	`900.0`	ml

Mix components thoroughly, bring to a boil for 5-10 seconds, then, cool down under a stream of H₂-CO₂ (80:20 v/v). Distribute the medium into suitable culture vessels (e.g., 4.6 ml medium in Hungate tubes) under the same gas mixture and seal with butyl rubber stoppers. Autoclave. After cooling, aseptically and anaerobically add per liter the following solutions (autoclaved or *filter-sterilized):

8% NaHCO₃ solution*	`70.0`	ml
1.0 M HEPES solution (pH 7.7)	`10.0`	ml
Vitamin solution (see Medium No. 898)	`1.0`	ml

Prior to inoculation, add per liter the following solution (freshly prepared and filtersterilized):

0.1 M Dithiothreitol solution 10.0 ml

Adjust pH 7.7, if necessary. Pressurize the inoculated vessels to 200 kPa H₂-CO₂ (80:20v/ v).