Medium data

Search for medium no. = [355].

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

355 METHANOSPHAERA MEDIUM

Rumen fluid, clarified (see below)	`100.0`	ml
Yeast extract (BD-Difco)	`2.0`	g
Trypticase peptone (BD-BBL)	`2.0`	g
Sodium acetate	`0.5`	g
Sodium formate	`0.5`	g
Trace minerals (see Medium No. 151)	`10.0`	ml
Trace vitamins (see Medium No. 197)	`1.0`	ml
Na₂SeO₄	`0.19`	mg
NiCl₂·6H₂O	`0.7`	mg
FeSO₄·7H₂O	`3.0`	mg
KH₂PO₄	`2.8`	g
K₂HPO₄	`0.6`	g
(NH₄)₂SO₄	`0.3`	g
NH₄Cl	`1.0`	g
NaCl	`0.6`	g
MgSO₄·7H₂O	`0.15`	g
CaCl₂·2H₂O	`0.076`	g
Methanol	`5.0`	ml
Resazurin	`1.0`	mg
L-Cysteine·HCl·H₂O	`0.875`	g
Na₂S·9H₂O	`0.375`	g
Distilled water	`900.0`	ml

Mix ingredients, except methanol, L-cysteine·HCl·H₂O and Na₂S·9H₂O, and adjust pH to 7.0. Bring to a boil for 5-10 sec, then, cool down under a stream of N₂ and add methanol to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in a 100-ml serum bottle) under a stream of N₂ and seal. After autoclaving, stand the medium overnight. Separately autoclave L-cysteine·HCl·H₂O and Na₂S·9H₂O as 5% solutions, respectively, under a N₂ atmosphere. Prior to inoculation, replace the gas phase with H₂-CO₂ (80:20, v/v), and add the L-cysteine·HCl·H₂O and Na₂S·9H₂O solutions aseptically and anaerobically. Pressurize the inoculated bottles to 200 kPa H₂-CO₂ (80:20, v/v). To prepare the clarified rumen fluid, preheat rumen content at 120C for 15 min. Use the supernatant after centrifuging at 25,000 x g for 15 min.