Medium data

Search for medium no. = [266].

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

266 METHANOMICROBIUM MOBILE MEDIUM

Rumen fluid, clarified (see below)	`300.0`	ml
KH₂PO₄	`0.3`	g
K₂HPO₄	`0.3`	g
(NH₄)₂SO₄	`0.3`	g
NaCl	`0.6`	g
MgSO₄·7H₂O	`0.13`	g
CaCl₂·2H₂O	`8.0`	mg
Trace minerals (see Medium No. 151)	`10.0`	ml
Trace vitamins (see Medium No. 197)	`10.0`	ml
FeSO₄·7H₂O	`2.0`	mg
NaHCO₃	`2.0`	g
Yeast extract (BD-Difco)	`1.0`	g
Trypticase peptone (BD-BBL)	`1.0`	g
Fatty acid mixture (see below)	`20.0`	ml
Resazurin	`1.0`	mg
Na₂S·9H₂O	`0.5`	g
L-Cysteine·HCl·H₂O	`0.5`	g
Distilled water	`660.0`	ml

Mix ingredients, except NaHCO₃, L-cysteine·HCl·H₂O and Na₂S·9H₂O, and adjust pH to 6.5. Bring to a boil for 5-10 sec, then, cool down under a stream of H₂-CO₂ (80:20, v/v) and add NaHCO₃ to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H₂-CO₂ (80:20, v/v) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine·HCl and Na₂S·9H₂O as 5% solutions, respectively, under a N₂ atmosphere. Prior to inoculation, aseptically and anaerobically add the L-cysteine·HCl·H₂O and Na₂S·9H₂O solutions. 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.
Fatty acid mixture:

Valeric acid	`0.5`	g
Isovaleric acid	`0.5`	g
α-Methylbutyric acid	`0.5`	g
Isobutyric acid	`0.5`	g
Distilled water	`20.0`	ml

Adjust pH to 7.5 with concentrated NaOH solution.