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Main sol. J355

Internal Solution-ID: S4074

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Rumen fluid , clarified (see below)	100.000	ml	-	-
Yeast extract (BD-Difco)	2.000	g	1.969	-
Trypticase peptone (BD-BBL)	2.000	g	1.969	-
Sodium acetate	0.500	g	0.492	5.999
Sodium formate	0.500	g	0.492	7.236
Trace minerals	10.000	ml	-	-
Trace vitamins	1.000	ml	-	-
Na₂SeO₄	0.190	mg	1.9e-4	9.9e-4
NiCl₂ x 6 H₂O	0.700	mg	6.9e-4	0.005
FeSO₄ x 7 H₂O	3.000	mg	0.003	0.011
KH₂PO₄	2.800	g	2.756	20.251
K₂HPO₄	0.600	g	0.591	3.391
(NH₄)₂SO₄	0.300	g	0.295	2.235
NH₄Cl	1.000	g	0.984	18.401
NaCl	0.600	g	0.591	10.105
MgSO₄ x 7 H₂O	0.150	g	0.148	0.599
CaCl₂ x 2 H₂O	0.076	g	0.075	0.509
Methanol	5.000	ml	-	-
Resazurin	1.000	mg	9.8e-4	0.004
L-Cysteine HCl x H₂O	0.875	g	0.861	-
Na₂S x 9 H₂O	0.375	g	0.369	1.537
Distilled water	900.000	ml	-	-
1 Mix ingredients, except methanol, L-cysteine x HCl x H₂O and Na₂S x 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 x HCl x H₂O and Na₂S x 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 x HCl x H₂O and Na₂S x 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.

Compound

Amount

Unit

Conc. [g/L]

Conc. [mM]

Rumen fluid , clarified
(see below)

100.000

Yeast extract
(BD-Difco)

2.000

1.969

Trypticase peptone
(BD-BBL)

2.000

1.969

Sodium acetate

0.500

0.492

5.999

Sodium formate

0.500

0.492

7.236

Trace minerals

10.000

Trace vitamins

1.000

Na₂SeO₄

0.190

1.9e-4

9.9e-4

NiCl₂ x 6 H₂O

0.700

6.9e-4

0.005

FeSO₄ x 7 H₂O

3.000

0.003

0.011

KH₂PO₄

2.800

2.756

20.251

K₂HPO₄

0.600

0.591

3.391

(NH₄)₂SO₄

0.300

0.295

2.235

NH₄Cl

1.000

0.984

18.401

NaCl

0.600

0.591

10.105

MgSO₄ x 7 H₂O

0.150

0.148

0.599

CaCl₂ x 2 H₂O

0.076

0.075

0.509

Methanol

5.000

Resazurin

1.000

9.8e-4

0.004

L-Cysteine HCl x H₂O

0.875

0.861

Na₂S x 9 H₂O

0.375

0.369

1.537

Distilled water

900.000

1 Mix ingredients, except methanol, L-cysteine x HCl x H₂O and Na₂S x 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 x HCl x H₂O and Na₂S x 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 x HCl x H₂O and Na₂S x 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.

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Main sol. J355

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Main sol. J355

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