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Main sol. 78c

Internal Solution-ID: S133

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Ground beef (fat free)	500.0	g	586.166	-
NaOH (1 N)	25.0	ml	-	-
Casein peptone (BD BACTO)	30.0	g	35.17	-
Yeast extract	5.0	g	5.862	-
K₂HPO₄	5.0	g	5.862	33.654
Sodium resazurin (0.1% w/v)	0.5	ml	5.9e-4	0.002
L-Cysteine HCl x H₂O	0.5	g	0.586	3.337
Na-fumarate	6.0	g	7.034	43.953
Na-formate	6.0	g	7.034	103.43
Clarified rumen fluid , clarified	50.0	ml	-	-
Horse serum (inactivated, INVITROGEN)	50.0	ml	-	-
Fatty acid mixture	20.0	ml	-	-
Wolin's vitamin solution (10x)	1.0	ml	-	-
Haemin solution (0.05% w/v)	5.0	ml	-	-
Vitamin K1 solution (0.05% w/v)	2.0	ml	-	-
DL-Dithiothreitol (DTT)	0.3	g	0.352	2.279
Distilled water	700.0	ml	-	-
1 Use lean beef or horse meat. Remove fat and connective tissue before grinding. Mix meat, water and NaOH, then boil for 15 min with stirring. Cool to room temperature, skim fat off surface, and filter, retaining both meat particles and filtrate. To the filtrate add Casitone, yeast extract, hydrogen phosphate and resazurin, then add water to a final volume of 700 ml. Sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic, then add cysteine and adjust pH to 7.0. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes containing meat particles (use ca. 7 ml liquid medium for 1 - 2 g meat particles), then autoclave at 121°C for 20 min. After autoclaving add fumarate, formate, rumen fluid, horse serum, fatty acids, vitamins, haemin, vitamin K₁ and DTT rom sterile anoxic stock solutions prepared under 100% N₂ gas atmosphere and sterilized by filtration.

Compound

Amount

Unit

Conc. [g/L]

Conc. [mM]

Ground beef
(fat free)

500.0

586.166

NaOH
(1 N)

25.0

Casein peptone
(BD BACTO)

30.0

35.17

Yeast extract

5.0

5.862

K₂HPO₄

5.0

5.862

33.654

Sodium resazurin
(0.1% w/v)

0.5

5.9e-4

0.002

L-Cysteine HCl x H₂O

0.5

0.586

3.337

Na-fumarate

6.0

7.034

43.953

Na-formate

6.0

7.034

103.43

Clarified rumen fluid , clarified

50.0

Horse serum
(inactivated, INVITROGEN)

50.0

Fatty acid mixture

20.0

Wolin's vitamin solution (10x)

1.0

Haemin solution
(0.05% w/v)

5.0

Vitamin K1 solution
(0.05% w/v)

2.0

DL-Dithiothreitol
(DTT)

0.3

0.352

2.279

Distilled water

700.0

1 Use lean beef or horse meat. Remove fat and connective tissue before grinding. Mix meat, water and NaOH, then boil for 15 min with stirring. Cool to room temperature, skim fat off surface, and filter, retaining both meat particles and filtrate. To the filtrate add Casitone, yeast extract, hydrogen phosphate and resazurin, then add water to a final volume of 700 ml. Sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic, then add cysteine and adjust pH to 7.0. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes containing meat particles (use ca. 7 ml liquid medium for 1 - 2 g meat particles), then autoclave at 121°C for 20 min. After autoclaving add fumarate, formate, rumen fluid, horse serum, fatty acids, vitamins, haemin, vitamin K₁ and DTT rom sterile anoxic stock solutions prepared under 100% N₂ gas atmosphere and sterilized by filtration.

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Main sol. 78c

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