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Main sol. 294

Internal Solution-ID: S561

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
KH₂PO₄	0.20	g	0.199	1.465
NH₄Cl	0.25	g	0.249	4.66
NaCl	1.00	g	0.997	17.06
MgCl₂ x 6 H₂O	0.40	g	0.399	1.962
KCl	0.50	g	0.499	6.687
CaCl₂ x 2 H₂O	0.15	g	0.15	1.017
Trace element solution SL-10	1.00	ml	-	-
Selenite-tungstate solution	1.00	ml	-	-
Yeast extract	1.00	g	0.997	-
Sodium resazurin (0.1% w/v)	0.50	ml	5.0e-4	0.002
Na₂CO₃	1.50	g	1.496	14.11
Seven vitamins solution	1.00	ml	-	-
Gentisic acid , sodium salt (2,5-Dihydroxybenzoic acid)	0.30	g	0.299	1.941
Na₂S x 9 H₂O	0.10	g	0.1	0.415
Distilled water	1000.00	ml	-	-
1 Dissolve ingredients (except carbonate, vitamins, gentisic acid and sulfide), then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Dispense under same gas atmosphere in culture vessels and autoclave. Add vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas and carbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Add gentisic acid (2,5-dihydroxybenzoic acid) from a sterile and neutralized 200 mM stock solution prepared under 100% N₂ gas atmosphere. If necessary, adjust the final pH of the medium to 7.2.
2 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N₂ and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum. Some cultures are shipped in semi-solid medium which stimulates growth at the beginning. For agar stabs 3.00 g/l agar are added to the complete medium from a sterile anoxic stock solution (2% w/v). Upon receipt add anoxically 1 - 2 ml of the recommended freshly prepared liquid medium to the agar tube and incubate for 3 - 5 days. After incubation transfer 0.5 ml of the resulting cell suspension in the liquid phase to tubes with liquid medium.

Compound

Amount

Unit

Conc. [g/L]

Conc. [mM]

KH₂PO₄

0.20

0.199

1.465

NH₄Cl

0.25

0.249

4.66

NaCl

1.00

0.997

17.06

MgCl₂ x 6 H₂O

0.40

0.399

1.962

KCl

0.50

0.499

6.687

CaCl₂ x 2 H₂O

0.15

1.017

Trace element solution SL-10

1.00

Selenite-tungstate solution

1.00

Yeast extract

1.00

0.997

Sodium resazurin
(0.1% w/v)

0.50

5.0e-4

0.002

Na₂CO₃

1.50

1.496

14.11

Seven vitamins solution

1.00

Gentisic acid , sodium salt
(2,5-Dihydroxybenzoic acid)

0.30

0.299

1.941

Na₂S x 9 H₂O

0.10

0.1

0.415

Distilled water

1000.00

1 Dissolve ingredients (except carbonate, vitamins, gentisic acid and sulfide), then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 - 45 min to make it anoxic. Dispense under same gas atmosphere in culture vessels and autoclave. Add vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas and carbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Add gentisic acid (2,5-dihydroxybenzoic acid) from a sterile and neutralized 200 mM stock solution prepared under 100% N₂ gas atmosphere. If necessary, adjust the final pH of the medium to 7.2.

2 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N₂ and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum. Some cultures are shipped in semi-solid medium which stimulates growth at the beginning. For agar stabs 3.00 g/l agar are added to the complete medium from a sterile anoxic stock solution (2% w/v). Upon receipt add anoxically 1 - 2 ml of the recommended freshly prepared liquid medium to the agar tube and incubate for 3 - 5 days. After incubation transfer 0.5 ml of the resulting cell suspension in the liquid phase to tubes with liquid medium.

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Main sol. 294

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Main sol. 294

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