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DSMZ

DSMZ 397 . FERULATE MEDIUM

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Equipment needed: Autoclave Filter Gassing station Hungate tubes
Final gas composition: 80 % N2 20 % CO2 read more
Final pH: n.d.
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ml
Compound Amount Unit
Solution A 882 ml
Solution B 30 ml
Solution D 1 ml
Solution E 10 ml
Solution F 10 ml
Sodium ferulate 10 mM
1 Add and dissolve ingredients of solution A and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes and autoclave. Solution B is prepared under 80% N2 and 20% CO2 gas atmosphere and autoclaved. Solutions E and F are autoclaved under 100% N2 gas atmosphere.
2 Solution D is prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated.
3 Note: 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.
4 Add sodium ferulate to 10 mM final concentration from filter-sterilized anaerobic stock solution.

Solution A #

882
Compound Amount Unit
KH2PO4 0.50 g
MgCl2 x 6 H2O 0.33 g
NaCl 0.40 g
NH4Cl 0.40 g
CaCl2 x 2 H2O 0.05 g
Trace element solution SL-10 1.00 ml
Selenite-tungstate solution 1.00 ml
Clarified rumen fluid , clarified 50.00 ml
Trypticase peptone
(BD BBL) 		1.00 g
Sodium resazurin
(0.1% w/v) 		0.50 ml
Distilled water 830.00 ml

Solution B #

30
Compound Amount Unit
Na2CO3 1.5 g
Distilled water 30.0 ml

Solution D #

1
Compound Amount Unit
Seven vitamins solution 1 ml

Solution E #

10
Compound Amount Unit
L-Cysteine HCl x H2O 0.3 g
Distilled water 10.0 ml

Solution F #

10
Compound Amount Unit
Na2S x 9 H2O 0.3 g
Distilled water 10.0 ml

Trace element solution SL-10 #

1000
Compound Amount Unit
HCl
(25%) 		10.0 ml
FeCl2 x 4 H2O 1.5 g
ZnCl2 70.0 mg
MnCl2 x 4 H2O 100.0 mg
H3BO3 6.0 mg
CoCl2 x 6 H2O 190.0 mg
CuCl2 x 2 H2O 2.0 mg
NiCl2 x 6 H2O 24.0 mg
Na2MoO4 x 2 H2O 36.0 mg
Distilled water 990.0 ml
1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.

Selenite-tungstate solution #

1000
Compound Amount Unit
NaOH 0.5 g
Na2SeO3 x 5 H2O 3.0 mg
Na2WO4 x 2 H2O 4.0 mg
Distilled water 1000.0 ml

Seven vitamins solution #

1000
Compound Amount Unit
Vitamin B12 100 mg
p-Aminobenzoic acid 80 mg
D-(+)-biotin 20 mg
Nicotinic acid 200 mg
Calcium pantothenate 100 mg
Pyridoxine hydrochloride 300 mg
Thiamine-HCl x 2 H2O 200 mg
Distilled water 1000 ml

Clarified rumen fluid #

1000
1 Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C.