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DSMZ

DSMZ 843 . HELMUTKOENIGIA MEDIUM

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Equipment needed: Autoclave Centrifuge Filter Gassing station Hungate tubes
Final gas composition: 80 % N2 20 % CO2 read more
Final pH: 7.2 - 7.4
Select a strain (optional):

ml
Compound Amount Unit
NaCl 1.00 g
KCl 0.50 g
MgCl2 x 6 H2O 0.40 g
CaCl2 x 2 H2O 0.10 g
NH4Cl 0.30 g
KH2PO4 0.20 g
Na2SO4 0.15 g
Trace element solution SL-10 1.00 ml
Selenite-tungstate solution 1.00 ml
Trypticase peptone
(BD BBL) 		1.00 g
Sodium resazurin
(0.1% w/v) 		0.50 ml
Na2CO3 1.50 g
Sugar mix 30.00 ml
Yeast autolysate 50.00 ml
Seven vitamins solution 1.00 ml
Co-factors solution 20.00 ml
DL-Dithiothreitol
(DTT) 		0.40 g
Distilled water 900.00 ml
1 Dissolve ingredients (except carbonate, sugar mix, yeast autolysate, vitamins, co-factors, and DTT), sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume (e.g., 4.5 ml per Hungate-type tube) and autoclave. After sterilization add carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere and sugar mix, yeast autolysate, vitamins, co-factors and DTT from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and sterilized by filtration. Check pH of complete medium and adjust to 7.2 - 7.4, if necessary.
2 Note: Basal medium and yeast autolysate have to be prepared freshly and cannot be stored for longer periods.

Sugar mix #

1000
Compound Amount Unit
Cellobiose 56.7 g
Sucrose 56.7 g
Maltose x H2O 60.0 g
Trehalose 63.3 g
D-Xylose 25.0 g
Distilled water 1000.0 ml

Yeast autolysate #

1000
Compound Amount Unit
Yeast extract
(dry baker's yeast, SIGMA YSC-1) 		280 g
Distilled water 1000 ml
1 Suspend dry baker's yeast (SIGMA YSC-1) in distilled water and allow cells to autolyse for 48 h at 55°C. Thereafter, cell debris are removed by centrifugation (3.700 x g for 30 min) and the amber-colored supernatant is stored in aliquots at -20°C.

Co-factors solution #

1000
Compound Amount Unit
Thiamine pyrophosphate 2.50 g
Pyridoxal hydrochloride 0.25 g
Pyridoxal phosphate 0.25 g
Ca-folinate 0.05 g
ß-NAD 0.05 g
Coenzyme A 0.05 g
FAD 0.05 g
Haemin
(dissolved separately in 10 mM NaOH) 		65.00 mg
Nicotinamide 25.00 mg
Folic acid 2.50 mg
Riboflavin 0.50 mg
Distilled water 1000.00 ml
1 Adjust pH to 7.0 with 1 N NaOH against indicator paper, filter sterilize and store under 100% N2 gas atmosphere.

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