MediaDive DSMZ
Content
Media Solutions Ingredients Strains Steps Gas
Search
Medium finder Solution finder Taxonomy search Isolation sources
Tools
Medium builder NEW Unit converter Compare media Compare solutions Prediction SOON
DSMZ

DSMZ 334a . METHANOTHRIX SOEHNGENII MEDIUM

What is this page?
Equipment needed: Autoclave Centrifuge Filter Gassing station Serum bottle
Final gas composition: 80 % N2 20 % CO2 read more
Final pH: 7.2
Select a strain (optional):

ml
Compound Amount Unit
Solution A 942 ml
Solution B 25 ml
Solution C 10 ml
Solution D 10 ml
Solution E 1 ml
Solution F 1 ml
Solution G 1 ml
Solution H 10 ml
1 Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, dispense under same gas atmosphere into anoxic serum vials or balch tubes (e.g., 20 ml medium in 50 ml bottles) and autoclave. Prior to inoculation complete the medium by adding sterile solutions C - H prepared under 100% N2 gas and solution B prepared under 80% N2 and 20% CO2 gas atmosphere. Solutions E, F and G are sterilized by filtration. Adjust the pH of the complete medium to 7.2.
2 Notes: Medium is only stable for a few weeks and has to be prepared freshly. Use 20% (v/v) inoculum. Sodium sulfide should be as pure as possible (use only clean crystals for the preparation of stock solutions). It has been noted that impurities of sulfide stock solutions can inhibit growth.

Solution A #

942
Compound Amount Unit
KH2PO4 0.22 g
Na2HPO4 x 2 H2O 0.86 g
Na-acetate 6.80 g
Na2-EDTA
(0.1% w/v) 		1.00 ml
Trace elements solution 10.00 ml
Sodium resazurin
(0.1% w/v) 		0.50 ml
Cell-free culture supernatant of S. associata 100.00 ml
Distilled water 830.00 ml

Solution B #

25
Compound Amount Unit
Na2CO3 1.25 g
Distilled water 25.00 ml

Solution C #

10
Compound Amount Unit
NaCl 0.30 g
CaCl2 x 2 H2O 0.11 g
MgCl2 x 6 H2O 0.10 g
Distilled water 10.00 ml

Solution D #

10
Compound Amount Unit
NH4Cl 0.3 g
Distilled water 10.0 ml

Solution E #

1
Compound Amount Unit
Wolin's vitamin solution (10x) 1 ml

Solution F #

1
Compound Amount Unit
Butyl vinyl ether
(ALDRICH 110299) 		5 mg
Methanol 1 ml

Solution G #

1
Compound Amount Unit
L-α-Phosphatidylcholine
(SIGMA P3556) 		5 mg
Methanol 1 ml

Solution H #

10
Compound Amount Unit
Na2S x 9 H2O 0.5 g
Distilled water 10.0 ml

Trace elements solution #

1000
Compound Amount Unit
Nitrilotriacetic acid
(NTA) 		12.80 g
FeCl3 x 6 H2O 1.35 g
MnCl2 x 4 H2O 0.10 g
CoCl2 x 6 H2O 0.03 g
CaCl2 x 2 H2O 0.10 g
ZnCl2 0.10 g
CuCl2 0.03 g
H3BO3 0.01 g
Na2MoO4 x 2 H2O 0.03 g
NiCl2 x 6 H2O 0.12 g
NaCl 1.00 g
Na2SeO3 x 5 H2O 0.03 g
Distilled water 1000.00 ml
1 First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 ml.

Cell-free culture supernatant of S. associata #

1000
1 Cultivate Sphaerochaeta associata DSM 26261 in DSM medium 119 supplemented with 1.00 g/l glucose at 28°C until stationary phase is reached (7-10 days). Centrifuge culture at 3500 xg for 30 min, then sterilize supernatant by filtration and add it to the anoxic solution A before autoclaving.
2 Note: It is also possible to add 50.00 ml/l of a grown S. associata culture to solution A before autoclaving, provided bacterial DNA from dead cells is not a problem in the downstream processing of the Methanothrix culture.

Wolin's vitamin solution (10x) #

1000
Compound Amount Unit
Biotin 20 mg
Folic acid 20 mg
Pyridoxine hydrochloride 100 mg
Thiamine HCl 50 mg
Riboflavin 50 mg
Nicotinic acid 50 mg
Calcium D-(+)-pantothenate 50 mg
Vitamin B12 1 mg
p-Aminobenzoic acid 50 mg
(DL)-alpha-Lipoic acid 50 mg
Distilled water 1000 ml