Medium data

Search for medium no. = [228].
Unless otherwise stated, sterilize media by autoclaving at 121°C for 15 min.

228   METHANOCOCCUS McC MEDIUM

General salts solution (see below)  500.0     ml
Trace minerals solution (see below)  10.0     ml
Iron stock solution (see below)  5.0     ml
NaCl  22.0     g
K2HPO4  0.14    g
Sodium acetate  1.36    g
Yeast extract (BD-Difco)  2.0     g
Resazurin  1.0     mg
NaHCO3  5.0     g
L-Cysteine·HCl·H2O  0.5     g
Na2S·9H2O  0.5     g
Distilled water  485.0     ml
Mix ingredients, except cysteine·HCl and Na2S·9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of N2-CO2 (80:20). Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-CO2 (80:20), seal with butyl rubber stoppers, and autoclave. Separately autoclave cysteine·HCl and Na2S·9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine·HCl and Na2S·9H2O solutions. Pressurize the inoculated vessels to 200 kPa H2-CO2 (80:20).
General salts solution:
MgCl2·6H2O  5.5     g
MgSO4·7H2O  6.9     g
NH4Cl  1.0     g
KCl  0.67    g
CaCl2·2H2O  0.28    g
Trace minerals solution:
Nitrilotriacetic acid  1.5     g
Fe(NH4)2(SO4)2·6H2O  0.2     g
Na2SeO3  0.1     g
Na2MoO4·2H2O  0.1     g
Na2WO4·2H2O  1.0     g
MnSO4·xH2O  0.1     g
ZnSO4·7H2O  0.1     g
NiCl2·6H2O  0.025   g
CuSO4·5H2O  0.01    g
Distilled water  1.0     L
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH. Then proceed to add minerals.
Adjust final pH to 7.0.
Iron stock solution:
Dissolve 0.2 g of Fe(NH4)2(SO4)2·6H2O in 5 ml of distilled water containing 2 drops of concentrated HCl. When the Fe(NH4)2(SO4)2·6H2O crystal have dissolved, bring volume up to 100 ml.



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