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|Dissolve ingredients (except thiosulfate, bicarbonate and vitamins), boil medium for 1 min., then cool to room temperature under 80% N2 and 20% CO2 gas atmosphere. Dispense under same gas atmosphere in culture vessels (up to a volume of 20%) and autoclave. Add vitamin solution, Na2S2O3 x 5 H2O and NaHCO3 to the autoclaved medium from sterile, anoxic stock solutions. Solutions of vitamins and thiosulfate are sterilized by filtration and stored under N2, whereas the solution of bicarbonate is prepared under 80% N2 and 20% CO2 gas mixture and autoclaved. Adjust the final pH of the medium to 6.7. After inoculation pressurize vessels to 0.5 bar overpressure with 80% N2 and 20% CO2 gas mixture and add sterile air in an amount that is equivalent to a volume of 20% of the headspace.|
|CaCl2 · 2 H2O||0.14||g|
|MgSO4 · 7 H2O||3.40||g|
|MgCl · 6 H2O||4.18||g|
|NiCl2 · 6 H2O||0.50||mg|
|Na2SeO3· 5 H2O||0.50||mg|
|Fe(NH4)2(SO4)2 x 6 H2O||0.01||g|
|Trace element solution||10.00||ml|
|Na2S2O3 · 5 H2O||1.50||g|
Cultivation medium for strain SW7.
For the pure culture of strain SW7, we are using MJY medium with NaHCO3 consists of MJ systhetic seawater (Sako et al. 1996; see KCTC medium 934) with yeast extract (Difco), 1 gL-1 and NaHCO_3_ 1g L-1. Final pH is expected between 6-7. pH range for stable growth is very narrow.
1. After adding resazurin, medium except for Na2S uder N2/CO2 gas mixture(80-20) or N2 gas is autoclaved.
2. Pressurize the medium with N2/CO2 gas mixture(80:20) at 1.5-2 atm. Final pH is expected to be between 6 and 7
3. Add neutralized Na2S solution
4. Growth will be observed after 1-3days of incubation. However, after the first inoculation from storage culture, it will take a week
Note: We use tube culture, but it is sometimes difficult ro oberve the growth because of salt precipitation. Cultivation using a serum vial is comfortable for growth observation