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Main sol. 1267

Internal Solution-ID: S2523

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Solution A	901	ml	-	-
Solution B	1	ml	-	-
Solution C	100	ml	-	-
1 Preparation of the bottom layer: Mix 1 volume of solution A (except MES buffer, NaHCO₃, and agarose) with 1 volume of solution C and add 1% (w/v) agarose type 1, low EEO. After autoclaving, aseptically fill 1 ml of the suspension in sterile Hungate-type tubes (15 ml total volume). The bottom layer solidifies in approx. 30 min.
2 Preparation of the top layer: Add MES buffer, bicarbonate and low melt agarose to solution A and autoclave. Let the sterile solution cool to 40°C and add 10.00 ml/l of solution B. Sparge solution with sterile CO₂ gas until a pH of 6.1 - 6.4 is reached. Then, aseptically pipette aliquots of 5.0 ml over the bottom layer of each tube under 100% N₂ gas atmosphere and let medium equilibrate for at least three hours, but not longer than 12 hours.
3 Inoculation: Inoculate the semisolid top layer under a stream of 80% N₂ and 20% CO₂ gas mixture using a sterile Pasteur pipette that is inserted just above the FeS layer; the pipette tip is drawn upward as the inoculum is dispensed. After inoculation close the tube and add 1.00 ml of sterile air.
4 Note: Some hints on the inoculation of Hungate-tubes under anoxic conditions can be found in the Video tutorial on the „Proper Handling of the Double-Vial Glass Ampoules (anaerobe)" (www.dsmz.de/shop-1/faqs/video-tutorials).

Compound

Amount

Unit

Conc. [g/L]

Conc. [mM]

Solution A

901

Solution B

Solution C

100

1 Preparation of the bottom layer: Mix 1 volume of solution A (except MES buffer, NaHCO₃, and agarose) with 1 volume of solution C and add 1% (w/v) agarose type 1, low EEO. After autoclaving, aseptically fill 1 ml of the suspension in sterile Hungate-type tubes (15 ml total volume). The bottom layer solidifies in approx. 30 min.

2 Preparation of the top layer: Add MES buffer, bicarbonate and low melt agarose to solution A and autoclave. Let the sterile solution cool to 40°C and add 10.00 ml/l of solution B. Sparge solution with sterile CO₂ gas until a pH of 6.1 - 6.4 is reached. Then, aseptically pipette aliquots of 5.0 ml over the bottom layer of each tube under 100% N₂ gas atmosphere and let medium equilibrate for at least three hours, but not longer than 12 hours.

3 Inoculation: Inoculate the semisolid top layer under a stream of 80% N₂ and 20% CO₂ gas mixture using a sterile Pasteur pipette that is inserted just above the FeS layer; the pipette tip is drawn upward as the inoculum is dispensed. After inoculation close the tube and add 1.00 ml of sterile air.

4 Note: Some hints on the inoculation of Hungate-tubes under anoxic conditions can be found in the Video tutorial on the „Proper Handling of the Double-Vial Glass Ampoules (anaerobe)" (www.dsmz.de/shop-1/faqs/video-tutorials).

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Main sol. 1267

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