Medium data

Search for medium no. = [301].

Unless otherwise stated, sterilize media by autoclaving at 121°C for 15 min.

301 THERMODESULFOBACTERIUM HYDROGENOPHILUM MEDIUM

NaCl	`30.0`	g
MgCl₂·6H₂O	`3.0`	g
Na₂SO₄	`4.0`	g
KCl	`0.5`	g
NH₄Cl	`0.25`	g
KH₂PO₄	`0.2`	g
PIPES	`3.46`	g
CaCl₂·2H₂O	`0.15`	g
Yeast extract (BD-Difco)	`0.5`	g
Sodium acetate	`0.83`	g
Trace element solution (see below)	`1.0`	ml
Na₂WO₄·2H₂O	`0.2`	mg
Na₂SeO₄	`0.05`	mg
Trace vitamins (see Medium No. 197)	`10.0`	ml
Vitamin B₁₂	`0.05`	mg
Growth stimulating factors (see below)	`1.0`	ml
Resazurin	`1.0`	mg
Na₂S·9H₂O	`0.5`	g
Distilled water	`1.0`	L

Mix ingredients, except Na₂S·9H₂O, and adjust pH to 6.5 with HCl. Distribute the medium into culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H₂-CO₂ (4:1, v/v) gas mixture and seal with butyl rubber stoppers, and autoclave. Neutralize Na₂S·9H₂O as a 5% solution, and autoclave under a N₂ atmosphere. Add the sterile Na₂S solution to the medium, and pressurize the bottles to 200 kPa H₂-CO₂ (4:1, v/v). After incubation at 70C for 1 hr, readjust pH to 6.5 with a sterile and anaerobic NaHCO₃ stock solution.
Trace element solution:

Nitrilotriacetic acid	`12.8`	g
FeSO₄·7H₂O	`0.42`	g
H₃BO₃	`0.01`	g
MnCl₂·4H₂O	`0.1`	g
CoCl₂·6H₂O	`0.17`	g
CuCl₂·2H₂O	`0.02`	g
Zn₂SO₄·7H₂O	`0.21`	g
Na₂MoO₄·2H₂O	`0.01`	g

Dissolve nitrilotriacetic acid in 800 ml distilled water and adjust pH to 6.5 with NaOH. Then proceed to add the minerals and bring volume to 1.0 L.
Growth stimulating factors:

Isobutyric acid	`0.5`	g
Valeric acid	`0.5`	g
2-Methyl-butyric acid	`0.5`	g
3-Methyl-butyric acid	`0.5`	g
Caproic acid	`0.2`	g
Succinic acid	`0.6`	g
Distilled water	`100.0`	ml

Adjust pH to 9.0 with NaOH.