Medium data

Search for medium no. = [284].

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

284 SI MEDIUM

Solution A:

Yeast extract (BD-Difco)	`0.1`	g
KH₂PO₄	`0.14`	g
MgCl₂·6H₂O	`0.2`	g
CaCl₂·2H₂O	`0.15`	g
NH₄Cl	`0.54`	g
Trace vitamins solution (see below)	`2.0`	ml
Trace element solution (see below)	`1.0`	ml
Se/W solution (see below)	`1.0`	ml
NaHCO₃	`2.5`	g
Resazurin	`1.0`	mg
Distilled water	`900.0`	ml

Mix components except NaHCO₃ and adjust pH to 6.5. Bring to a boil for 5-10 sec, then, cool down under a stream of N₂-CO₂ (80:20, v/v) and add NaHCO₃ to the solution. Dispense 0.9 volume of the medium into culture vessels (e.g., 18 ml of the medium in 50 ml serum bottle) under a stream of N₂-CO₂ (80:20, v/v) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight.
Solution B:

Sodium pyruvate	`2.2`	g
Distilled water	`100.0`	ml

Adjust pH to 7.0 and filter-sterilize. Keep the solution under a N₂ atmosphere.
To complete the medium, add 0.1 volume of Solution B to the culture vessels containing Solution A. Prior to inoculation, aseptically and anaerobically add 0.01 volume of L-cysteine·HCl·H₂O and Na₂S·9H₂O solutions to the medium (3% solutions, autoclaved and stored under a N₂ atmosphere).
Comment: For strain JCM 10971, incubate with a 10% inoculum.
Trace vitamins solution:

Biotin	`4.9`	mg
Folic acid	`8.8`	mg
Pyridoxine·HCl	`4.1`	mg
Thiamine·HCl	`6.7`	mg
Riboflavin	`7.5`	mg
Nicotinic acid	`2.5`	mg
DL-Calcium pantothenate	`9.5`	mg
Vitamin B₁₂	`27.1`	mg
p-Aminobenzoic acid	`2.7`	mg
Lipoic acid	`4.1`	mg
Distilled water	`1.0`	L

Trace element solution:

Nitrilotriacetic acid	`12.8`	g
FeCl₃·6H₂O	`1.35`	g
MnCl₂·4H₂O	`0.1`	g
CoCl₂·6H₂O	`0.024`	g
CaCl₂·2H₂O	`0.1`	g
ZnCl₂	`0.1`	g
CuCl₂·2H₂O	`0.025`	g
H₃BO₃	`0.01`	g
Na₂MoO₄·2H₂O	`0.024`	g
NiCl₂·6H₂O	`0.12`	g
Distilled water	`1.0`	L

Dissolve nitrilotriacetic acid first in 200 ml of distilled water and adjust pH to 6.5 with KOH. Then add the remaining salts and bring volume to 1.0 L. Readjust pH to 7.0 with KOH.
Se/W solution:

Na₂SeO₃·5H₂O	`4.0`	mg
Na₂WO₄·2H₂O	`4.0`	mg
Distilled water	`1.0`	L