Medium data

Search for medium no. = [600].

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

600 G5G6 MEDIUM

Solution A:

Na₂HPO₄·12H₂O	`0.5`	g
KH₂PO₄	`0.65`	g
FeCl₂ solution (see Medium No. 187)	`1.0`	ml
Trace element solution (see below)	`1.0`	ml
Resazurin	`0.5`	mg
Distilled water	`880.0`	ml

Solution B:

CaCl₂·2H₂O	`0.11`	g
MgCl₂·6H₂O	`0.1`	g
Distilled water	`50.0`	ml

Solution C:

NH₄HCO₃	`0.44`	g
NaHCO₃	`3.73`	g
L-Cysteine·HCl·H₂O	`0.175`	g
Distilled water	`50.0`	ml

Prepare all solutions and autoclave under a N₂ atmosphere. After cooling, combine the solutions and replace the gas phase with a N₂-CO₂ (4:1, v/v) gas mixture. Then, add the following autoclaved or *filter-sterilized solutions to the medium:

1.0 M NaNO₃ solution	`10.0`	ml
Trace vitamins* (see Medium No. 197)	`10.0`	ml
Vitamin B₁₂ solution* (see Medium No. 403)	`1.0`	ml

Check the final pH to be around 7.3.
Distribute appropriate amount of the medium into culture vessels under the same gas mixture (e.g., 40.0 ml in 120 ml serum bottle) and seal with Viton rubber stoppers. Finally add per liter the following solution from an anaerobic stock (autoclaved):

5.0 mM Toluene (water-saturated) 40.0 ml

Trace element solution:

Trace element solution (see Medium No. 187)	`1.0`	L
AlCl₃·6H₂O	`10.0`	mg

Comment: For strain JCM 14632, incubate with a 5-10% inoculum. When toluene is depleted, re-add the toluene solution (normally every 3 to 4 days, until enough cell growth is seen).