Medium data

Search for medium no. = [747].

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

747 FRESHWATER MEDIUM WITH TOLUENE

Solution A:

Iron(III) citrate	`3.0`	g
NaCl	`1.0`	g
KCl	`0.5`	g
MgCl₂·6H₂O	`0.4`	g
NH₄Cl	`0.25`	g
KH₂PO₄	`0.2`	g
CaCl₂·2H₂O	`0.1`	g
Na₂SO₄	`1.42`	mg
Sodium acetate·3H₂O	`1.36`	g

First dissolve iron(III) citrate in 800 ml distilled water and adjust pH to 7.0 with 10 N NaOH. Then, add the remaining minerals, bring volume to 970 ml and autoclave under a N₂ atmosphere. Add aseptically and anaerobically the following filter-sterilized solutions:

8.5% NaHCO₃ solution	`30.0`	ml
FeCl₂ solution (see Medium No. 187)	`1.0`	ml
Trace element solution (see Medium No. 187	`1.0`	ml
Vitamin solution (see Medium No. 403)	`1.0`	ml
Thiamine solution (see Medium No. 403)	`1.0`	ml
Vitamin B₁₂ solution (see Medium No. 403)	`1.0`	ml
Selenite-tungstate solution (see Medium No. 431)	`1.0`	ml

Distribute the medium into sterile culture vessels (e.g., 50 ml per 120 ml bottle) under a N₂-CO₂ (4:1, v/v) gas mixture, and seal with sterile butyl rubber stoppers. Then add per 50 ml the following solutions from anaerobic stocks:

100 mM FeCl₂ solution	`0.5`	ml
Toluene	`5.0`	µl

Check the final pH of the medium to be around 7.0.