Medium data

Search for medium no. = [915].

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

915 SEMI-SOLID AUTOTROPHIC GROWTH MEDIUM FOR MAGNETOCOCCUS MARINUS

NaCl	`16.4`	g
MgCl₂·6H₂O	`3.49`	g
Na₂SO₄	`2.74`	g
KCl	`0.47`	g
CaCl₂·2H₂O	`0.39`	g
NH₄Cl	`0.3`	g
Modified Wolfe's mineral solution (see below)	`5.0`	ml
Resazurin	`0.4`	mg
Distilled water	`1.0`	L
Agarose	`2.0`	g

Mix components thoroughly, except agar, and adjust pH to 7.0. Add agarose to the medium and autoclave under a N₂ gas atmosphere. After cooling to 50C, add the following solutions to the medium aseptically and anaerobically (autoclaved or *filter-sterilized):

0.5 M Potassium phosphate buffer (pH 7.0)	`1.8`	ml
40% Na₂S₂O₃·5H₂O solution	`3.0`	ml
Vitamin solution* (see below)	`0.5`	ml
5% L-Cysteine·HCl·H₂O solution	`4.0`	ml
8% NaHCO₃ solution*	`2.35`	ml

Keep the medium temperature at 50C until the medium is distributed into sterile screw-capped test tubes as described below.
Adjust pH to 7.0 - 7.2 with sterile HCl or NaOH solution. Then, aseptically and anaerobically, add the following solution (autoclaved):

0.01 M FeSO₄·5H₂Osolution (in 0.02 N HCl) 2.5 ml

Dispense the medium to sterile screw-capped test tubes (typically 12 ml medium into 125 by 15 mm test tubes) under air and cap. Allow to stand until the medium is solidified and a O₂-gradient formed (as evidenced by a pink upper oxic zone and a colorless lower anoxic zone). Inoculate cells just below the pink-colorless interface in the medium tube using a syringe.
Modified Wolfe's mineral solution:
Use trace minerals (see Medium No. 151) with final 0.4 g/L Na₂MoO₄·2H₂O, 0.02 g/L CuSO₄·5H₂O and 0.01 g/L NiCl₂·6H₂O.
Vitamin solution:

Thiamine·HCl	`90.0`	mg
myo-Inositol	`40.0`	mg
Calcium pantothenate	`4.0`	mg
p-Aminobenzoic acid	`5.0`	mg
Vitamin B₁₂	`5.0`	mg
Pyridoxine·HCl	`4.0`	mg
Nicotinic acid	`4.0`	mg
Biotin	`0.1`	mg
Folic acid	`2.0`	mg
Distilled water	`100.0`	ml

Comment: The medium tubes can be stocked under a N₂ gas atmosphere. In this case, prior to use, fill the head space with air and allow to stand until the O₂ gradient is formed.