Medium data

Search for medium no. = [1431].

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

1431 SEMI-SOLID AUTOTROPHIC GROWTH MEDIUM FOR GAMMAPROTEOBACTERIA

NaCl	`37.8`	g
MgCl₂·6H₂O	`5.4`	g
Na₂SO₄	`5.4`	g
KCl	`0.9`	g
CaCl₂·2H₂O	`0.5`	g
NH₄Cl	`0.3`	g
NaHCO₃	`1.26`	g
Modified Wolfe’s mineral solution (see Medium No. 915)	`5.0`	ml
Resazurin	`0.2`	mg
Distilled water	`1.0`	L
Agarose	`1.6`	g

Mix components thoroughly, except agarose, and adjust pH to 7.0. Add agarose to the medium and autoclave under a N₂ gas atmosphere. After cooling to 50℃, add the following solutions to the medium aseptically and anaerobically (autoclaved or *filter-sterilized). Keep the medium temperature at 50℃ until the medium is distributed into sterile screw-capped test tubes as described below:

0.5 M Potassium phosphate buffer (pH 7.0)	`1.8`	ml
Vitamin solution* (see Medium No. 915)	`0.5`	ml
0.01 M FeSO₄·7H₂O solution (in 0.02 N HCl)	`3.0`	ml
40% Na₂S₂O₃·5H₂O solution	`3.0`	ml
5% L-Cysteine·HCl·H₂O solution	`8.0`	ml

Adjust pH to 7.0 with sterile HCl or NaOH solution. Dispense the medium to sterile screw-capped test tubes (typically 11 ml medium into sterile Hungate 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.
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.