Medium data

Search for medium no. = [628].

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

628 FRESHWATER IRON-OXIDIZING BACTERIA MEDIUM

The culture medium consists of two layers to create opposing gradients of O₂ and Fe(II). The top layer is the semisolid mineral salts medium and the bottom layer serves as the iron source.
Bottom layer:

FeS solution (see below)	`50.0`	ml
Modified Wolfe's solution (see below)	`50.0`	ml
Agarose	`1.0`	g

Top layer:

Modified Wolfe's solution (see below)	`100.0`	ml
Trace minerals (see Medium No. 197	`1.0`	ml
MES	`0.195`	g
NaHCO₃	`0.042`	g
Agarose	`0.15`	g

Adjust pH to 6.2.
Autoclave both layer solutions. Shortly after autoclaving, pipette the bottom layer into each culture vessel (typically 0.75 ml in 10 ml glass vials, use pipette tips that have been cut to enlarge the opening). The bottom layer is allowed to cool for at least 30 min to ensure its solidification. Cool the top layer to 35 - 40C, add 1 ml Trace vitamins (see Medium No. 197) and adjust pH to 6.1 - 6.4 by bubbling with sterile CO₂ gas. Slowly overlay the top layer onto the solidified bottom layer (3.75 ml in the 10 ml vial). Allow to solidify the top layer a minimum of 3 hr to a maximum of overnight. To inoculate, 10 µl of the inoculum is drown into a pipette tip and inject just above the bottom layer. Seal vials with butyl rubber stoppers and replace the gas phase with a N₂-CO₂-O₂ (8:1:1, v/v) gas mixture.
FeS solution:

FeSO₄·7H₂O	`46.2`	g
Na₂S·9H₂O	`39.6`	g
Distilled water	`300.0`	ml

Prewarm distilled water at 50C in a beaker with a stir bar. While rapidly starring the water, add FeSO₄·7H₂O, followed immediately by Na₂S·9H₂O. A thick black precipitate will form instantly. Stir continuously for 2 to 3 min. Remove the mixture to a narrow-mouthed glass bottle (500 ml) that can be tightly stopped. The bottle is filled to the top with distilled water and capped. Allow to settle for several hours, decant and replace the overlaying water. Repeat this procedure at least five times to wash the FeS precipitate. Check pH to be close to neutral. To stock the FeS solution, fill the bottle with distilled water and cap. Remove the overlaying water when prepare the bottom layer.
Modified Wolfe's mineral solution:

NH₄Cl	`1.0`	g
MgSO₄·7H₂O	`0.2`	g
CaCl₂·2H₂O	`0.1`	g
KH₂PO₄	`0.05`	g
Distilled water	`1.0`	L