Medium data

Search for medium no. = [1067].

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

1067 FRESHWATER IRON-OXIDIZING BACTERIA MEDIUM-II

Bottom layer:

FeS solution (see Medium No. 628)	`50.0`	ml
Modified Wolfe's solution (see Medium No. 628)	`50.0`	ml
Agar, Noble (BD-Difco)	`1.5`	g

Top layer:

Modified Wolfe's solution (see Medium No. 628)	`1.0`	L
Trace minerals (see Medium No. 151)	`10.0`	ml

Autoclave both layer solutions. Shortly after autoclaving, pipette the bottom layer into each culture vessel (typically 5 ml in 60 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.
After cooling of the top layer solution, add per liter the following solutions (filter-sterilized):

Trace vitamins (see Medium No. 197)	`10.0`	ml
8% NaHCO₃ solution	`5.25`	ml

Add the top layer solution in 5 to 6 times the volume of the bottom layer to the culture vessels containing the bottom layer (e.g., 25 to 30 ml in 60 ml glass vials with 5 ml of the bottom layer).
Then, cap the culture vessels with silicone rubber stoppers, and replace the gas phase with a N₂-CO₂ (4:1, v/v) gas mixture.
Comment: The medium can be stored in culture vessels for a certain time by capping with butyl rubber stoppers, instead of silicon rubber stoppers and keeping anoxic in the vessels. Silicone rubber stopper is O₂ permeable and creates a mircoaerophilic condition in the culture vessels.