Nitrobacter sp.

1. Rehydrate entire vial contents with 0.5 mL of #480 broth.  Aseptically transfer this aliquot to 5.0 mL of the broth contained in a large test tube (20 X 150 mm).
2. Incubate tube at 26°C, static, in the dark, and in a slanted position to increase surface area.
3. Monitor culture growth daily by assaying for the disappearance of NO₂ in the culture medium.  Withdraw 0.1 mL from the culture and place in a small test tube.  Add one drop of NO₂ Reagent A followed by one drop of NO₂ Reagent B, mixing after each addition.  Pink color development occurs within 10 minutes and is directly related to amount of NO₂ present.  Initial tests will probably be dark magenta reflecting the 20 mM NO₂ concentration of medium #480; but as culture grows and NO₂ is oxidized, color will become magenta, pink and finally clear.  This process can take from 3 to 20 days, or possibly longer, depending on the viability of the stock.
4. When all NO₂ has been oxidized, inoculate a 250 mL flask containing 100 mL of medium #480 with the 5.0 mL tube culture.  Wrap the flask in foil to protect from light and incubate at 26°C with gentle shaking.  Monitor NO₂ concentration daily.
5. When NO₂ is again depleted in the culture medium, either transfer the culture to fresh media or feed the culture by adding additional NO₂.  A 10% inoculum is recommended for transfers.  To feed cultures, aseptically add sterile NaNO₂ solution, returning NO₂ concentrations to approximately 10 mM (3.5 mL of 2% NaNO₂ per 100 mL culture).  Cells should be harvested or sub-cultured to fresh media when cultures can utilize 10 mM NO₂ in less than 24 hours and cell numbers are greater than 10 cells per field at 1000X magnification.
6. Cells may be harvested by centrifugation.  For long-term storage, 1.0 mL aliquots of concentrated cells can be frozen with glycerol as the cryoprotectant at a final concentration of 10%.  The frozen vials should be stored at –-70°C or colder for best preservation.

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