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Pono Pono
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A week ago
Dear all,

I've obtained a freeze-dried sample of Komagataeibacter xylinus from DSMZ (German equivalent of ATCC). According to the provided instructions, I will revive the culture in 1000ml of liquid medium. The colony will stay in that medium for 2-3 days.

I'd like to store 250ml of that original bacteria broth in 4°C to keep the culture intact. There are a few questions bugging me right now:

1. How often should I subculture that 250ml colony?
2. What's the volume (in ml) of bacteria broth that I should transfer to fresh medium?
3. Should the fresh medium be again 1000ml in volume?

Essentially, it all boils down to: how do I keep my original culture active and healthy for indefinite periods of time?

Thank you!
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A week ago
Short-term storage

Working bacterial stocks can be streaked onto agar plates and stored at 4°C for daily or weekly use. Culture dishes should be wrapped with laboratory sealing film (plastic or paraffin) and stored upside down (agar side up) to minimize contamination and to keep both the culture and agar properly hydrated. Some bacterial strains can be stored for up to 1 year at 4°C in agar stab cultures.

 Stab cultures are prepared by first sterilizing strain-compatible agar (e.g., lysogeny broth [LB] agar for E. coli) and then transferring the warm liquid agar to screw-cap vials using the appropriate aseptic technique. After the agar has solidified, a single colony is picked from an actively growing culture using a sterile, straight wire. The wire with the bacteria is then plunged deep into the soft agar several times, and the vial is incubated at 37°C for 8–12 hours with the cap slightly loose. The vial is then sealed tightly and stored in the dark at 4°C.

Long-term storage

This involves freezing, of which is not necessary if you plan to use the sample regularly. The greater the cell density, the better the recovery is after thawing the cells. For most bacteria, a density of 107 cells/mL will result in adequate recovery if all conditions are properly maintained.

Quote
3. Should the fresh medium be again 1000ml in volume?

This depends on the cell count. Start with a cell-count of the original sample. After adding 1000 mL, take a drop of that to count the number of cells. You should maintain this for the next round after storing.

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