Methylosinus trichosporium (type II, strain NCIMB 11131T)

(Cryopreservation of methane-oxidizing bacteria Methylosinus trichosporium)

Intro/Description:

For the preservation of methane-oxidizing bacteria (MOB) to increase viability and culturability 3 to 12 months after cryopreservation. Adapted from Hoefman et al. (2012).

Materials:

• DSM1180 media
• Trypticase
• Trehalose
• Cryppreservation tubes
• Liquid nitrogen
• Dimthylsulfoxide (DMSO) or gylcerol

Protocol:

1. Culture strains in diluted Nitrate Mineral Salts (dNMS) + TT buffer (Trypticase (1:10) + 1% trehalose) to early stationary phase.

2. Centrifuge cultures at 6000 x g for 15mins. Discard supernatant.

3. Resuspend cultures in dNMS + TT buffer.

4. In a cryopreservation tube, mix 500µl sample with 500ul 5% DMSO, or 15% gylcerol in TT buffer (the cryoprotection media).

5. Freeze and store samples in liquid nitrogen.

6. To resuscitate, thaw tubes at 37°C.

7. Transfer immediately to centrifuge tubes, centrifuge at 6000 x g for 15mins. Discard supernatant.

8. Resuspend culture in fresh media (dNMS, of TT buffer). Allow to grow for 1hr at RT, then determine amount of viable cells recovered.

References:

Hoefman, S., K. Van Hoorde, N. Boon, P. Vandamme, P. De Vos, and K. Heylen. 2012. Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. PLoS ONE. 7:e34196.

Added by: Carlton Hoyt on July 25, 2012

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