Methylosinus trichosporium (type II, strain NCIMB 11131T)

(Cryopreservation of methane-oxidizing bacteria Methylosinus trichosporium)


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).


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


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.


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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