We have been preserving vibrios for more than 20 years without problems, we use glass beads in the cryovials (TSB + 2.0% NaCl + 15% glycerol), inoculate a loop, mix thoroughly allow them to settle for about 5 min, remove the broth and store them directly at -80C. To recover just collect a bead and streak it in TSA +2.0% NaCl or put it in a similar broth.

all the best

Hi

Simply, instead of streaking on solid media inoculated the stored culture in broth media.

Hope this will work for you.

Vibrio, as most of the G- are vulnerable for long-term deep freezing because of the fragility of their cell wall. So, the first thing to be sure - is that you mixed very well the culture with glycerol before you put it into the freezer. If it was done properly and you are using the same medium, on which it was grown - it should be able to recover, albeit slowly than normally, because even when it was done correctly, a substantial part of the cells are still damaged. Also, normally, I remove glycerol by centrifugation of the thawed culture stock in sterile 2 ml epps before putting the cells into the fresh medium. Sometimes all this do not help and the culture is not growing after the deep freezing. One thing which helped me once in that case - addition of 10% skimmed milk as a solid phase is addition to glycerol

Your glycerol should have low level of CFUs. The best that you can do is to inoculate the whole tubes (normally 1 ml) in 10 ml of SOC medium (adjusted pH, divalent cations, almost isotonic, glucose and so on). Incubate overnight. After that, if you have growth, you have to streak the bacteria in a plate to have single colonies and make sure that it is Vibrio (you know, the culture of pure culture). Pick a single colony a prepare a new cryotube per specimen, and I would use DMSO 12 % instead of glycerol. I recommend to thaw the tubes on ice before inoculation and pipette them carefully to the inoculation tubes. If you have at least one CFU per tube, you should recover them. Hope this help.      

We have been preserving vibrios for more than 20 years without problems, we use glass beads in the cryovials (TSB + 2.0% NaCl + 15% glycerol), inoculate a loop, mix thoroughly allow them to settle for about 5 min, remove the broth and store them directly at -80C. To recover just collect a bead and streak it in TSA +2.0% NaCl or put it in a similar broth.

all the best

Try giving it a heat shock say at 60C for varying amount of time.

Inoculate good medium (TSB or BHI0, incubate @ 37 C, streak on good medium, MEA or BHI .I preserved cultures for years by lyophilization.