Storing Photobacterium Phosporeum

[SMJW]

Howdy,

I have cultivated some fairly hardy strains of photobacterium phos. over the last months but wont be able to reculture them for a few weeks and am worried that I'll now lose my babies. I've read that the populations will start to diminish after about 5 days so what might be the best way of storing them? I've read making some glycerol solution and freezing is the way to go but I dont have access to a freezer that goes less than -15 degrees celcius.

Thanks heaps :D 

[Mega [Andreas Stuermer]]

Agar plates in the fridge should still be viable after weeks. At least with E coli and agrobacterium even months in my experience. Or try making stab culture (in eppis). You can freeze them in 10% glycerol or just LB at -15 for less than a year probably at -15°C. I think people underestimate the hardiness of bacteria. On the other hand, a marine bacterium may not have evolved antifreeze mechanisms like a soil/land bacterium

[SMJW]

Great! Thanks for the reply and peace of mind :)

[Elisabeth Steel]

I agree, I have been able to use E.coli after a few weeks stored on agar in the fridge, or out of 10% glycerol/LB stocks out of -20 C.

Take 500 ul of your batch culture and put it in 500 ul of 50% glycerol (this is for 25% glycerol concentration, adjust as you see fit). 

Freeze. 

Subsequent freeze thaw cycles will reduce the viability of your culture so when you go to recover bacteria from your stock, don't thaw the whole volume. Just take a scraping off the top and inoculate a plate. 

Good luck! 

[Cathal Garvey]

I've lost every stock of Vibrio phosphoreum* I ever made at -20, as has
an academic colleague who made stocks at -20. I'm actually concerned it
might be the opposite of what you say Andreas; because V.phosphoreum is
very cold-tolerant, I have a mild suspicion that it continues a very
basal metabolism when antifrozen at -20, enough to kill itself? I can't
imagine why else it appears so brittle.

I may just be unlucky. BCCing in another DIYbioer who I know to have
isolated these guys who might have had better luck..

* Originally termed "Photobacterium" but since renamed

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[SMJW]

Thanks so much everyone!

Someone sent me a message recommending that I inject the culture 20mm into an agar slant with regular medium, allow that to rest for 2 days before capping it and placing it in the refrigerator.

This should hold for several months at least.

Reinoculate from the cloudy area in the afore pierced area of the slant. Collect bacon.

[John Griessen]

On 09/21/2015 10:29 PM, SMJW wrote:
> Reinoculate from the cloudy area in the afore pierced area of the slant. Collect bacon.

Bacon! Hahahha! :-)