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Making glycerol stock of bacteria - storage issue - (Apr/29/2010 )

I wanted to preserve slants, so i made a sterile solution of 25-30% glycerol and filled my tubes with it.
I put them in the freezer (an ordinary freezer)... my professor told me i wasnt supposed to freeze them, and they are to be put in the fridge. So i took them out of the freezer and put them in the fridge. I dont know which way is correct...anyone tried either of these ways?
I will go crazy if my samples die!!!

-maryjo-

I don't know about slants but the glycerol stocks I use are all liquid cultures with 10-15% glycerol added and frozen at -80. In my experience agar will freeze at -20 and become mush on thawing, so I suspect that slants should be stored in the fridge - which means you will need to subculture them every so often to keep them viable/prevent contaminants taking over the slant.

-bob1-

Glycerol is used as a cryoprotectant -- why add it if you're not going to freeze your samples? What are your samples?

I've heard of storing bacterial cultures for a year or so as stab cultures, but not as slants...

-HomeBrew-

I have a large variety of samples of a number of different species (Staph., Bacillus, E. coli, Pseudomonas...etc)
Using broth instead of agar is what i read about after i had already done it with slants :P
My professor has no experience in preservation techniques, he said that we cover slants with glycerol so they dont dry...

When u say freeze at -80 degrees...is an ordinary freezer suitable?

And concerning stab slants, what organisms will survive for months?

-maryjo-

An ordinary freezer will do if it's not frost-free. But you should go in about every 6 months and reculture and refreeze them.

-microgirl-

maryjo on Thu Apr 29 20:30:16 2010 said:


I wanted to preserve slants, so i made a sterile solution of 25-30% glycerol and filled my tubes with it.
I put them in the freezer (an ordinary freezer)... my professor told me i wasnt supposed to freeze them, and they are to be put in the fridge. So i took them out of the freezer and put them in the fridge. I dont know which way is correct...anyone tried either of these ways?
I will go crazy if my samples die!!!

The best is to keep them at -80 degree Celsius.
The worst possible thing you can do is to keep them at 0 to -4 degree Celsius freezer. At around 0 degree, the water freezes but forms really large crystals of ice. These crystals rupture the cells, or any biological tissue too.
If -80 freezer is not available, keep it at 4 degree for short term storage. NEVER KEEP BIOLOGICAL SAMPLES AT 0 DEGREE.
Hope this helps.

-baccilus-

OK so like they say, you dont know if something works until you try it! I made regular slants and put 30% glycerol over them and put them in the fridge. They have been in the fridge since May...and still as good as new!

-maryjo-

I have put 30% glycerol on my slants and frozen the tubes at -80 C, but the how do i thaw it afterwards??? I mean should i leave it to melt and then use my cultures or what!!!

-Preetam Akki-

Ok fellas, its been over a year and my bacterial slants are still preserved in the fridge (not freezer)! Good luck to u all :)

-maryjo-