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Making glycerol stock of bacteria - storage issue


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8 replies to this topic

#1 maryjo

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Posted 29 April 2010 - 12:30 PM

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!!!

#2 bob1

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Posted 29 April 2010 - 04:26 PM

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.

#3 HomeBrew

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Posted 29 April 2010 - 07:07 PM

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...

#4 maryjo

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Posted 30 April 2010 - 12:39 PM

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?

#5 microgirl

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Posted 16 June 2010 - 09:23 AM

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.

#6 baccilus

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Posted 24 August 2010 - 11:27 PM

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.

#7 maryjo

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Posted 07 September 2010 - 01:57 PM

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!

#8 Preetam Akki

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Posted 16 June 2011 - 10:21 AM

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!!!

#9 maryjo

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Posted 22 June 2011 - 10:49 AM

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




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