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Frozen bacterial stock


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#1 sching

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Posted 31 March 2009 - 01:21 PM

Hi

When making frozen bacterial stock, you do a flash freeze as soon as you add DMSO before putting into -80 freezer. But i was told that you need to vortex the mix after adding the DMSO. So am i suppose to flash freeze first and then vortex? or vortex first and then flash freeze? My guess would be vortex and then flash freeze, but i am unsure about it.

Sometime i don't get the purpose of doing a frozen stock, because i have been streaking plates every week in duplicates. :)

Edited by sching, 31 March 2009 - 01:23 PM.


#2 bob1

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Posted 31 March 2009 - 04:25 PM

You need to vortex then flash freeze, if you freeze before vortxing, you won't mix the DMSO in. The DMSO acts as a cryo-protectant stopping ice crystal formation so that the cells aren't ruptured by the freezing/thawing process.

The purpose of a frozen stock is to prevent genetic drift, which is potentially a serious problem with bacteria due to the short generation time - your frozen stocks are not actively dividing, therefore they are not likely to lose or mutate the plasmids/genes being studied, meaning that you can go back to a frozen stock many times and still get the same results from it.

#3 sching

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Posted 31 March 2009 - 09:21 PM

You need to vortex then flash freeze, if you freeze before vortxing, you won't mix the DMSO in. The DMSO acts as a cryo-protectant stopping ice crystal formation so that the cells aren't ruptured by the freezing/thawing process.

The purpose of a frozen stock is to prevent genetic drift, which is potentially a serious problem with bacteria due to the short generation time - your frozen stocks are not actively dividing, therefore they are not likely to lose or mutate the plasmids/genes being studied, meaning that you can go back to a frozen stock many times and still get the same results from it.



Thanks, if that is the case, that would mean at certain point of time, i have to streak plates using the so called "working stock" that was made from the original stock. So when is it an appropriate time? like once in a month???

#4 pito

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Posted 01 April 2009 - 06:25 AM

You need to vortex then flash freeze, if you freeze before vortxing, you won't mix the DMSO in. The DMSO acts as a cryo-protectant stopping ice crystal formation so that the cells aren't ruptured by the freezing/thawing process.

The purpose of a frozen stock is to prevent genetic drift, which is potentially a serious problem with bacteria due to the short generation time - your frozen stocks are not actively dividing, therefore they are not likely to lose or mutate the plasmids/genes being studied, meaning that you can go back to a frozen stock many times and still get the same results from it.



Thanks, if that is the case, that would mean at certain point of time, i have to streak plates using the so called "working stock" that was made from the original stock. So when is it an appropriate time? like once in a month???



What do you mean exactly?

You mean streak plates and then work with those plates and trow away. Or you mean streak plates, let it grow and then freeze those?

If it is the first one: do it whenever you need it. (you can store it for a very long time so no need to worry really)

Second one: its a good idea to store enough for your research so that you do not need to grow then to freeze them again and repeat that many times... thus making it easier to mistakes to happen.
(ex. if you need to test it each month for a year, then freeze 15 vials (12+3 reserves) so you can use 1 vial each month.


The more you work with a bacteria the more prone it is for defects to happen or contamination or...

Thats why you store them in liquid nitrogen or freezedry them.. so that the bacteria "stays" the same.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





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