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Preparing bacterial stock culture
Posted by: fr0gjaywalker (IP Hidden, New member, 3)
Date: July 2, 2005 12:56AM
Hello I'm a HS senior working at a biochem lab. Many of the procedures are new to me and I have many question. I really hope that someone can help me out here.
Question: Why do I need to freeze the bacteria at -70 deg. C after I suspended it in some Kphosphate-EDTA buffer? I'm going to be putting the bacteria through the French pressure chamber. And also, my professor did not mention adding some glycerol, so my mixture did not contain it. What is the function of the glycerol? Is it an essential component in the mixture to be frozen?
Re: Preparing bacterial stock culture
Posted by: fr0gjaywalker (IP Hidden, New member, 3)
Date: July 4, 2005 01:44AM
Hello? Can someone please help me?
Re: Preparing bacterial stock culture
Posted by: lvmont (IP Hidden, Unregistered user, )
Date: July 4, 2005 09:38AM
fr0gjaywalker,
You need to add 20% sterile glycerol to freeze bacterial. It is a cryoprotectant agent that can protect cell membrane from damage when freeze.
Re: Preparing bacterial stock culture
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 7, 2005 09:26AM
without glycerol your suspension will lose most if not all of its viability when frozen. -70C will keep help prevent DNA, RNA and protein degradation- at least one of which you will be trying to extract through the french press. If you are interested in protein extraction, you may wish to add protease inhibitors (sigma makes an easy to use cocktail containing several inhibitors required for different proteases) and PMSF before lysing the cells to prevent protein degradation.
Re: Preparing bacterial stock culture
Posted by: midhilapadman (IP Hidden, Unregistered user, )
Date: July 29, 2005 01:08AM
hello sir i was a microbiology student and though i cant remember well i will try to answer your question i think the role of freezing is to preserve the bacteria by stopping its enzymatic activities so that its stable the role of glycerol may be to stabilize it providing a buetiful environment for it sorry sir if am giving a very immature answer
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