Glycerol stock preparation - (May/16/2007 )
I am really interested to hear the different methods people use to prepare their glycerol stocks…
Currently I am sterilising about 10 ml of glycerol and then transferring 0.15ml (with snipped nib pipettes) into sterile microcentrifuge tubes. (ready prepared for addition of my culture).
Does anyone know if it is ok to add the glycerol to the microcentrifuge tubes and then autoclave them?
Any advice would be greatly appreciated....
Many thanks!
Currently I am sterilising about 10 ml of glycerol and then transferring 0.15ml (with snipped nib pipettes) into sterile microcentrifuge tubes. (ready prepared for addition of my culture).
Does anyone know if it is ok to add the glycerol to the microcentrifuge tubes and then autoclave them?
Any advice would be greatly appreciated....
Many thanks!
I always autoclave the glycerol (50%). Put 0.8ml of this and 1ml of culture into the sterile tube, mix well and place at -80oC.
I never sterilise the glycerol, and I make glycerol stocks on the bench, in ordinary tubes with ordinary pipette tips. I just put 800ul of culture (in LB+Amp) in the tube and add 200ul of glycerol, give it a good shake and put it in the -80 (no need for liquid nitrogen). I've never had any problems with my glycerol stocks.
I don't think they need to be perfectly sterile. They'll be frozen at -80! Nothing's going to grow in them! When inoculating plates or cultures from a glycerol stock, use a sterile toothpick and get a visible chunk of the 'ice'. Assuming you're using selective media, there's really no way that any contamination is going to compete with your cells.
My old supervisor is pedantic about glycerols. We add glycerol solution to wheaton vials and autoclave. You would have to jam microcentrifuge tubes shut or they would pop open. Then we scrape nearly a whole plate!!!! of bacteria into the vial and vortex. We freeze instantly in a dry ice/ethanol bath and keep in dry ice when removing stocks from the freezer. I have heard of people spinning down overnight broths and resuspending in glycerol, which would be heaps easier. In the lab where I work now, my colleague only adds a loopfull and then vortexes, but it is a bit hit and miss. If you have then energy/time making two stocks for each isolate is good.
I think it's important to keep everything sterile and not contaminate your stocks, so I would always autoclave at least before you pippette out into tubes. It can take considerable work if they do get contaminated.
Tom
I don't think they need to be perfectly sterile. They'll be frozen at -80! Nothing's going to grow in them! When inoculating plates or cultures from a glycerol stock, use a sterile toothpick and get a visible chunk of the 'ice'. Assuming you're using selective media, there's really no way that any contamination is going to compete with your cells.
This is fine when you have transformed bacteria. But if you work with other bacteria than an antibiotic resistant E coli, you need to have every thing sterile.
In my previous lab, we used to aliquote glycerol in cryovials, and autoclave (don't close completely the cap, and cover the rack with aluminium foil).
It's much more convenient to aliquote and then sterilize, because it's quite boring to aliquote glycerol, it takes so much time, and you might not do it really in a sterile way.
In a 100ml reagent bottle I autoclave 50% glycerol and keep. So just mix 700ul logphase culture and 300ul 50% glycerol, votex and store it in -80. For inoculation just scrape the frozen surface with a sterile inoculation loop and inoculate into the media which is enough and thawing of the stock is always avoided.
Absolutely! I should have mentioned that in my post. Antibiotic resistant E coli is probably the easiest organism of all to use (because you can be lazy). I do my PCR colony screens on the bench now, because the laminar flow is in another room and I got lazy once, but didn't have any problems with it...
But green algae (my other organism) is quite a bit more painful to use!
We use glycerol as it is for transformed bacteria. No sterilization.
I don't think they need to be perfectly sterile. They'll be frozen at -80! Nothing's going to grow in them! When inoculating plates or cultures from a glycerol stock, use a sterile toothpick and get a visible chunk of the 'ice'. Assuming you're using selective media, there's really no way that any contamination is going to compete with your cells.
Yeah I feel the same way. My old supervisor used to make us prepare them over the flame with sterilized glycerol and then immediately flash freeze them in nitrogen. Personally I find this quite silly, nothing grows at -80. Now I autoclave my 50% glycerol, and mix 250ul of it with 750ul of overnight on my bench and then put it in the -80. Like something is going to grow in the time I put the glycerol in and put it into the freezer. Haha. Of course it is good to avoid contamination wherever possible but a little common sense goes along way.
i don't sterilize my glycerol.
I use clean 50ml tube to do an aliquot.
I think that as your plating your bacterias to recover them, this should be enough for avoiding contamination.