Glycerol for freezer stocks - What percentage do you use? (Aug/13/2005 )

Just curious as to what glycerol percentage everyone uses when preparing bacterial freezer stocks. I have some stocks in 60% glycerol and -80 C right now, but I've run across another protocol that recommends 80%. Anyone ever notice differerences when trying to use freezer stocks stored in various glycerol concentrations to seed cultures?

-Hank

I keep my frozen stock in 40% glycerol. Is that why the cells are not growing very well after keeping in -20 or -80 deg Cel?

I am purifying restriction enzymes nowadays, I use 40% glycerol for my REs to prevent them from frozen in -70 degree celcius. 40% is adequate and good enough. I try to avoid myself from giving more than that to my REs as too much glycerol will cause star activities to my REs (which I am not sure how, I am happy if someone can pass some information). Hope this help.

I use 50% glycerol. People in my lab seem to do that for ages.

Freiberger

50%? So I should fine then.

It took forever to get this strain in and the last thing I need to do is murder it by storing it wrong :-/

Thanks!
-Hank

Most bacterial species will be alright in 15% (w/v) glycerol solution. I have been using this concentration for years and so did my previous lab. We basicly made a 75% (w/v) glycerol stock and added 0.2 mL of glycerol stock per 0.8 mL of bacterial culture.

My new lab used 25% (v/v). I have not notice any difference between stocks if they are prepare from cell that are in the exponential phase (i.e. in good shape).

hi
1ml of bacterias / 500µl of glycerol.
That's 33%... and ok

It's very difficult to pipet 100% glycerol. Even an 80% glycerol solution is dramatically easier to work with than 100%, and almost as useful in preparing frozen cultures.

This is something we've been doing for ages but i don't know how it will affect your bacteria: we store our freezer stocks in 20% DMSO...they plate out onto plates pretty well and seem to have no adverse effect. another alternative is 60% glycerol.

I've been storing and using thousands of E.coli for environmental samples (and some may be a -relative- pain to grow in the lab) and stored them in 15% glycerol, and never had a problem. In one lab I was working with they were making a (30% glycerol+ 4g/l Na MetaPhosphate) solution to be used as 2x preservative, it keeps osmotic pressure right and is said to be better in the long run. You can replace 4g/l Na MetaPhosphate by 1.6% NaCl, or MgCl2 (I do not remember which concentration is best...) or any of all those bacterial diluent solution.