I usually use 5% DMASO and never got problem with it. I also tried 10% glycerol, it was worke good to me. But prefer DMSO for culture work and glycerol for microbiology work.

I usually prepare my GS with a final glycerol concentration of 25 %. At -80°C the stocks last for at least 3 years. Often you can recover the bacteria even after a decade. However, I would prepare new stocks latest after 5 years.

You can make glycerol stocks with 60% culture and 40% glycerol. Flash freeze them in liquid nitrogen and store in aliquots at -80degrees. While reviving, thaw partially and add ~50ul to 10ml LB medium. Dont refreeze the stock. Such glycerol stocks can be revived after 3-4years also.

While glycerol works okay, there is a loss in viability associated with it, particularly when exposed to freeze- thaw cycles (happens during handling if stocks or when freezer breaks down ). I prefer using skim milk at a final concentration of 5%. This is based on an article that was published after hurricane Katrina comparing the viability of stocks that were kept in glycerol versus skim milk (after thawing due to lack of power) and skim milk was fount the best...

if it's for yeast we use 50% glycerol for -80C storage in cryovials

Final concentration of glycerol around 24% with 0.1 M CaCl2

I use a final glycerol concentration of 20% for -80°C storage

I agree with Dr. Kuldeep Patel

Glycerol concentration around 24% is suitable for preservation of microbial cultures.

Excellent protocols in the Bible of Molecuar Biology "Sambrook, Maniatis, Molecular clonging". :-) http://www.molecularcloning.com/

25% is suitable and the cells can be kept minus 80 degrees for up to 5 years

Down to 10% glycerol works fine. We usually have either LB or H2O/glycerol 30% and mix it 1:1 with a sample from the culture. They last for many years at -80C.

I always prefer to use 30% glycerol stock for longer use.

Hi Raksha, the suggestion from Karin Sauer is good. I found the final concentration of glycerol is 10-15% with 0.1M CaCl2 working good for E. coli. But the range vary from 10-80% depending on the cell & culture type.

I agree with all of them using glycerol concentration around 10% with 0.1 M CaCl2 is suitable for preservation of most of bacterial cultures. In this way, the culture could even last for at least 5 years at -80C.

40% Sterlised Glycerol  is ideal & used as  1:1 (sample : 40% Glycerol) ratio  with generally log phase pure bacterial culture cells grown in LB/NB/TYG.

I have seen many people commenting about storing microbial cells in -80 for several years. However, is this is strain specific? I guess for some strains (eg: microalgae) storing in -80 is not enough,may be they are required to store in liquid nitrogen (-130) ?  Any suggestions are welcome.. 

You can use 10-12 % for shortime storage and 20-25% for little longer storage

what are the alternatives to -800c? 

We use 18% glycerol and store at -80C, the viability might depend on the media used for the culture, fresh media is better.

I use a solution of 50mM CaCl2 and 15% glycerol, for storage at -80ºC. Now, I have a question myself. Would this solution affect my bacteria in a negative way, if they are kept overnight at 4ºC in it? I made this mistake myself, and my cells did not become competent, so I'm wondering if this would be the cause.

Using glycerol concentration up to 30% at -80 C. is suitable for preservation of most bacterial cultures for at least 5 years.

I agree with all of them using glycerol concentration around 10% with 0.1 M CaCl2 is suitable for preservation of most of bacterial cultures. In this way, the culture could even last for at least 5 years at -80C.

If you had E. Coli glycerol stocks that are repeatedly used. Removed from -80 into a Nalgene labtop cooler that was stored in -80 and returned to -80 in less than 5 minutes. They aren't thawed but still they are subjected to some temperature change. Some of our lab does 25% while others does 40%. Would having a higher glycerol % make a noticable difference in viability over time?

20 per cent but long term storage is not advisable

Generally, 20% glycerol is used and store this 20% glycerol stock culture at -80 degree.