It really doesn't matter too much. I think 5% is the minimum, I usually use about 10-15%. You can use much higher as well without problem. However when you get to higher concentrations then the stock is more likely to stay liquid and the cells may settle out. At 10-15% it remains a slushy solid at -80.

It really doesn't matter too much. I think 5% is the minimum, I usually use about 10-15%. You can use much higher as well without problem. However when you get to higher concentrations then the stock is more likely to stay liquid and the cells may settle out. At 10-15% it remains a slushy solid at -80.

Me, I always used 25% (1/4 v/v)

Hi there,

10-15% vol / vol final concentration is OK. The essential step is to homogenize the mixture and to flash freeze the samples before any sedimentation of cells.

I routinely use 20% v/v and store at -80°C for months.

I appreciate 15-20% v/v glycerol concentration for long term preservation.

 Thanks all

Finally, I decided to use 10-15%vol / vol  final concentration.

Between 15 and 20%.

We are using 30% v/v glycerol concentration for long term preservation of Actinobacteria. However, with any concentration you finally choose, keep in mind that give 30 min to bacteria to adsorb glycerol so it can protect them. Then, flash freeze them by dipping them in liquid nitrogen and preserve them in -80 C. Moreover, always keep the cells frozen even when you are streaking them on plates.

Good LucK

I usually used 15% concentration. 

We use 20%. It works perfect.

15% works perfect. But 10-15% is also perfect as suggested by Michael. Higher concentration t will only increase your cost.

We used 20% for years. Now we are using 35% and it works great too.

We use 30% and it works fine. However 10-20% is also applicable. It all depends on your resource. 

i have always used 10% and it works well for me

You get rid off the exhausted medium before storing the culture at 20% glycerol in culture broth at -80oC.

Hi Dear 

the optimum is 15%

BR

I always use 20% and it works great. You want to keep thawing the cultures to a minimum as the freeze-dry cycle will decrease viability over time.

I always use 10% and it works great. This percentage may be change depend upon the temperature storage. So you will prepare your stock percentage depend upon temperature. I used 10% under the freezing temperature.

I apologize that this topic is out of my research focus because my researches are related to aspects of conservation of heritage buildings.

My dear, 

We use 20% of glycerol . It works perfect.

A final concentration of 30% glycerol works very nicely.  Use an autoclaved 80% glycerol solution in water for easier aliquoting.  Add 0.6mL of 80% glycerol to 1mL of bacterial culture and freeze immediately.

20-50% glycerol in 2X nutrient broth

We always use 15%

I used to use 30% glycerol at -80, and it worked perfectly.

You can use 15-20% glycerol for storage. Also 10-15% is okay as mentioned by M. Benedik