Generally, freezing at -80 gives you longer term confidence of being able to recover viable cells than freezing at -20. It is recommended for long term storage when you don't expect to utilize the stocks frequently. You want to avoid opening and closing the -80 freezer doors, for the sake of the freezer itself, as it takes a lot of energy to re-cool down to temp after the door has been opened and the cold air has flowed out like a waterfall (you can see it). -20 storage is for when you will occasionally be accessing the stocks, and you should make fresh cultures (from -80 stocks) and refreeze at -20 every 6 or 12 mo or so. So you should probably make fresh cultures (from single colonies), freeze some at -80 and some at -20, then use the -80 stocks to replenish the -20's every so often. I think the standard glycerol concentration for freezing is 15%, and I usually froze my stocks in LB or nutrient broth rather than PBS.
Since glycerol is so hard to pipet, my protocol was to make a sterile stock of "freezing medium," which was 30% warmed-up sterile glycerol (50 degrees C, makes it flow better) in sterile broth. Then when I wanted to freeze an overnight broth culture, I'd mix 400 ul culture with 400 ul freezing medium, and the final glycerol conc. would be 15%. There was apparently no harm if I placed the new stocks in a -20 first, then took them upstairs to the -80 a few days later. There are probably lots of minor variations on this protocol and they probably all work. There are commercial freezing media, too. One is called "skim milk broth" and one lab I worked in prepared vials of the broth, autoclaved them, stored them in the fridge, then added bacteria from a single colony with an inoculating loop as needed. No glycerol required. Worked fine.
good luck, stay safe!
Edited by OldCloner, 01 May 2020 - 11:52 AM.