Glycerol stocks are for the long term storage of microbial strains at extremely low temperatures (-70 to -80°C). These are prepared by growing cells at an exponential rate and adding sterile glycerol, a cryoprotectant, to the culture to a final volume of 25% glycerol. The addition of the glycerol reduces the proportion of cells that lyse due to the formation of crystalline lattice structures when the water in and around the cells freezes. ## Handling Glycerol stocks should be stored at -70 to -80°C and, when removed, should be kept on dry ice.[^1] Time out of the freezer should be minimized and the stocks should not be allowed to thaw, as each subsequent freeze-thaw cycle decreases the number of remaining viable cells. Thus, when streaking plates from glycerol stocks simply use a sterile loop to scrape a small amount of frozen material from the stock onto the plate and immediately return the glycerol stock to the dry ice or freezer - it is not necessary (and is actively harmful) to wait for any portion of the stock to thaw. ## Making a glycerol stock 1. Obtain a fresh plate of the desired bacterial strain. 2. Grow your desired strain into the late exponential phase, $OD_{600} \ge 0.8$, retaining some media for blanking a spectrophotometer the following day.[^2][^3] 3. Add 500 µL of sterile 50% glycerol to a sterile freezer vial, pre-labeled. 50% glycerol prepared for this purpose is good for 1 year at room temperature. 4. Add 500 µL of the bacterial suspension to the vial. 5. Vortex the suspension for 15 seconds. 6. Immediately place into the -80°C freezer and record the location of the strain (box and position) in your laboratory's records. ## Preparing sterile 50% glycerol 1. Combine glycerol and water 1:1, stir. 2. Autoclave or filter sterilize the mixture using a 0.2 µm syringe filter in the biosafety cabinet. 3. Store up to 1 year at room temperature. [^1]: **Lab specific note:** Dry ice can be found on the third floor of BSBW. [^2]: An overnight culture with 10 mL of broth in a 15 mL conical tube is often suitable, but match all growth conditions to your organism. [^3]: Overnights are not necessarily a requirement if you have fresh cells on a plate and want to suspend them and let them grow for a few hours to produce a glycerol stock same-day.