BISC209/F13: Freezing Isolates

Freezing Pure Culture Isolates

FREEZING “PURE” BACTERIAL CULTURES FROM STOCKS

• microfuge tubes
• 1000µL Pipetman
• microfuge tube labels
• sterile 20% glycerol stock
• sterile toothpicks
• young culture (~24 hr) of student pure isolates grown on plates
• Google Doc or Excel Spreadsheet for Strain Database

Remember that your frozen stocks are potentially the one and only source you have for that bacterial species. Therefore be especially careful with your sterile technique when creating and working with your stocks, and make sure you are vigilant about keeping your strain list up-to-date and correct

Wear gloves to avoid contamination and work near flame (watch your hair and hands!)

Thoroughly wash your hands and decontaminate the bench before beginning

Make sure not to place tube cap on any surface (to keep it sterile)

Create a strain database file

1. Give each isolate a strain name: your initials followed by a number
2. Use the 209 lab Google Doc spreadsheet and fill in all the information listed below
   

• Strain name
• Bacterial species (if known)
• The source of the bacterial strain (person or environment)
• Any known genotype information or mutations
• The date the stock was made
• Information about where in your notebook you first discuss this strain
• The source of the bacterial strain (person or environment)
• Any known genotype information or mutations
• The date the stock was made
• Information about where in your notebook you first discuss this strain

Making the Cell Stock
1. Label the side of the cryo-tube with the cell strain name (and any additional info you want)
2. Add scotch tape over the label to ensure it doesn’t rub off
3. If the cryo-tube has a place to put a top label on, prepare that also

NOTE: The cryo-tubes typically do not fit into the standard eppendorf racks; a good alternative is to use the styrofoam base that 15 ml conicals come in—those openings are large enough for the cryo-tubes to rest securely in but not so deep you cannot remove and replace the cap easily

4. Screw open the tube but leave the cap sitting loosely on top
5. Add 800 ul of 20% glycerol in LB to the cryo-tube using P1000 pipette (be careful to not set cap down or let it touch any surface)
6. Scrape up cells from the plate with a flat toothpick—try not to gouge agar, just remove cells
7. Open cap and swirl toothpick in the media/glycerol to remove cells
8. Place 2 – 3 good-sized scrapings into the glycerol media
9. Pipette gently with P1000 to break up chunks of cells and disperse them
10. Mixture should be opaque—if not, add more cells
11. Place in -80 degree freezer in labeled freezer box

Be sure to label both the lid and the tube of a glycerol stock before you place the sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube!

Tips and FAQ

The optimal concentration of long-term glycerol storage is unknown. Most labs store bacteria in 15-25% glycerol.

Try not to freeze/thaw your glycerol stock too many times. If you need to thaw frequently than make a new glycerol stock replacement from a freshly grown culture.

 

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10