Freezing Aliquots

This is a generalized protocol for bacteria. Please use the correct type of plates and broth for the aliquots that you are preparing..

• Day 1

1. Streak cells from the original stock from the -80° freezer onto an agar plate. Use blood plates for Streptococcus without plasmids; use plates with antibiotics for any strains that should have a plasmid that confers antibiotic resistance.
2. Incubate plate overnight at 37°. Do the resulting colonies look correct? Are they all one colony type? Did they lyse blood, where appropriate?
3. If you have not gotten approval from Eric, have him train you on how to use the electronic pipette, which will make your future work much easier.

• Day 2 — Streptococcus

1. Fill sterile 13mm test tube with 8ml of TSB (S. pneumoniae) or BHI (S. mutans).
2. Add a large amount of cells from the plate — enough to see a change in absorbance (such as up to 0.05), but not enough to raise the absorbance over ~ 0.075. Be sure to vortex well before measuring.
3. Vortex to mix, and put 4ml of this mixture into another sterile 13mm test tube.
4. Incubate both tubes at 37° (with 5% CO2, where possible and appropriate).
5. Label a large number of 0.6ml tubes. There will not be time for this later. Consider coloring the tubes on top and providing a color.
6. Check periodically until cells are at 0.225 O.D. (0.2925 absorbance in 13mm tube). Be sure to vortex before measuring! Do not allow cells to go significantly past this absorbance; 0.25 O.D. (0.325 absorbance) is too high.
7. Pipet 2666µl of 80% sterile glycerol into a sterile 50ml erlenmeyer flask. Add the bacteria from each of the tubes. There should now be a bit over 10ml of liquid in the flask.
8. Mix well. The final glycerol concentration should be 20%.
9. Pipette 225µl into sterile micro-centrifuge tubes using an electronic pipette. Set the electronic pipette to 4 x 225µl
10. Immediately freeze in the -80° freezer. Even better — ask Eric about using liquid nitrogen to freeze the aliquots.

• Day 3 (or longer) — Streptococcus

1. Thaw the aliquot using a ice box from the -20 freezer.
2. Put 200µl of one aliquot into appropriate broth.
3. Incubate at 37°.
4. Time how long until 0.300 O.D. (0.39 absorbance in 13mm tube). Record this time for future reference.

• Day 2 (E. coli)

1. Fill sterile flask with 10ml LB + antibiotics. Innoculate with a single colony from the plate started on Day 1. Incubate overnight at 37 degrees.
2. Have 800ml of freshly-made LB autoclaved into a 2L flask, for each strain you want to freeze down.
3. Label a large number of 0.6ml tubes. There will not be time for this later. Consider coloring the tubes on top and providing a color.
4. Ask Eric to reserve the ultracentrifuge for the next day.

• Day 3 (E. coli)

1. Add antibiotics to the 800ml of LB.
2. Add all 10ml of overnight culture into this 2L flask.
3. Immediately after this, move the 0.6ml tubes into the 3rd floor cold room.
4. Prepare 30ml LB + 10ml 80% glycol into a 50ml Falcon tube. Keep on ice for the next 3-5 hours.
5. Incubate with shaking for at least 3 hours.
6. Remove 3ml and add to a 13mm tube. Check the absorbance; we are aiming for 0.8-0.9OD (1.0Ab - 1.17Ab)
7. Split into 3 centrifuge tubes; balance to the nearest 0.1g, and show Eric these weights.
8. Have Eric spin down cells using the untracentrifuge: 10 minutes at 9,000g.
9. Resuspend incredibly well in the chilled 30ml LB + 10ml 80% glycerol.
10. Combine all cells into a new, 50ml sterile flask.
11. Make 225ul aliquots (easiest with an electronic pipettes) into the labeled 0.6ml tubes. Freeze in liquid nitrogen.
12. Transfer to the -80 degree freezer.