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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
- 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.
- Incubate plate overnight at 37°. Do the resulting colonies look correct? Are they all one colony type? Did they lyse blood, where appropriate?
- If you have not gotten approval from Eric, have him train you on how to use the electronic pipette, which will make tomorrow's work much easier.
- Day 2
- Fill sterile 13mm test tube with 8ml of broth (TSB for Streptococcus; LB for E. coli).
- 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.
- Vortex to mix, and put 4ml of this mixture into another sterile 13mm test tube.
- Incubate both tubes at 37° (with 5% CO2, where possible and appropriate).
- 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. If so, then amend the strain guide on the -20 freezer.
- 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.
- 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.
- Mix well. The final glycerol concentration should be 20%.
- Pipette 225µl into sterile micro-centrifuge tubes using an electronic pipette. Set the electronic pipette to 4 x 225µl
- Immediately freeze in the -80° freezer across the hall. Even better — ask Eric about using liquid nitrogen to freeze the aliquots.
- Day 3 (or longer)
- Transfer to the -20° freezer in the lab.
- Day 4 (or longer)
- Thaw the aliquot using a ice box from the -20 freezer.
- Put 200µl of one aliquot into appropriate broth.
- Incubate at 37° in a water bath.
- Time how long until 0.215 O.D. (0.2795 absorbance in 13mm tube). Record this time, and write it on the -20 aliquot sheet.