Freezing Aliquots

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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
  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 tomorrow's work much easier.

  • Day 2
  1. Fill sterile 13mm test tube with 8ml of broth (TSB for Streptococcus; LB for E. coli).
  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. If so, then amend the strain guide on the -20 freezer.
  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 across the hall. Even better — ask Eric about using liquid nitrogen to freeze the aliquots.

  • Day 3 (or longer)
  1. Transfer to the -20° freezer in the lab.

  • Day 4 (or longer)
  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° in a water bath.
  4. 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.