Freezing Aliquots

From Microbial Ecology and Evolution Lab Wiki
Jump to: navigation, search

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 previous aliquot tube (or better, from -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 large, sterile test tube with 9ml of broth (TSB for Streptococcus; LB for E. coli).
  2. Add a large amount of cells from the plate, but not enough to raise the OD to 0.1
  3. Vortex to mix, and put 4ml of this mixture into each of 2 sterile, small test tubes.
  4. Incubate 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!
  6. Check periodically until cells are at 0.3 OD. Be sure to vortex before measuring! Do not allow cells to go significantly past 0.3 OD.
  7. Add 666.67µl of 80% sterile glycerol twice to each tube (so, 1333.3µl total per tube). Vortex to mix well. The final glycerol concentration should be 20%.
  8. Pipette 225µl into sterile micro-centrifuge tubes using an electronic pipette.
  9. Immediately freeze in the -80° freezer across the hall.

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

  • Day 4 (or longer)
  1. Put 200µl of one aliquot into appropriate broth. Thaw the aliquot on ice.
  2. Incubate at 37° in a water bath.
  3. Time how long until OD 0.3. Record this time, and write it on the -20 aliquot sheet