Measuring the Rate of Attrition in Frozen or Cooled Samples

Plates stored at 4C

1. Day 1: Inoculate strain in approximately 5mL of growth medium in shaking incubator.
2. Day 2: Precondition strain by transferring 10uL of the overnight culture to a 50mL flask containing 10mL of growth medium, and incubate in shaker overnight.
3. Day 3: 24 (+/- 1) hours later, dilute the culture 1:1,000,000 by first transferring 10uL of the preconditioned cells into 10mL of sterile saline, vortex, and then transfer 10uL of that into another 10mL of fresh sterile saline. Plate 100uL of 1:1,000,000 dilution and incubate overnight.
4. Day 4: ~24 hours later, pick one full colony (do this by cutting the agar out from below the colony and scooping the whole thing up), and re-suspend in 10mL of sterile saline and vortex. Using a sterile toothpick is recommended. Transfer 100uL of suspended colony into another tube containing 10mL of sterile saline (I'm not entirely sure what the exact dilution factor is for this, but I believe this is what we did the first time when Brian was testing this). Then plate 100uL of this onto LB only (this will be your day 0 plate because the cells have been freshly cultured and haven't been in the fridge yet), and put in the incubator for counting the next day. Repeat this whole step as many times as you need replicates for, and be sure to pick the same sized colonies every time. Place the original plate with the colonies on it back in the fridge (you will keep picking colonies from this same plate each day and resuspending them then counting the next day to see how many are living compared to the previous day.

-- Main.AurkoDasgupta - 06 Jun 2012