Measuring the Rate of Attrition in Frozen or Cooled Samples

Plates stored at 4C

1. Day 1: Inoculate strain in approximately 5mL of LB in shaking incubator at 30C.
2. Day 2: Precondition strain by transferring 10uL of the overnight culture to a 50mL flask containing 10mL of LB, and incubate in shaker overnight 30C.
3. Day 3: 24 (+/- 1) hours later, transfer 10uL of the preconditioned cells into 10mL of saline, vortex, and then transfer 10uL from that into another 10mL of fresh saline. Plate 100uL of 1:1,000,000 dilution and incubate overnight 30C.
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 a tube (A) containing 10mL of saline and vortex. Using a sterile toothpick is recommended. Transfer 20uL of suspended colony into another tube (B) containing 10mL of saline. Then plate 50uL of this onto LB. Label these plates T=0 and incubate overnight 30C. At least 5 repetitions on different plates are recommended.
5. Day 5 onwards: On day 5, first count the plates. Next, find a number n (equal to the number of intended repetitions) of colonies on each plate and circle them on the plates. Pick colonies that are all as similar in size as possible. Once this is done, repeat the suspension, dilution and plating as on day 4. Each following day, count the preceding day's plates and make another set of plates in the same method. Make sure to keep the T=0 plates at 4C throughout, as the attrition of cells within the colonies at 4C is the subject of the experiments.

This dilutions used should result in 10 colonies for every 1 million cells in a colony. Thus, a colony with 5 million cells will produce 50 colonies and one with 25 million cells will produce 250 colonies.

Liquid culture stored at 4C

1. Day 1: Inoculate strain in approximately 5mL of LB in shaking incubator 30C.
2. Day 2: Precondition strain by transferring 10uL of the overnight culture to a 50mL flask containing 10mL of LB, and incubate in shaker overnight 30C.
3. Day 3: 24 (+/- 1) hours later, transfer 10uL of preconditioned cells into 10mL of LB in a 50mL flask, then transfer 10uL from the flask into a test tube with 10mL of saline. Plate 100uL from this tube and incubate the plate at 30C and store the preconditioned flask at 4C overnight.
4. Day 4: Count the plates from the day before. This can be used to calculate the T=0 concentration of cells. Use the refrigerated flask to produce another 1:1,000,000 dilution into a saline tube then once again plate 100uL. Repeat for as many days as desired.

From this flask, transfer 10uL into a test tube with 10mL of saline. Plate 100uL from this tube onto LB plates. Incubate the plates and flasks overnight 30C. At least 5 repetitions are recommended.

Due to the similarity of the solid and liquid attrition quantification procedures, it is recommended to do them at the same time. The first two days are identical for both sets of experiments, and the following days involve highly similar procedures.

-- Main.AurkoDasgupta - 06 Jun 2012