Select relevant cultures

After overnight incubation the flasks seeded at high concentrations will likely be in stationary or death stage. The goal is to identify the flask that has just reached stationary phase as this is where you will find the highest concentration of culturable cells. To save time, initially inspect and discard flasks that seem irrelevant before measuring concentrations in the remaining ones.

In this example we will only keep vials -4 to -8. Below we will explain why.

1

Transfer culture to centrifuge vial

Use a sterile serological pipette to transfer 5 mL of each culture to a sterile 15 mL vial.

2

Remove vials with low turbidity

Remove the vials with very low turbidity because they likely have very low bacterial concentrations. In this example, we remove vials -9 and -10.

3

Remove vials with high initial seeding concentration

Remove vials with high initial seeding concentration because these will likely contain a significant number of non-culturable cells.

• In this example, vial -1 to -6 have similar turbidity.

• We remove vials -1, -2, and -3 because these cultures are likely already deep in stationary or death stage.

Choose relevant incubation time

• The incubation time will strongly affect the cell concentration. Choose the same incubation time as you expect to use for your process.

• Here we incubated the E. coli culture for 11 hours. Typically an overnight culture is started in the afternoon and analyzed in the morning, i.e. a normal incubation time is approximately 16 hours.

Summary

We have chosen the relevant cultures from the shake flask dilution series. In the present example we removed half of the vials leaving only vials -4 to -8.

In the next step, Check lowest culture, we will measure the cell concentration in these cultures.