Choose best seeding ratio

The measurements are now completed and it is time to choose the inoculation ratio that results in the best High-quality culture. In this 11-hour E. coli example, the 10^-5 cryo stock dilution produces a culture that has likely just arrived at stationary stage. The 10^-4 cryo stock dilution has a similar cell concentration but is less suitable due to being in the stationary phase longer. It therefore likely contains a higher proportion of cells that have lost culturability.

The inoculation ratio is only valid for the given cryo stock and growth parameters.

Conduct a new optimization experiment if there are any changes to cryo stocks, vessel type, broth, or incubation time.

How to prepare high dilutions of the cryo stock

In the present example, a 10^-5 dilution resulted in the best High-quality culture. This ratio is equivalent to a 1:100,000 dilution of the cryo stock, i.e substantially higher dilution than the typical 1:100 volumetric seeding ratio.

For future 11-hour overnight cultures, this would correspond to adding just 1 µL cryo stock to 100 mL (100,000 µL) growth medium. Because it it tricky to accurately pipette 1 µL we recommend that you first prepare a 1:100 dilution and then dilute this 1:1 000 for the final 1:100,000 dilution:

1. Prepare 1:100 dilution: Transfer 0.1 mL cryo stock to 9.9 mL sterile growth medium

2. Prepare 1:100,000 dilution: Transfer 0.1 mL 1:100 dilution to ~100 mL sterile growth medium in shake flask

Summary and next steps

We hope that you are enjoyed the insights from this step by step guide. We are confident that your new and shiny High-quality culture will improve your future bioprocesses.

Now that you have the conditions for a great starter culture try to Track growth curves for it. For example, you could compare the High-quality culture head-to-head with your previous starter culture protocol. You will likely observe a shorter lag phase duration for the High-quality culture.