Optimize starter culture

MPD-6. Get a high-quality overnight culture after the desired incubation time.

Skill level	Time to complete (E. coli)	Hands-on time	HW requirement
Basic microbiology	16 hours	2 hours	7.6a

Get a high-quality starter culture

Most bioprocesses are initiated from a cryo stock. The cryo stock is thawed at room temperature and a volume is added to growth medium in a shake flask for overnight incubation. This starter culture will later be used to inoculate a bioreactor. The ratio between volume of cryo stock to volume of growth medium is called inoculation ratio (or seeding ratio).

A typical seed train for a bioprocesses. The initial step is the reactivation and expansion of bacteria from a cryo stock.

A common pitfall at this stage is overseeding. Too much salt ruins your food; overseeding ruins your culture. In practice high inoculation ratio may cause the starter culture to enter death phase prematurely. As a result, the subsequent expansion suffers from a prolonged lag phase, requiring extended incubation times before the culture is ready for transfer to the next bioreactor. To achieve a high-quality culture, it is crucial to establish the correct inoculation ratio.

Introducing OptiSeeding

OptiSeeding is a step-by-step workflow designed to help you select the optimal seeding ratio for the initial cryo stock expansion. The workflow is essentially a screening experiment with 1:10 dilution series prepared directly in shake flasks.

High-level view of OptiSeeding: Dilution series are prepared directly in shake flasks and then incubated overnight.

OptiSeeding ensures efficient culture expansion, and thereby expedites process completion. The explainer Overnight cultures presents proof of principle for the OptiSeeding approach with investigation of culturability using different initial inoculation ratios.

Step by step overview

In this how to example, we use E. coli in a shake flask with tryptic soy broth (TSB) as the growth medium. We illustrate the procedure using Access for automated dilution factor calculations. You can also use the BactoBox® buttons for measurements and perform the calculations manually.

The general workflow follows the below steps

1. Get things ready for the experiment.

2. Prepare flasks and media for the overnight cultures.

3. Create measurement group to link the measurements.

4. Measure on cryo stock to determine initial total cell concentration.

5. Prepare dilution series directly in shake flasks and incubate flasks overnight.

6. Select relevant cultures by visual inspection.

7. Check lowest culture by 1:1 000 dilution and BactoBox® measurement.

8. Check remaining cultures by 1:10 000 dilution and BactoBox® measurement.

9. Choose best seeding ratio for future overnight cultures.

Summary

You are now ready to start your journey to get a high-quality culture. Let's jump into it. First we will get things ready.