# Optimize starter culture

| Skill level                                        | Time to complete (E. coli)                       | Hands-on time                                   | Requirements                                                                                                                                                                                                 |
| -------------------------------------------------- | ------------------------------------------------ | ----------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| <i class="fa-medal">:medal:</i> Basic microbiology | <i class="fa-hourglass">:hourglass:</i> 16 hours | <i class="fa-stopwatch">:stopwatch:</i> 2 hours | <i class="fa-hard-drive">:hard-drive:</i> [7.6a](/encyclopedia/item-register/bactobox-r/bactobox-r-hardware-changelog.md) <i class="fa-floppy-disk">:floppy-disk:</i> [v2026.02a](/encyclopedia/software.md) |

## **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).

<figure><img src="/files/kAONNihW7KOmaBzaoqYJ" alt=""><figcaption><p>A typical seed train for a bioprocesses. The initial step is the reactivation and expansion of bacteria from a cryo stock.</p></figcaption></figure>

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](/mpd/encyclopedia/high-quality-culture.md), 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.

<figure><img src="/files/72HVDNWcE1rh1b9GaUWx" alt=""><figcaption><p>High-level view of OptiSeeding: Dilution series are prepared directly in shake flasks and then incubated overnight.</p></figcaption></figure>

OptiSeeding ensures efficient culture expansion, and thereby expedites process completion. The explainer [Overnight cultures](/mpd/encyclopedia/overnight2.md) 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](https://help.sbtinstruments.com/encyclopedia/software/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](/mpd/workflows/identify-optimal-inoculum-size/get-things-ready.md) for the experiment.
2. [Prepare flasks and media](/mpd/workflows/identify-optimal-inoculum-size/prepare-flasks-and-media.md) for the overnight cultures.
3. [Create measurement group](/mpd/workflows/identify-optimal-inoculum-size/create-measurement-group.md) to link the measurements.
4. [Measure on cryo stock](/mpd/workflows/identify-optimal-inoculum-size/measure-on-cryo-stock.md) to determine initial total cell concentration.
5. [Prepare dilution series](/mpd/workflows/identify-optimal-inoculum-size/prepare-dilution-series.md) directly in shake flasks and incubate flasks overnight.
6. [Select relevant cultures](/mpd/workflows/identify-optimal-inoculum-size/select-relevant-cultures.md) by visual inspection.
7. [Check lowest culture](/mpd/workflows/identify-optimal-inoculum-size/check-lowest-culture.md) by 1:1 000 dilution and BactoBox® measurement.
8. [Check remaining cultures](/mpd/workflows/identify-optimal-inoculum-size/check-remaining-cultures.md) by 1:10 000 dilution and BactoBox® measurement.
9. [Choose best seeding ratio](/mpd/workflows/identify-optimal-inoculum-size/choose-best-seeding-ratio.md) for future overnight cultures.

## Summary

You are now ready to start your journey to get a [high-quality culture](/mpd/encyclopedia/high-quality-culture.md). Let's jump into it. First we will [get things ready](/mpd/workflows/identify-optimal-inoculum-size/get-things-ready.md).


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