> For the complete documentation index, see [llms.txt](https://help.sbtinstruments.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.sbtinstruments.com/mpd/workflows/identify-optimal-inoculum-size.md). # Optimize starter culture | Skill level | Time to complete (E. coli) | Hands-on time | Requirements | | -------------------------------------- | ------------------------------------------------ | ----------------------------------------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | :school\_satchel: No skill requirement | :hourglass: 16 hours | :stopwatch: 2 hours | :hard-drive: [7.6a](/item-register/bactobox-r/bactobox-r-hardware-changelog.md) :floppy-disk: ≥ v2026.02a | ## **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](/mpd/cell-growth/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.

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](/mpd/cell-growth/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/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/cell-growth/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). --- # Agent Instructions This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com. ## Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter, and the optional `goal` query parameter: ``` GET https://help.sbtinstruments.com/mpd/workflows/identify-optimal-inoculum-size.md?ask=&goal= ``` `ask` is the immediate question: it should be specific, self-contained, and written in natural language. `goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.