# Choose best seeding ratio

The measurements are now completed and it is time to choose the inoculation ratio that results in the best [https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md](https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md "mention"). In this 11-hour *E. coli* example, the 10<sup>-5</sup> cryo stock dilution produces a culture that has likely just arrived at stationary stage. The 10<sup>-4</sup> 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.

<figure><img src="/files/nThfdK5rOBQIfuDpsF1m" alt=""><figcaption></figcaption></figure>

{% hint style="warning" %}

#### 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.
{% endhint %}

## How to prepare high dilutions of the cryo stock

In the present example, a 10<sup>-5</sup> dilution resulted in the best [https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md](https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md "mention"). 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 [https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md](https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md "mention") will improve your future bioprocesses.

Now that you have the conditions for a great starter culture try to [https://github.com/sbtinstruments/help--mpd/blob/main/workflows/track-growth-curves/README.md](https://github.com/sbtinstruments/help--mpd/blob/main/workflows/track-growth-curves/README.md "mention") for it. For example, you could compare the [https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md](https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md "mention") head-to-head with your previous starter culture protocol. You will likely observe a shorter lag phase duration for the [https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md](https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md "mention").


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