# 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](https://help.sbtinstruments.com/encyclopedia/bacterial-growth/high-quality-culture "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.&#x20;

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FlUWHNA9CkRfaJ7Xpbavj%2FOptiSeeding_BarChart.png?alt=media&#x26;token=9d7b0558-f003-4ff6-9770-9f3766b616c0" 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 [high-quality-culture](https://help.sbtinstruments.com/encyclopedia/bacterial-growth/high-quality-culture "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.&#x20;

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:&#x20;

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](https://help.sbtinstruments.com/encyclopedia/bacterial-growth/high-quality-culture "mention") will improve your future bioprocesses.

Now that you have the conditions for a great starter culture try to [track-growth-curves](https://help.sbtinstruments.com/protocols/optimize-bioprocesses/track-growth-curves "mention") for it. For example, you could compare the [high-quality-culture](https://help.sbtinstruments.com/encyclopedia/bacterial-growth/high-quality-culture "mention") head-to-head with your previous starter culture protocol. You will likely observe a shorter lag phase duration for the [high-quality-culture](https://help.sbtinstruments.com/encyclopedia/bacterial-growth/high-quality-culture "mention").&#x20;