# Inoculate liquid culture

Use the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) to determine the volume needed to seed the growth medium. A typical overnight seeding concentration is 1 x 10<sup>6</sup> cells/mL.

{% hint style="success" %}

#### Use Optiseeding workflow to determine suitable seeding concentration

The seeding concentration may need adjustment to get a [high-quality culture](/mpd/encyclopedia/high-quality-culture.md). Do a screening experiment with a broad 1:10 dilution series to find a suitable seeding concentration. See [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size.md) for more details.
{% endhint %}

{% stepper %}
{% step %}
**Open the subcultivation calculator**

Open the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) in a browser.
{% endstep %}

{% step %}
**Calculate inoculation volume**

* Fill in cells/mL of the capture vial in the first field.
* Add volume information in the second field. In this case it is 40 mL
* Set the desired inoculation concentration in the final field. In this case 1 × 10<sup>6</sup> cells/mL
* Click <kbd>Calculate V\_add</kbd> to get the result.
  {% endstep %}

{% step %}
**Add starter culture to growth curve flask**

Transfer starter culture to growth curve flask and swirl aggressively to disperse the inoculum.

Record the inoculation time. Record it on the autoclave tape as a backup, ensuring you have a reference if you forget to document it elsewhere.

<figure><img src="/files/JnH6zTG6bm1XBzex7UZp" alt=""><figcaption><p>Inoculation of shake flask at well-defined concentration. In this video the 50 mL vial with the red cap represents the capture vial.</p></figcaption></figure>
{% endstep %}
{% endstepper %}

## Summary

You have now inoculated the overnight culture at a well-defined starting concentration of 1 <sup><sub>x<sub></sup> 10<sup>6</sup> cells/mL. Next step is [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-crm-product/determine-concentration-after-incubation.md).


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