# Inoculate culture

Once you know the cell concentration of the inoculum you can calculate the volume necessary to hit a target seeding concentration (here 1 × 10<sup>7</sup> cells/mL). Use the [subcultivation calculator tool](https://www2026.sbtinstruments.com/wp-content/uploads/2025/12/202512-BactoBox-sub-cultivation-calculator.html) for the calculations.

{% hint style="success" %}

## Use Optiseeding workflow to determine suitable seeding concentration

The seeding concentration may need adjustment to get a [Broken mention](broken://pages/vrWP9ojZJ21IAYmCChgF). Do a screening experiment with a broad 1:10 dilution series to find a suitable seeding concentration. See [Broken mention](broken://pages/HCyA5smADMmJsn8WcnzH) 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 inoculum in the first field.
* Add volume information in the second field. In this case it is 50 mL
* Set the desired inoculation concentration in the final field. In this case 1 × 10<sup>7</sup> cells/mL
* Click <kbd>Calculate V\_add</kbd> to get the result.

<figure><img src="/files/4wsg2MOPepn3yLOJUyGJ" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}
**Add inoculum culture to growth medium**

Transfer the calculated volume of inoculum to growth curve flask and swirl aggressively to disperse the inoculum.

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

<figure><img src="/files/OuerBsPVgLOtWaRO9aKX" alt=""><figcaption><p>Inoculation of shake flask at well-defined concentration. In this video the 50 mL vial with the red cap represents the inoculum.</p></figcaption></figure>
{% endstep %}
{% endstepper %}

## Summary

Once the growth medium is inoculated, proceed to [Determine initial concentration](/mpd/workflows/get-starter-cultures/from-liquid-culture/determine-initial-concentration.md).


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