# Prepare flasks and media

{% stepper %}
{% step %}
**Prepare growth medium**

Dissolve 500 mL culture medium in an autoclavable bluecap flask as recommended by the manufacturer.
{% endstep %}

{% step %}
**Get flasks ready**

Loosen the lids on 10 shake flasks slightly to allow steam entry.

Place a piece of autoclave tape between the flask and the cap.

Label each flask clearly with a permanent marker on the autoclave tape.

* Label the first flask 10<sup>-1</sup><sup><sub>.<sub></sup>
* Proceed with the remaining flasks until reaching 10<sup>-10</sup>.
  {% endstep %}

{% step %}
**Sterilize growth medium and flasks**

Sterilize the bluecap and shake flasks according to the medium manufacturer's autoclaving instructions: usually at 121 °C and \~15 psi for 15 minutes.
{% endstep %}

{% step %}
**Transfer growth medium to each flask**

Once the flasks and cultivation medium have cooled, transfer 9 mL growth medium to each flask using proper aseptic technique.
{% endstep %}
{% endstepper %}

{% hint style="info" icon="cloud-fog" %}

#### Liquid evaporates during the autoclave process

For a reliable dilution series, we need the volume of growth medium in each flask to be accurate.

To ensure consistent liquid levels and reliable dilutions, we autoclave the growth medium separately before transferring it to shake flasks. Autoclaving directly in flasks can cause unpredictable evaporation.
{% endhint %}

## Summary

Shake flasks and growth medium is ready for the experiment.\
Next step: [Create measurement group](/mpd/workflows/identify-optimal-inoculum-size/create-measurement-group.md).


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