# 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.&#x20;
{% 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.&#x20;

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](https://help.sbtinstruments.com/protocols/optimize-bioprocesses/identify-optimal-inoculum-size/create-measurement-group "mention").