# Measure starter culture

You need a starter culture with a known concentration. From this, we calculate the volume required to reach a final concentration of 1 × 10⁷ cells/mL in the flask.

Here we have already prepared the overnight culture based on the guidance provided in [Broken mention](broken://pages/SVUVO8i4W6FOh9xhJAUr).

{% stepper %}
{% step %}
**Retrieve a sample of the starter culture**

1. Use a serological pipette to aseptically transfer 10 mL of the overnight culture to a sterile vial.
2. Vortex thoroughly to disperse any cell clumps. For an *E. coli* culture it is usually sufficient to vortex 30 seconds at max speed.

<figure><img src="/files/KEGu03nejnOanvu0yBjP" alt=""><figcaption></figcaption></figure>

{% hint style="warning" %}

## Proper vortexing is important for reliable results!

Ensure that you see a proper vortex forming. Poor vortexing can cause inadequate disaggregation, leading to low precision in replicates and non-linearity in dilution series.

Different pedestals exists for different sizes of vials. In the video, we show a multipurpose pedestal that works for many types of vials. Ensure that your pedestal is compatible with the given vial.
{% endhint %}
{% endstep %}

{% step %}
**Prepare the sample for BactoBox® measurement**

1. Prepare a 1:10 000 dilution using two sequential 1:100 dilution steps.
   1. First make a 1:100 dilution:
      1. Transfer 101 µL of your sample to 10 mL of diluent.
      2. Vortex 10 seconds at maximum speed.
   2. Then make the 1:10 000 dilution:
      1. Transfer 101 µL of the 1:100 dilution to 10 mL diluent.
      2. Vortex 10 seconds at maximum speed.
2. Transfer the tubing kit to the 1:10 000 vial.

<figure><img src="/files/te1PLPBqwVPcl27psgEp" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}
**Measure the diluted sample on BactoBox®**

1. Transfer the tubing kit to the 1:10,000 vial.
2. Click <kbd><mark style="background-color:purple;">Measure<mark style="background-color:purple;"></kbd> to start a new measurement. Notice that there is a group icon on the button because you are within a measurement group.
   1. Dilution: <kbd>10 000</kbd>
   2. Label: <kbd>Overnight\_culture</kbd>
3. After completing the measurement, Access will automatically calculate the bacterial concentration in the shake flask. To view the full table, drag the panel sideways.
   {% endstep %}

{% step %}
**Clean the device**

1. Transfer the tubing kit to the disinfection vial.
2. Click <kbd>Clean</kbd>. The button in Access works the same way as the one on BactoBox®.

<figure><img src="/files/jtZ8ZHpF58dtXtJB2ZpQ" alt=""><figcaption></figcaption></figure>
{% endstep %}
{% endstepper %}

## Summary

Now you know the bacterial concentration of your starter culture. Proceed to the next step to calculate the volume of starter culture needed to inoculate the growth curve flask.


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