# Determine concentration after incubation

Now, you will determine the concentration after overnight incubation. Cell concentration is typically in a ball-park range of 1 × 10<sup>10</sup> cells/mL, and therefore a dilution of 1:10,000 is usually suitable. This dilution is obtained by two sequential 1:100 dilutions. Below we demonstrate this step by step using the Access interface. You can also use BactoBox® as a standalone device and do the calculations manually.

{% stepper %}
{% step %}
**Retrieve and disaggregate a sample of the overnight culture**

Use a sterile serological pipette to transfer 10 mL of the overnight culture to a sterile vial.

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/MGwE2U3Smfapw0hNK5AG" 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 %}
**Dilute 1:100**

Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.

<figure><img src="/files/Tvwnw2tdSque6o1nLWlY" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}
**Dilute 1:10 000**

Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.

<figure><img src="/files/KEqpWgk16dqxaq3GYVRR" alt=""><figcaption></figcaption></figure>
{% endstep %}

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

Transfer the tubing kit to the 1:10,000 vial.

Press <kbd><mark style="background-color:purple;">Measure<mark style="background-color:purple;"></kbd> in Access to start a new measurement.

* Dilution: <kbd>10 000</kbd>
* Label: <kbd>Overnight\_culture</kbd>

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 %}
{% endstepper %}

## Summary

Now you know the bacterial concentration of your overnight culture. With cells from an agar plate as input material, you now have a liquid starter culture with a well-defined cell concentration.


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