# Determine concentration after incubation

For every e.g. 2 hours, pull a sample to check if the cell concentration has increased. The starter culture is ready when the cell concentration is at least ten-fold higher.

{% hint style="info" icon="arrow-up-right-dots" %}

#### Extend incubation until cell concentration is at least ten-fold higher compared to the T<sub>0</sub> result

* A cryo stock contains a complex mix of culturable, viable-but-non-culturable, and dead cells. The initial BactoBox® cell concentration right after inoculation detects all these variants and does therefore not only reflect the concentration of culturable cells.
* Incubate the culture until the cell concentration is at least a 10× higher. At this point at least 90% of the detected cells are culturable.
  {% endhint %}

## Step by step

{% stepper %}
{% step %}
**Retrieve a sample after 2 hours**

After 2 hours of incubation, use a serological pipette to pull 1 mL sample. Transfer to e.g. a 5 mL centrifuge vial.
{% endstep %}

{% step %}
**Disaggregate cell clumps**

Vortex the sample 1 min at max speed to disaggregate cell clumps.
{% endstep %}

{% step %}
**Dilute 1:100**

Transfer 101 µL of T0 culture to 10 mL of diluent. Vortex 10 sec. at max speed.
{% endstep %}

{% step %}
**Measure**

Transfer tubing kit to the diluted sample. Press <kbd><mark style="background-color:purple;">Measure<mark style="background-color:purple;"></kbd> and wait for the result to appear on the BactoBox® screen.
{% endstep %}

{% step %}
**Calculate concentration**

Multiply the cells/mL result with the dilution factor, in this case 100, to get the cell concentration in the culture at T<sub>0</sub>.
{% endstep %}

{% step %}
**Stop or repeat**

Your starter culture is ready when the cell concentration is at least ten-fold higher than at T<sub>0</sub>,

If the concentration is still too low, extend the incubation by two hours and repeat steps 1 to 5 until ten-fold increase is obtained.
{% endstep %}
{% endstepper %}

## Summary

The starter culture is ready once the cell concentration has increased by at least 10-fold. You can now use the culture for experiments or to continue with the next step in your seed train.


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