# Determine initial concentration

We determine the concentration immediately after inoculation, T<sub>0</sub>, to know the starting concentration. This number will help compare post-incubation results to determine if cell concentration increases. Use a 1:100 dilution before performing a BactoBox® measurement, as the cryo stock is already diluted in the growth medium.

## Step by step

{% stepper %}
{% step %}
**Pull a sample at T0**

Use a sterile serological pipette to collect 1 mL sample immediately after inoculation.
{% 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 T<sub>0</sub> 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 %}
**Incubate**

Place the shake flask at desired growth conditions.
{% endstep %}
{% endstepper %}

## Summary

After determining the concentration at T<sub>0</sub>, proceed to [Determine concentration after incubation](/mpd/workflows/get-starter-cultures/from-cryo-stock/determine-concentration-after-incubation.md).


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