> For the complete documentation index, see [llms.txt](https://help.sbtinstruments.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.sbtinstruments.com/mpd/workflows/identify-optimal-inoculum-size/measure-on-cryo-stock.md).
# Measure on cryo stock
The cryo stock is the starting point for preparing the dilution series. It will likely contain a mix of culturable, viable-but-non-culturable (VBNC), and dead cells. Knowing the cell concentration helps determine how far the dilution can go before cells are virtually absent in the growth medium. We will use a 1:1 000 dilution in two sequential dilution steps: An initial 1:100 dilution followed by a additional 1:10 dilution.
{% stepper %}
{% step %}
**Disaggregate cell clumps**
Allow the cryo stock to thaw at room temperature.
Vortex the cryo stock 1 minute at maximum speed to disaggregate cell clumps.
{% endstep %}
{% step %}
**Dilute 1:100**
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Dilute 1:1000**
Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.
Transfer 1 mL of the 1:100 dilution to the vial containing 9 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure on the diluted sample**
Transfer the tubing kit to the 1:1 000 vial.
Add the metadata:
* Dilution: 1 000
* Label: E\_coli\_cryo
Then press Measure in Access to start a new measurement.
After completing the measurement, Access will automatically calculate the bacterial concentration in the cryo stock in the field Cells/mL × Dil.
{% endstep %}
{% endstepper %}
## Summary
Now you know the bacterial concentration of your cryo stock.\
Next step: [Prepare dilution series](/mpd/workflows/identify-optimal-inoculum-size/prepare-dilution-series.md).
---
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