> 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/encyclopedia/optimize-starter-culture-old-steps/check-remaining-cultures.md).
# Check remaining cultures
A 1:1 000 dilution worked well for the -8 culture. The remaining cultures from -7 to -4 were seeded with more cells and therefore a dilution factor of 1:10 000 will be used for these overnight cultures. This is achieved through two sequential 1:100 dilutions.
{% stepper %}
{% step %}
**Disaggregate cell clumps**
Vortex the vial 1 minute at maximum speed to disaggregate[^1] cell clumps. See [Break up clumps and chains](/advanced/advanced-sample-preparation/break-up-clumps-and-chains.md) if your bacterium needs more aggressive disaggregation[^1].
{% 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 1:10 000**
Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
Transfer the tubing kit to the 1:10 000 vial.
Add the metadata:
* Dilution: 10 000
* Label: overnight\_-7 , overnight\_-6 , overnight\_-5 or overnight\_-4 ,
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 %}
{% step %}
**Check remaining cultures**
Repeat above steps with 1:10 000 dilutions of the remaining cultures, in this case vial -6, -5, and -4.
{% endstep %}
{% endstepper %}
## Summary
You have now determined the cell concentration in the relevant cultures.\
Proceed to the next step: [Choose best seeding ratio](/mpd/encyclopedia/optimize-starter-culture-old-steps/choose-best-seeding-ratio.md).
[^1]: Break up clumps or chains of cells
---
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