> 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/inoc/workflows/crosscheck-cfu-and-cells.md).
# Crosscheck CFU and cells
In the below step by step instructions we demonstrate how to compare the real-time BactoBox® result to the final plate count.
For *in vivo* experiments, the bacterial culture will is often harvested by centrifugation and resuspended in 1× PBS to remove the growth medium components. Disaggregate carefully to convert the centrifugation pellet into a single-cell suspension.
{% hint style="success" icon="arrows-to-circle" %}
## Always disaggregate - especially after centrifugation
Bacteria tend to clump. Especially when centrifugation is used to harvest the culture into a neat pellet. Vortex thoroughly to get a single-cell suspension. This is needed for reliable BactoBox® and plate count results.
{% endhint %}
## Step by step
{% stepper %}
{% step %}
### Prepare the sample
Vortex the resuspended pellet thoroughly 1 min at max speed. Use a suitable mixer platform and ensure you see a proper vortex forming in the tube
{% endstep %}
{% step %}
### Create a *basic* measurement group
Connect your BactoBox® to a computer and create a *basic* measurement group. See [Do measurements in Access](/measurement-essentials/do-measurements-in-access.md) for more information.
{% endstep %}
{% step %}
### Dilute the sample for BactoBox measurements
Make a 1;10 000 dilution. Keep both the 1:100 and 1:10,000 dilution. See [Hit the right concentration](/measurement-essentials/hit-the-right-concentration.md) for more information.
{% endstep %}
{% step %}
### Measure on BactoBox®
First run the 1;10,000 dilution dilution on BactoBox®. Add sampling time, dilution factor, and sample description to the Access fields. Then click Measure to run the measurement.
If the result is <30,000 cells/mL then run the 1:100 dilution. The Cells/mL × Dil. field in the table shows you the bacterial concentration in the 1× PBS vial.
Add information on your sample and click Measure .
{% endstep %}
{% step %}
### Do two additional BactoBox® measurements
Make a fresh dilution series to reach the dilution that worked in step 4. Then measure.
Repeat this step for a total of 3 BactoBox® repeats.
{% endstep %}
{% step %}
### Prepare plate counts in triplicates
Dilute and plate to determine CFUs. Prepare three separate dilution series to include the variability involved with dilution, mixing, and plating. Incubate at optimal growth conditions.
{% hint style="info" icon="ring" %}
## Plate smarter
Use the cell concentrations determined via BactoBox® analyses to calculate suitable dilutions for plate counts. See the workflow [Plate smarter](/inoc/workflows/plate-smarter.md) for more information.
{% endhint %}
{% endstep %}
{% step %}
### Determine CFUs
Count colonies on the plates that contain between \~25–250 colonies. Multiply with the dilution factor to get the concentration in the non-diluted 1× PBS vial.
{% endstep %}
{% step %}
### Add the CFU data to Access
Add the CFU data to the CFU/mL × Dil. fields in Access that correspond to either of the BactoBox® repeats.
{% endstep %}
{% step %}
### Compare results
Click Export CSV to export the data as a csv file.
Use a spreadsheet or AI tool to determine the arithmetic mean, standard deviation, coefficient of variation (CV), and log difference between methods.
A log difference of 0 corresponds to a perfect agreement between the methods. BactoBox®-to-CFU log difference is typically within ±0.3, this corresponds to a two-fold difference. In the below calculation example the log difference 1.15.
| Method | Mean | SD | CV |
| ---------------- | ------------------- | ------------------- | ----- |
| BactoBox® | 1.21×108 | 0.08×108 | 6.6% |
| CFU | 1.05×108 | 0.12×108 | 11.4% |
| {% endstep %} | | | |
| {% endstepper %} | | | |
## Summary
With this step by step guide you know the essentials to compare BactoBox® and CFU methods.
{% hint style="info" icon="handshake" %}
## SBT offers custom solutions
Contact SBT if your results fall outside the accepted range when compared with the final CFU results. Together we can explore solutions to improve the agreements between results. For more information, see [What is a custom solution?](/advanced/what-is-a-custom-solution.md).
BactoBox® has high precision and reproducibility. Plate count procedures differs from lab to lab and it is impossible to make a single measurement program that accounts for this. That said, it is possible to apply empirical correction factors or customize the measurement programs.
{% endhint %}
---
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