> 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/not-in-the-new-structure/measure-vitroid-tm-growth-curve/1-10-000-dilutions.md).
# 1:10,000 dilutions
When the culture reaches the deceleration and stationary growth phases you should switch to a 1:10,000 dilution.
The rule of thumb still applies when deciding whether to increase the dilution factor: switch to the next dilution scheme if the measurement obtained approximately 30 minutes earlier was ≥ 1,500,000 total cells/mL.
## How to make 1:10,000 dilutions
A 1:10,000 dilution is obtained by two sequential dilutions. First dilution is the standard 1;100 dilution done by transferring 101 µL to 10 mL of diluent. Second dilution is an additional 1:100 dilution from the first 1;100 vial.
## Step by step
{% hint style="success" icon="bacteria" %}
Remember to disaggregate[^1]: Vortex each culture sample for 1 min at max speed.
{% endhint %}
{% hint style="success" icon="stopwatch" %}
Set a 30 min timer after each sampling point.
{% endhint %}
{% stepper %}
{% step %}
### Label two dilution vials
Label first dilution vial 1:100.
Label second dilution vial 1:10,000.
{% endstep %}
{% step %}
### Dilute 1:100
Transfer 101 µL of your sample to the 1:100 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Dilute 1:10,000
Transfer 101 µL of the 1:100 vial to the 1:10,000 vial.
Suspend ×10 to rinse the pipette tip for residual bacteria.
Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Measure the diluted sample on BactoBox®
Transfer the tubing kit to the 1:10,000 vial.
Press Measure to start a new measurement and wait for the results.
Add your results to the measurement table.
{% endstep %}
{% step %}
### Clean the device
Transfer the tubing kit to the disinfection vial and press Clean .
{% endstep %}
{% step %}
### Repeat 1:10,000 dilutions for subsequent samples
Repeat the sampling, disaggregation[^1], 1:10,000 dilution, measurement, and cleaning procedure for the next samples.
Conclude the sampling when two subsequent data points are within ±25%.
{% endstep %}
{% endstepper %}
## Summary
In deceleration and stationary phase 1;10,000 dilutions are necessary to hit the right concentration. By now you have gathered sufficient data points to plot a nice growth curve and calculate the growth rate. Proceed to [Data analysis](/not-in-the-new-structure/measure-vitroid-tm-growth-curve/data-analysis.md).
[^1]: Break up clumps or chains of cells
---
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