> 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/tutorials/measure-vitroid-tm-growth-curve/1-1-000-dilutions.md).
# 1:1,000 dilutions
As the bacteria multiply, the cell concentration increases, and a 1:100 dilution may eventually exceed the maximum concentration of 5,000,000 total cells/mL permitted for a BactoBox® measurement.
At this point, switch from the initial 1:100 dilution to a 1:1,000 dilution.
{% hint style="success" %}
## Rule of thumb for when to switch to 1:1,000 dilutions
To avoid the “too many particles” error during the exponential growth phase, follow this simple rule of thumb: if the previous 1:100 dilution measurement, taken approximately 30 minutes earlier, exceeded **1,500,000 cells/mL**, switch to a 1:1,000 dilution for the next measurement.
{% endhint %}
## How to make 1:1,000 dilutions
A 1:1,000 dilution is prepared by performing two sequential dilution steps. The first step is the standard 1:100 dilution, which is prepared by transferring 101 µL of sample to 10 mL of diluent.
The second step is a 1:10 dilution of the first vial. To prepare this dilution, transfer 1 mL of the 1:100 dilution to a second dilution vial. Because the dilution vials contain a standard volume of 10 mL, you must first discard 1 mL of diluent from the second vial.
## Step by step
{% hint style="success" icon="bacteria" %}
Remember to disaggregate: 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 %}
### Dilute 1:100
Label a dilution vial 1:100 .
Vortex sample vial briefly (it has already been thoroughly vortexed when sampling).
Transfer 101 µL of your sample to the 1:100 vial. Pipette up and down ×10 in the 1:100 vial to rinse the pipette tip for residual bacteria.
Cap tightly and vortex 10 seconds at maximum speed.
{% endstep %}
{% step %}
### Dilute 1:1,000
Label a second dilution vial 1:1,000.
Aspirate and discard 1 mL diluent from the vial labeled 1:1,000
Transfer 1 mL of the 1:100 vial to the 1:1,000 vial. Pipette up and down ×10 in the 1:1,000 vial 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 1:1,000 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 1:1,000 dilutions for subsequent samples
Repeat the sampling, disaggregation, 1:1,000 dilution, measurement, and cleaning procedure for the next samples.
Proceed to the [1:10,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-10-000-dilutions.md) when the measured cell concentration of the last sample is ≥ 1,500,000 cells/mL.
{% endstep %}
{% endstepper %}
## Summary
Once the cell concentration increased it was necessary to switch from 1:100 to 1:1,000 dilutions. As the cell concentration continues to rise you will need to use [1:10,000 dilutions](/tutorials/measure-vitroid-tm-growth-curve/1-10-000-dilutions.md).
---
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