# 1:1 000 dilutions
As the bacteria multiply, the concentration increases, and a 1:100 dilution is no longer suitable. The maximum concentration in the diluted sample is 5 000 000 total/mL. If this limit is exceeded, an error will display instead of a concentration. In such cases, clean the setup and measure a more diluted sample.
{% hint style="info" %}
#### Avoid getting *too many particles.*
To prevent the "too many particles" error, follow this simple rule of thumb during the exponential growth stage: If the 1:100 diluted sample was above 1 500 000 at the last measurement \~30 minutes ago, it is time to start using a 1;1 000 dilution.
This rule works for all bacteria as long as the sampling frequency is set to 1.5 x generation time.
{% endhint %}
{% hint style="success" %}
#### :spray-can: Do a *Clean* program after each measurement.
{% endhint %}
{% hint style="success" %}
#### :hourglass: Start a 30-minute countdown to remind you to collect the next sample.
{% endhint %}
{% stepper %}
{% step %}
**Prepare the sample for BactoBox® measurement**
1. Use a serological pipette to transfer a sample from the shake flask to a vial.
2. Use a permanent marker to label the vial cap with the sampling time.
3. Return the shake flask to the incubator shaker.
4. Start a 30-minute countdown to remind you to collect the next sample.
5. Vortex 30 seconds at max speed to disaggregate bacteria.
6. Make a 1:1 000 dilution
1. First make a 1:100 dilution:
1. Transfer 101 µL of your sample to 10 mL of diluent.
2. Vortex 10 seconds at max speed.
2. Then make the 1:1 000 dilution:
1. Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.
2. Transfer 1 mL of the 1:100 dilution to the vial containing 9 mL of diluent.
3. Vortex 10 seconds at max speed.
7. Transfer the tubing kit to the 1:1 000 vial.
{% hint style="info" %}
## Pro tip: 1-step 1:1 001 dilution.
The described 2-step procedure for a 1:1,000 dilution is reliable with high precision. However, it demands a longer sample preparation time. If you have good pipetting skills, you can save time, sample and diluent by doing a 1-step dilution. Simply transfer 10 µL of sample to 10 mL of diluent to [get a 1:1 001 dilution](https://github.com/sbtinstruments/docsites/blob/main/help/protocols/prepare-your-sample/hit-the-right-concentration/README.md).
{% endhint %}
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
1. Click the Measure button in Access.
2. Add metadata:
1. Dilution: 1 000.
2. Label: E\_coli\_growth.
{% endstep %}
{% step %}
**Clean the device**
1. Transfer the tubing kit to the disinfection vial.
2. Click Clean.
{% endstep %}
{% endstepper %}
## Summary
The 1:1 000 dilution scheme works well for mid- to late-exponential stage.
Monitor when the diluted sample's concentration surpasses 1,500,000. When this happens, jump to the next section where we will detail how to dilute the sample at a ratio of 1:10 000.
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