# 1:10 000 dilutions
Use a 1:10,000 dilution if the 1:1,000 dilution results in more than 1,500,000 total/ml, as observed in the sample analyzed approximately 30 minutes ago.
{% 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. Prepare a 1:10 000 dilution using two sequential 1:100 dilution steps.
1. First make a 1:100 dilution:
1. Transfer 101 µL of your sample to 10 mL of diluent.
2. Vortex 10 seconds at maximum speed.
2. Then make the 1:10 000 dilution:
1. Transfer 101 µL of the 1:100 dilution to 10 mL diluent.
2. Vortex 10 seconds at maximum speed.
7. Transfer the tubing kit to the 1:10 000 vial.
{% endstep %}
{% step %}
**Measure the diluted sample on BactoBox®**
8. Click the Measure button in Access.
9. Add metadata:
1. Dilution: 10 000.
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
You have now tracked the concentration through lag, exponential, deceleration and stationary stage. The final step is to determine when to conclude the growth curve experiment.
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