# 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" %}

## <i class="fa-spray-can">:spray-can:</i> Do a *Clean* program after each measurement.&#x20;

{% endhint %}

{% hint style="success" %}

## <i class="fa-hourglass">:hourglass:</i> 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.&#x20;
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:&#x20;
      1. Transfer 101 µL of your sample to 10 mL of diluent.&#x20;
      2. Vortex 10 seconds at maximum speed.
   2. Then make the 1:10 000 dilution:&#x20;
      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.

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FvaZfg6ZOqoIsMqlfbTbY%2F1%3B10000_prepare.avif?alt=media&#x26;token=f1a417b1-1865-40f0-bfe3-1631d84f467a" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}

### Measure the diluted sample on BactoBox®

8. Click the  <kbd><mark style="background-color:purple;">Measure<mark style="background-color:purple;"></kbd> button in Access.&#x20;
9. Add metadata:&#x20;
   1. Dilution: <kbd>10 000</kbd>.
   2. Label: <kbd>E\_coli\_growth</kbd>.&#x20;

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FseSlDIg4OsySqW1Rzvf3%2F1%3B10000_access.avif?alt=media&#x26;token=43a216fb-b204-41d3-8f4b-beeca384efa0" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}

### Clean the device

1. Transfer the tubing kit to the disinfection vial.
2. Click <kbd>Clean</kbd>.&#x20;
   {% 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.&#x20;