# 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.

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

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## <i class="fa-hourglass">:hourglass:</i> Start a 30-minute countdown to remind you to collect the next sample.

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### 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. 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.&#x20;
      2. Vortex 10 seconds at max speed.
   2. Then make the 1:1 000 dilution:&#x20;
      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.

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## Pro tip: 1-step 1:1 001 dilution.&#x20;

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://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-concentration).
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<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2Fb1YB458hXVjIdGCSvu77%2F1%3B1000_prepare.avif?alt=media&#x26;token=89e51b53-d075-479a-ba14-fdef55fc7674" alt=""><figcaption></figcaption></figure>
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{% step %}

### Measure the diluted sample on BactoBox®

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

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FvHfbo3YyQdbuOUKUQoeR%2F1%3B1000_access.avif?alt=media&#x26;token=20e4badf-0bdc-4874-ab8c-2fd75dac4d9d" alt=""><figcaption></figcaption></figure>
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{% step %}

### Clean the device

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

The 1:1 000 dilution scheme works well for mid- to late-exponential stage.&#x20;

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.