# 1:100 dilution post-inoculation

Once the shake flask is inoculated, immediately collect a sample to obtain a measurement as close to T<sub>0</sub> as possible.

{% 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. Dilute 1:100 in diluent by adding 101 µL of sample to 10 mL of diluent. Vortex 10 sec., max speed.
7. Transfer the tubing kit to the diluted sample.

{% hint style="success" %}

## Put the flask back in the incubator shaker immediately after collecting the sample.

Collect the sample quickly to ensure prompt return. Minimize the time the flask is outside the incubator shaker to avoid drops in temperature and dissolved oxygen levels.&#x20;
{% endhint %}

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2Fyvo1ZGsZ65GFtFu9wnMc%2FT0_prepare.avif?alt=media&#x26;token=297976a5-374a-41b6-91e9-bd92085521c7" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% 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>100</kbd>.
   2. Label: <kbd>E\_coli\_growth</kbd>.&#x20;
3. Once measurement is done, click the label <kbd>E\_coli\_growth</kbd> above the chart to visualize the growth curve data.

<figure><img src="https://4216107837-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FeYTtd7c0A325muFr5lMW%2Fuploads%2FjtNevBAxFQJYLIOYLYtq%2FT0_measure.avif?alt=media&#x26;token=95293164-1218-48d2-ac7b-3160727af01d" alt=""><figcaption></figcaption></figure>

{% hint style="danger" %}

## Do you get the error *conductivity too high*?

Some cultivation media have very high conductivity and a 1:100 dilution in standard diluent is not sufficient to bring the diluted sample within the accepted conductivity limits.&#x20;

You can typically solve this issue by using a 1:201 dilution: Simply add 50 µL of sample to 10 mL of diluent. For more information, please see the section [hit-the-right-concentration](https://help.sbtinstruments.com/protocols/prepare-your-sample/hit-the-right-concentration "mention").
{% endhint %}
{% endstep %}

{% step %}

### Clean the device

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

## Summary

You now know the bacterial concentration at T<sub>0</sub>. Next we will collect a sample for every 30 min and keep repeating the 1:100 dilution scheme for lag and early exponential growth stage.&#x20;