> For the complete documentation index, see [llms.txt](https://help.sbtinstruments.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.sbtinstruments.com/tutorials/measure-vitroid-tm-growth-curve/inoculate-lb-medium.md).

# Inoculate LB medium

The first objective is to inoculate the growth medium. The step-by-step procedure below provides two options. Click the tab headers to switch between them.

Our recommended approach is to inoculate the culture using a Vitroids™ disc. This method has been thoroughly tested, and the expected timeline is well established.

Alternatively, you can inoculate the culture using an in-house glycerol stock. Because we have no information about the characteristics or storage conditions of your glycerol stocks, we cannot guarantee either the timeline or the outcome. If you already have established protocols for preparing *E. coli* overnight cultures, feel free to follow those and use our tutorials as inspiration.

## Step by step

{% tabs %}
{% tab title="Preferred: Vitroids™ disc" icon="flying-disc" %}
{% stepper %}
{% step %}

### Start the inoculation early

Start the inoculation procedure below as early as possible during your workday.

Alternatively, have an <i class="fa-earlybirds">:earlybirds:</i> early bird colleague to complete these steps before you arrive.
{% endstep %}

{% step %}

### Label the shake flask

Use a marker to write <kbd>*E. coli*</kbd> <kbd>ATCC</kbd><sup><kbd>®<kbd></sup> <kbd>25922</kbd> and inoculation time on the 250 mL baffled shake flask.
{% endstep %}

{% step %}

### Transfer Vitroid&#x73;**™** disc

Use proper aseptic technique and transfer the disc to the shake flask. The flask already contains 50 mL of autoclaved LB medium.

The video below demonstrates manual shaking and a 5-minute dissolution step. You may skip these steps and allow the incubator shaker to dissolve the disc during the next step.

<figure><img src="/files/4tA6VtXuodUY6uYAhjvf" alt=""><figcaption><p>Demonstration of the dissolution of a Vitroids<strong>™</strong> disc in growth medium. You can proceed directly step 4 after transferring the disc (You don't need to wait for the dissolution to happen).</p></figcaption></figure>
{% endstep %}

{% step %}

### Start the incubation

Transfer the shake flask to the shaking incubator. Incubate at 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% endtab %}

{% tab title="Alternative: In-house glycerol stock" icon="snowflakes" %}
{% stepper %}
{% step %}

### Label the shake flask

Use a marker to write *E. coli,* in-house strain designation, and inoculation time directly on the 250 mL baffled shake flask containing 50 mL of LB medium.
{% endstep %}

{% step %}

### Thaw glycerol stock

Collect an *E. coli* glycerol stock from your -80 °C freezer and allow it to thaw for \~30 minutes at room temperature.
{% endstep %}

{% step %}

### Inoculate using \~0.1 % (V/V)

Transfer 50 µL glycerol stock to the flask.
{% endstep %}

{% step %}

### Start the incubation

Transfer the shake flask to the shaking incubator. Incubate at 37 °C, 200 RPM.
{% endstep %}

{% step %}

### Run BactoBox® measurements every 30 min

In the preferred version of this tutorial (Vitroids™ disc), the culture is incubated for \~5 hours to reach concentrations detectable by BactoBox® measurements.

**When using the in-house alternative (glycerol stock), perform measurements every 30 minutes.**
{% endstep %}
{% endstepper %}
{% endtab %}
{% endtabs %}

## Summary

You have now inoculated the LB growth medium. For the Vitroids™ disc procedure \~5 hours of incubation are required before the bacterial concentration is high enough for BactoBox® measurements. Proceed to [Prepare sample](/tutorials/measure-vitroid-tm-growth-curve/disaggregate-bacteria.md) for how to sample and disaggregate.


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