> 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-overnight-culture/inoculate-lb-medium.md).

# Inoculate LB medium

First objective is to start an overnight culture. We provide two options in the below step by step procedure. Click the tabs in the upper left corner to toggle between the two options.

The preferred route is to use a Vitroids™ disc for the inoculation. We have tested the robustness of this workflow and we know it works.

The alternative route is to use an in-house glycerol stock for the inoculation. We can't guarantee a successful outcome because we have no information on your glycerol stocks. If you have other guidelines for *E. coli* overnight cultures, feel free to use these.

## Step by step

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

### Start the culture in the afternoon

Wait until the end of the workday before inoculating the flask. Inoculate at 4:00 PM to ensure the overnight culture is ready by 8.00 AM the following morning.
{% 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 drop the disc in the shake flask that already contains 50 mL autoclaved LB medium.

<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 16 hours at 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% endtab %}

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

### Start the culture in the afternoon

Wait until the end of the workday before inoculating the flask. Inoculate at 4:00 PM to ensure the overnight culture is ready by 8.00 AM the following morning.
{% endstep %}

{% 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.
{% endstep %}

{% step %}

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

Transfer 50 µL *E. coli* glycerol stock directly to the shake flask. The flask already contains 50 mL LB medium and therefore this corresponds to \~0.1% volumetric seeding

{% hint style="info" %}

## How to get great overnight cultures

An *E. coli* culture inoculated at 0.1 % (V/V) is likely to be within stationary or even decline phase after 16 hours of incubation. The culture will be fine for this tutorial, but bad for e.g. bioprocess seed trains.

Check our workflow [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size.md) for how to get a [High-quality culture](/mpd/cell-growth/high-quality-culture.md).
{% endhint %}
{% endstep %}

{% step %}

### Start the incubation

Transfer the shake flask to the shaking incubator. Incubate 16 hours at 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% endtab %}
{% endtabs %}

## Summary

You inoculated the LB growth medium to get an overnight *E. coli* culture. Wait for \~16 hours and proceed to the next step [Prepare sample](/tutorials/measure-overnight-culture/disaggregate-bacteria.md).


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