> 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/not-in-the-new-structure/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 :earlybirds: early bird colleague to complete these steps before you arrive. {% endstep %} {% step %} ### Label the shake flask Use a marker to write *E. coli* ATCC® 25922 and inoculation time on the 250 mL baffled shake flask. {% endstep %} {% step %} ### Transfer Vitroids**™** 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.

Demonstration of the dissolution of a Vitroids™ 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).

{% 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](/not-in-the-new-structure/measure-vitroid-tm-growth-curve/disaggregate-bacteria.md) for how to sample and disaggregate[^1]. [^1]: Break up clumps or chains of cells --- # Agent Instructions This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com. ## Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation by asking a question. Perform an HTTP GET request on the following URL with the `ask` and `goal` query parameters: ``` GET https://help.sbtinstruments.com/not-in-the-new-structure/measure-vitroid-tm-growth-curve/inoculate-lb-medium.md?ask=&goal= ``` `ask` is the immediate question: it should be specific, self-contained, and written in natural language. `goal` is what the user is ultimately trying to achieve, the reason they need the answer. Sharing it helps GitBook give you a better, more relevant answer. A goal is most helpful when it describes the outcome the user wants rather than restating the question. For example, with `ask=how do I create an API token`, a goal like `automate deployments from our CI pipeline` lets GitBook tailor the answer to that use case. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.