> 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/inoc/workflows/get-exponential-phase-inoculum.md).

# Get exponential phase inoculum

Infectivity studies and other *in vivo* models are done using bacteria in exponential phase. Most overnight cultures are in stationary phase and therefore a short subculture step is needed. 

The step by step section below is a rough guide for fast-growing bacteria (generation time <30 min). Your culture is in exponential phase when you see see at least **10× increase in cell concentration**.&#x20;

If you are working with slow-growing bacteria it is necessary to adapt the workflow. [Contact SBT](/troubleshooting/contact-our-support-team/contact-our-support-team.md) for assistance.

{% hint style="success" icon="chalkboard-user" %}

## Sample preparation for BactoBox® analyses

BactoBox® gives you reliable results when you have a single-cell suspension and hit the right concentration range. Read [Measurement essentials](/measurement-essentials/simple-measurement.md) to avoid some of the common sample preparation pitfalls.
{% endhint %}

{% hint style="success" icon="pump-soap" %}
[Clean](/maintenance/clean-the-setup.md) your BactoBox® after each measurement.
{% endhint %}

## Step by step

{% stepper %}
{% step %}

### Start your culture

Use your standard laboratory protocol to inoculate an overnight culture from e.g. cryo stock, agar plate colony, or certified reference material (CRM).&#x20;
{% endstep %}

{% step %}

### Wait for it to grow overnight

Incubate at optimal growth parameters overnight. It is preferable to inoculate at the end of the workday so the overnight culture does not reach decline/death phase.&#x20;
{% endstep %}

{% step %}

### Make a subculture in the morning&#x20;

As early as possible, make a subculture in fresh growth medium, e.g. by transferring 0.5 mL overnight culture to \~50 mL growth medium (\~1% V/V).&#x20;

<figure><img src="/files/OuerBsPVgLOtWaRO9aKX" alt=""><figcaption><p>Inoculation of subculture. In this video the 50 mL vial with the red cap represents the overnight culture transferred from the original shake flask.</p></figcaption></figure>
{% endstep %}

{% step %}

### Measure starting concentration at T<sub>0</sub>

Use BactoBox to determine the starting concentration of bacteria, C<sub>0</sub>. A 1:100 dilution in BactoBox® diluent is usually suitable.&#x20;

<figure><img src="/files/bXKclJZrqlIFfa8jFBI4" alt="" width="531"><figcaption><p>1:100 dilution is done by a single transfer of 101 µL culture of 10 mL of diluent.</p></figcaption></figure>
{% endstep %}

{% step %}

### Calculate starting concentration, C<sub>0</sub>

To determine the concentration of the culture, multiply the cells/mL with the dilution factor.&#x20;

Example: C<sub>0</sub> = 540 000 cells/mL × 100 = 54 000 000 cells/mL = 5.4×10<sup>7</sup> cells/mL.

<figure><img src="/files/69rlQ3SGIYgK3lAmGska" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}

### Incubate two hours&#x20;

Incubate at optimal growth conditions for two hours and then retrieve a representative sample of the culture.&#x20;
{% endstep %}

{% step %}

### Measure concentration

Do a 1:10,000 dilution and measure bacterial concentration using BactoBox®. If the concentration is <30 000 cells/mL, use the 1:100 dilution from [step 4](#measure-starting-concentration-at-t0).

<figure><img src="/files/04FNIoxa26Qp0DnwekUU" alt=""><figcaption></figcaption></figure>
{% endstep %}

{% step %}

### Wait for at least 10× increase compared to C<sub>0</sub>

Repeat step 6 and 7 until you get at least <mark style="background-color:green;">10× increase in cell concentration</mark> compared to C<sub>0</sub>. \
In this example, the cell concentration in the flask should be at least 5.4×10<sup>8</sup> cells/mL.
{% endstep %}

{% step %}

### Next steps

Your culture is now in exponential phase and ready for your experiments.&#x20;
{% endstep %}
{% endstepper %}

## Related workflows

If you wan't to compare the CFU and BactoBox® results, see [Crosscheck CFU and cells](/inoc/workflows/crosscheck-cfu-and-cells.md).


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