> 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-for-fast-growing-bacteria.md).

# Get exponential phase inoculum for fast-growing bacteria

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 intended for fast-growing bacteria (generation time <30 min). Your culture is in exponential phase when you see see at least **10× increase relative to starting cell concentration**.&#x20;

If you work 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.

<div data-with-frame="true"><figure><img src="https://3226621149-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FuOGB3JuT2RALmbnviT9H%2Fuploads%2FtWXFj52BUlPedshi3PsO%2Fabstract-exponential-culture-combined.png?alt=media&amp;token=303ad63e-f976-4721-be2a-c5b4252a3057" alt=""><figcaption><p>Workflow illustration. Inoculate a subculture from an overnight culture at 1% (v/v). Then determine starting concentration (<mark style="background-color:$info;">grey line</mark>). After 2 hours of incubation, measurements follow every hour. The culture is in exponential phase when measured concentration exceeds 10× the starting concentration (<mark style="background-color:green;">green line</mark>). To remove growth medium substances, centrifuge the culture, resuspend, and adjust it to the target concentration with BactoBox®.</p></figcaption></figure></div>

{% 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 %}

## Step by step exponential culture

{% stepper %}
{% step %}

### Start an overnight culture in the afternoon

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[^1] 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;

An overnight culture is usually between 1×10<sup>9</sup>–1×10<sup>10</sup> cells/mL. The resulting subculture will therefore be \~1×10<sup>7</sup>–1×10<sup>8</sup> cells/mL.
{% endstep %}

{% step %}

### Measure starting concentration

Use a sterile serological pipette to retrieve a sample. Vortex 1 min at max speed to disaggregrate cell clumps and then prepare a 1:100 dilution in BactoBox® diluent. This should bring the cell concentration to \~1×10<sup>5</sup>–1×10<sup>6</sup> cells/mL.

Press <kbd>Measure</kbd> to start. Multiply cells/mL with the dilution factor to get the concentration, of the non-diluted culture.&#x20;

* Example: C<sub>0</sub> = 110 000 cells/mL × 100 = 11 000 000 cells/mL = 1.1×10<sup>7</sup> cells/mL.

<div data-with-frame="true"><figure><img src="https://3226621149-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FuOGB3JuT2RALmbnviT9H%2Fuploads%2FHNfqnHWph0MQbtfrEO7y%2Fillustration-1%3B100-new.png?alt=media&amp;token=03ca4ace-47c1-42f0-91a2-922bad269cd3" alt="" width="519"><figcaption><p>1:100 dilution is done by a single transfer of 101 µL culture of 10 mL of diluent.</p></figcaption></figure></div>
{% endstep %}

{% step %}

### Incubate and measure after two hours

The culture is likely in lag phase within the first hour of incubation and therefore cell concentration is expected to be constant.&#x20;

Instead, draw a sample after two hours of incubation, vortex, and use the 1:100 dilution scheme for the measurement.
{% endstep %}

{% step %}

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

Extend incubation and perform hourly measurements until you get at least **10× increase in cell concentration** compared to C<sub>0</sub>. You may need to switch to a [1:1,000](/measurement-essentials/hit-the-right-concentration.md#id-1-1-000-dilutions) or [1:10,000](/measurement-essentials/hit-the-right-concentration.md#id-1-10-000-dilutions) dilution.

In this example, the cell concentration should be ten times C<sub>0</sub>, i.e at least 1.1×10<sup>8</sup> cells/mL.

***

Your culture is now in exponential phase. The next steps differ from lab to lab, but they give a rough idea of the workflow. The goal is a washed cell suspension, adjusted to the target concentration.
{% endstep %}

{% step %}

### Harvest by centrifugation

The culture is "washed" to remove growth medium components.&#x20;

Centrifuge to collect the cells in a neat pellet, e.g. 5000×*g* for 5 min.&#x20;
{% endstep %}

{% step %}

### Resuspend cells

Resuspend cells in the buffer used for your experiments, e.g. 1×PBS. For the initial suspension, aspirate with a pipette. Then vortex thoroughly to get a single-cell suspension, e.g. 1 min at max speed.&#x20;
{% endstep %}

{% step %}

### Determine cell concentration

Use BactoBox® to determine cell concentration of the washed cell suspension.&#x20;
{% endstep %}

{% step %}

### Adjust cell concentration

Dilute with 1×PBS or similar to hit the target cell concentration for your experiments.
{% endstep %}
{% endstepper %}

## Related workflows

If you would like to compare CFU and BactoBox® results, see [Crosscheck CFU and cells](/inoc/workflows/crosscheck-cfu-and-cells.md).

[^1]: Decline phase is often referred to as death phase.


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