> 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/advanced/measurements-explained/gating/inactivated-bacteria/inactivation-by-heat.md).

# Inactivation by heat

Heat treatment has a profound effect on bacterial membranes. As discussed in [Inactivated bacteria](https://help.sbtinstruments.com/advanced/measurements-explained/inactivated-bacteria), heat exceeding \~75 °C causes the bilayer to collapse to a disordered state. Embedded proteins denature and leave gaps. Small molecules from the cytoplasm leak out.

{% hint style="info" %}

## Does [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md) count heat-inactivated bacteria as cells?

It depends. Some bacteria are still enumerated as cells by [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md), while heat-treatment causes other bacteria to change electrical properties and fall outside the [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md) cell-definition.

The typical scenario is that heat-treated Gram-negative bacteria are still counted as cells by [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md) while Gram-positive cells often fall outside the cell-definition. Some Gram-positive bacteria like *Lactobacillus* spp. still fall within the [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md) cell-definition after heat treatment.

You can include heat-inactivated populations by custom gating. This is an advanced feature. We only support advanced features through a [custom solution](/advanced/what-is-a-custom-solution.md). [Contact support](/troubleshooting/contact-support.md) for details.
{% endhint %}

## Heat-inactivated bacteria counted by [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md)

The default BactoBox® configuration uses [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md). Objects classified as cells are found within the region with lavender shading. The distributions correspond to cultures before (green) and after (red) boiling for 15 min.

Bacteria like *Escherichia coli*, *Klebsiella aerogenes*, *Lactobacillus bulgaricus*, *Salmonella enteritidis*, and *Pseudomonas fluorescens* will largely still be counted with [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md).

<figure><img src="https://2483617760-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FCaBGkox4Fd7NCFFCF47X%2Fuploads%2Fo1GK4PBiq11aFC0v9lS7%2Fheat_coli-klebsiella-bulgaricus-salmonella-fluorescens.png?alt=media&#x26;token=4aa05a68-e052-4f0f-9421-90f4a2502609" alt=""><figcaption><p>Examples of heat-inactivated bacteria that are still counted as cells with <a data-mention href="/advanced/measurements-explained/gating/bactotal-10.md">BacTotal-10</a>. Bacterial cultures were boiled for 15 min.</p></figcaption></figure>

## Heat-inactivated bacteria excluded by [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md)

Sometimes the heat-inactivation causes the electrical signatures to fall outside the [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md) cell-definition.

In the distributions below, *Avibacterium paragallinarium*, *Edwardsiella tarda*, *Mycoplasma*, and *Yersinia ruckeri* all become less conductive than their untreated counterparts. Heat-treated *Lactobacillus brevis* has populations both within the non-conductive and the highly conductive region.

<figure><img src="https://2483617760-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FCaBGkox4Fd7NCFFCF47X%2Fuploads%2Fwsdnx8yVS9SHdIjYtV35%2Fheat-avibacterium-tarda-mycoplasma-yersinia-brevis.png?alt=media&#x26;token=9602f04c-bf94-4a3c-bfbb-31c5d215fcc8" alt=""><figcaption><p>Examples of heat-inactivated bacteria that are not counted as cells with <a data-mention href="/advanced/measurements-explained/gating/bactotal-10.md">BacTotal-10</a>. Bacterial cultures were boiled for 15 min, except for <em>Mycoplasma</em> (65 °C, 30 min).</p></figcaption></figure>

The species below also fall outside the [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md) cell-definition after heat-inactivation: *Bacillus subtilis*, *Lactobacillus buchnerii*, *Lactobacillus plantarum*, *Pediococcus acidilactici*, and *Roseburia intestinalis*.

When subjected to boiling for 15 min these Gram-positive bacteria all shift to the highly conductive region. The increase in conductivity is likely because the thick sack of conductive cell wall is fully exposed with no membranes left to impede electricity. This is explained in more detail in [Heat](https://help.sbtinstruments.com/advanced/measurements-explained/inactivated-bacteria#heat).

<figure><img src="https://2483617760-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FCaBGkox4Fd7NCFFCF47X%2Fuploads%2FwNMCfdsTY7ocsWJLZsah%2Fheat_bacillus-buchnerii-plantarum-pediococcus-roseburia.png?alt=media&#x26;token=3aeb3186-bf45-48f5-8af4-638d18c88ed3" alt=""><figcaption><p>Examples of Gram-positive heat-inactivated bacteria that are not counted as cells by <a data-mention href="/advanced/measurements-explained/gating/bactotal-10.md">BacTotal-10</a>. Bacterial cultures were boiled for 15 min.</p></figcaption></figure>

## Summary

When cells are heat-inactivated they may move outside the [BacTotal-10](/advanced/measurements-explained/gating/bactotal-10.md) cell-definition. This is especially the case for most Gram-positive bacteria. With custom gating the cell-definition can be tailored so BactoBox® enumerates heat-inactivated cells.

{% hint style="info" %}

## Advanced feature 🧩

Custom gating is an advanced feature. We only support advanced features through a [custom solution](/advanced/what-is-a-custom-solution.md). [Contact support](/troubleshooting/contact-support.md) for details.
{% endhint %}


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