> 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/technical-documentation/gating/inactivated-bacteria/effects-of-aging.md).

# Effects of aging

In this section we investigate if BactoBox® enumerates bacteria in aged/senescent cultures.

{% hint style="info" %}

## Does [BacTotal-10](/technical-documentation/gating/bactotal-10.md) count aged bacteria as cells?

It depends. Most aged cultures of Gram-negative bacteria are still found within the [BacTotal-10](/technical-documentation/gating/bactotal-10.md) cell-definition. In contrast, some Gram-positive species may change electrical properties that differ from the [BacTotal-10](/technical-documentation/gating/bactotal-10.md) cell-definition.

You can use custom gating to include the aged cell populations. 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 %}

## Aged cultures and [BacTotal-10](/technical-documentation/gating/bactotal-10.md)

The default BactoBox® configuration uses [BacTotal-10](/technical-documentation/gating/bactotal-10.md). Objects classified as cells are found within the region with lavender shading. The distributions correspond to late-exponential phase (green) and aged (red) cultures.

Some aged cultures stay within the [BacTotal-10](/technical-documentation/gating/bactotal-10.md) cell-definition. This is the case for *Salmonella enteritidis* (2 days), *Escherichia coli* (5 days), *Lactobacillus rhamnosus* (8 days), *Lactobacillus casei* (6 days), and *Lactobacillus salivarius* (4 days). The distributions shift left upon aging, but they are still within the [BacTotal-10](/technical-documentation/gating/bactotal-10.md) cell-definition.

<figure><img src="https://1559892669-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FHvoombw1JBsKTJTfYyuJ%2Fuploads%2FoU1VrCAfzIet4wcH1wMU%2FAging_salmonella-rhamnosus-casei-salivarius-coli.png?alt=media" alt=""><figcaption><p>Examples of aged cultures that stay within the <a data-mention href="/technical-documentation/gating/bactotal-10.md">BacTotal-10</a> cell-definition.</p></figcaption></figure>

Cultures of some aged bacterial species move outside the [BacTotal-10](/technical-documentation/gating/bactotal-10.md) cell-definition. This is exemplified with *Bifidobacterium bifidum* (6 days), *Bifidobacterium longum* (3 days), *Pediococcus acidilactici* (6 days), *Lactobacillus plantarum* (5 days), and *Staphylococcus epidermidis* (2 days).

The aged cells of Gram-positive bacteria shift to the same conductive region as where we found the [alcohol-treated cells](/technical-documentation/gating/inactivated-bacteria/inactivation-by-alcohol.md). [BacTotal-10](/technical-documentation/gating/bactotal-10.md) will not count these objects as cells.

<figure><img src="https://1559892669-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FHvoombw1JBsKTJTfYyuJ%2Fuploads%2FsGiZov87CxM1v8MGS8H1%2FAging_bifidum-longum-pediococcus-plantarum-epidermidis.png?alt=media" alt=""><figcaption><p>Examples of aged cultures that move outside the <a data-mention href="/technical-documentation/gating/bactotal-10.md">BacTotal-10</a> cell-definition.</p></figcaption></figure>

### Why aged electrical signatures may differ between Gram-positive and Gram-negative bacteria

Autolysins are present in both Gram-negative and Gram-positive bacteria. These enzymes remodel the peptidoglycan cell wall during cell division and aging.

In Gram-positive cells the peptidoglycan cell wall is the only load-bearing structure. Nick it and the lipid membrane herniates through the breach and the membrane tears immediately. There is essentially no "damaged wall, intact membrane" intermediate state. The Gram-positive cells often become more conductive as they age.

Gram-negatives are different: the outer membrane plus a thin peptidoglycan layer means they can die non-lytically and persist for a long time. Some of the cytoplasm is lost during aging and the cell shrinks. Overall, the Gram-negative cells usually become less conductive during aging.

<figure><img src="https://1559892669-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FHvoombw1JBsKTJTfYyuJ%2Fuploads%2FX4thyrpfEkhCMKbMIvoF%2Faging-both.png?alt=media" alt=""><figcaption><p>Illustration of the fates of Gram-positive and Gram-negative bacteria during aging. Gram-positive bacteria often become highly conductive, while the Gram-negative bacteria often become less conductive.</p></figcaption></figure>

## Summary

Cells in aged cultures may move outside the [BacTotal-10](/technical-documentation/gating/bactotal-10.md) cell-definition. Custom gating may be required for BactoBox® to enumerate these objects.

{% 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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