> 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/advanced-sample-preparation/break-up-clumps-and-chains/avoid-cell-lysis.md). # Avoid cell lysis When your disaggregate chains or clumps of cells in your sample, you may accidentally *disintegrate* the cells themselves! This unintentional cell [lysis](https://en.wikipedia.org/wiki/Lysis) may lead to surprising measurement results with BactoBox®. BactoBox® (using [BacTotal](/advanced/measurements-explained/gating/bactotal.md)) does *not* count lysed cells towards cells/mL. Therefore, lysis is an important thing to avoid.

Bacterium with broken membrane.
BacTotal does *not* count these towards cells/mL.

Bacterium with intact membrane.
BacTotal counts these towards cells/mL.

## BactoBox® analysis Use BactoBox® to evaluate the disaggregation method. Look for an increase in the *cell/mL* concentration, which indicates successful disaggregation. Be aware of the *cell/total* ratio (*cell/mL* divided by *total/mL*), which should *not* decrease. If the *cell/total* ratio decreases, then this indicates cell lysis. Visualize the amplitude distribution in BactoBox® Explorer. A shift from high amplitude to smaller amplitude indicates that objects are now smaller. In other words, that the disaggregation method works. ## Complementary cell analysis methods Use a complementary set of cell analysis methods to detect cell lysis. This way, you know which disaggregation methods to avoid. We list a couple of complementary analysis method below. ### Fluorescence microscopy {% hint style="info" icon="puzzle-piece" %} ### Complementary cell analysis method This covers a non-BactoBox® method. We only support complementary cell analysis methods through a [custom solution](/advanced/what-is-a-custom-solution.md). Contact SBT for details. {% endhint %} Although microscopy is somewhat laborious, it is a fantastic method to evaluate the effect of disaggregation and lysis.

*Giardia muris* cysts stained with the reagents in the LIVE/DEAD® *Bac*Light™ Bacterial Viability Kit (Cat. nos. L7007, L7012). When incubated with the SYTO® 9 and propidium iodide nucleic acid stains provided in this kit, live bacteria with intact cell membranes fluoresce green and dead bacteria with compromised membranes fluoresce red.
Source: https://www.thermofisher.com/order/catalog/product/L7012?SID=srch-srp-L7012

Under a [fluorescence microscope](https://en.wikipedia.org/wiki/Fluorescence_microscope), it is easy to see if your sample contains chains or clumps in the first place. Moreover, you can count the clumps or chains vs. free cells. This gives you a direct way with to quantify the effect of the disaggregation method. Use the [LIVE/DEAD™ *Bac*Light™ Bacterial Viability Kits](https://www.thermofisher.com/order/catalog/product/L7012?SID=srch-srp-L7012) to quantify the extent of cell lysis. An uptake of propidium iodide and red-fluorescent cells indicate membrane rupture. {% hint style="success" %} ### Live cells grow The best way to know if a culture is alive is to [track its growth](https://help.sbtinstruments.com/mpd/workflows/track-growth-curve). A growing cell is an alive cell. See [How do I differentiate dead and alive cells?](/troubleshooting/faq/how-do-i-differentiate-dead-and-alive-cells.md) {% endhint %} ### Turbidity with OD600 {% hint style="info" icon="puzzle-piece" %} ### Complementary cell analysis method This covers a non-BactoBox® method. We only support complementary cell analysis methods through a [custom solution](/advanced/what-is-a-custom-solution.md). Contact SBT for details. {% endhint %}

Use [OD600](https://en.wikipedia.org/wiki/OD600) in combination with a [5 µm cell sieve](https://www.neolab.de/en/aktuelle-angebote/neoculture-zellsiebe-fur-spritzen-5-m-steril-c-8245) to evaluate the disaggregation method: 1. Measure the turbidity of your sample *before* you use the cell sieve. 2. Press the sample through the 5 µm cell sieve. 3. Dilute to get into the linear range from, e.g., 0.3–0.8 AU. 4. Measure the turbidity of your sample again Compare the turbidity results from step 1 and step 4. Lower OD600 in step 4 (after the cell sieve) indicates that a high proportion of the sample is stuck in the 5 µm cell sieve. In other words, that the disaggregation is not optimal. Remember that BactoBox® counts particles in the [0.5–5 µm range](/troubleshooting/faq/what-can-i-measure-on.md). If particles are stuck in the 5 µm cell sieve, they were too big for BactoBox® to count anyhow. --- # 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 dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter, and the optional `goal` query parameter: ``` GET https://help.sbtinstruments.com/advanced/advanced-sample-preparation/break-up-clumps-and-chains/avoid-cell-lysis.md?ask=&goal= ``` `ask` is the immediate question: it should be specific, self-contained, and written in natural language. `goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal. 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.