# 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® does *not* count cells with a broken membrane consistently. Therefore, lysis is an important thing to avoid.

Bacterium with broken membrane. E.g., due to lysis.
BactoBox® does *not* count these consistently.

Bacterium with intact membrane.
BactoBox® counts these consistently.

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. ## Live/dead fluorescence microscopy 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 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 [live/dead staining](https://www.thermofisher.com/order/catalog/product/L7012?SID=srch-srp-L7012) to quantify the extent of cell membrane lysis. An uptake of propidium iodide and red-fluorescent cells indicate membrane rupture. {% hint style="success" %} ## Use live/dead fluorescence microscopy Use a [fluorescence microscope](https://en.wikipedia.org/wiki/Fluorescence_microscope) and the [LIVE/DEAD™ *Bac*Light™](https://www.thermofisher.com/order/catalog/product/L7012?SID=srch-srp-L7012) staining kit to ensure that: * Bacteria are in single-cell form (no aggregates) * Bacteria are separate from other particles * Bacterial cell membrane is intact See [#live-dead-fluorescence-microscopy](#live-dead-fluorescence-microscopy "mention"). {% endhint %} ## 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. ## Turbidity with OD600

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](https://help.sbtinstruments.com/encyclopedia/measurements-explained/what-can-i-measure-on#particle-size). If particles are stuck in the 5 µm cell sieve, they were too big for BactoBox® to count anyhow.