Avoid cell lysis

Sample over time to investigate effectiveness and absence of cell lysis

When your disaggregate chains or clumps of cells in your sample, you may accidentally disintegrate the cells themselves! This unintentional cell 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® BacLight™ 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 to quantify the extent of cell membrane lysis. An uptake of propidium iodide and red-fluorescent cells indicate membrane rupture.

Use live/dead fluorescence microscopy

Use a fluorescence microscope and the LIVE/DEAD™ BacLight™ 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.

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

Cuvettes for OD600 analysis

Use OD600 in combination with a 5 µm cell sieve 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. If particles are stuck in the 5 µm cell sieve, they were too big for BactoBox® to count anyhow.