Live / Dead assay bacteria - Bacillus subtilis

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

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1 year ago

1 year ago by M. Daecher Germany

Live/dead assay Bacteria

Hello everyone! I am going to do a live/dead assay for my cells and I saw that I can use both fluorescence and absorbance as my detection method. Is there a difference in the results depending on the method? Is one method preferred over the other in certain situations?

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Upstream tips
re‐suspended cells in 0.9% NaCl containing 10 μg ml−1 nisin
Protocol tips
Incubate both samples at room temperature for 1 hour, mixing every 15 minutes.
Downstream tips
. Determine the optical density at 670 nm (OD670) for each bacterial suspension using a spectrophotometer.
Protocol tips
Add 3 µL of the dye mixture for each mL of the bacterial
suspension.

Mix thoroughly and incubate at room temperature in the dark for 15 minutes
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