Bacteria Live/Dead Staining Kit

Protocol tips

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

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.

Publication protocol

Bacterial cultures were cultivated as described above. One millimetre of cell culture at OD600 1.0 was harvested at 6000 × g for 2 m and re‐suspended in 0.9% NaCl containing 10 μg ml−1 nisin. Cells re‐suspended in 0.3 ml 0.9% NaCl without nisin served as controls. The cell suspension was stored at room temperature for 1 h with brief mixing at appropriate intervals (10 min). These cells were then washed and re‐suspended in 0.9% NaCl. Live–dead staining was performed using ‘Promokine Kit’ as per manufacturer's instructions. A 100× mixture of dyes containing one volume of DMAO and two volumes of EthD‐III was prepared. One hundred microlitres of cells were stained with 1 μl of the 100× dye for 15 min in the dark. For microscopy, 3 μl of stained cells were transferred on a 1% agarose pad for microscopic visualization.

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