LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit, for flow cytometry

SYTO 9 dye will enter all cells, live or dead. PI only enters cells with compromised membranes (dead cells or damaged cells). First, perform single color staining and examine under both filter sets. Longpass filters or the use of too much dye may result in excessive bleedthrough of the green dye emission into the red channel and the emission of PI in the green channel. Use narrower bandpass filters if possible, and use lower concentrations of the dye. Some live cells may take up PI by engulfment; avoid extended incubation times with the dye. Apply PI to a live cell culture and optimize incubation times to limit engulfment.

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Oxygen content should not affect the binding of propidium iodide and SYTO 9 to nucleic acids. SYTO 9 will label all cells, and propidium iodide will label only dead cells or cells with a compromised membrane.

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Unfortunately, no. SYTO 9 will label the nuclei of live or dead cells, including the eukaryotic cells. Propidium iodide is cell impermeant, and will only enter dead cells. If the eukaryotic cells are dead, they will label with propidium iodide as well. If the eukaryotic cells are alive, propidium iodide will not be able to enter and thus will not label the bacteria inside, whether the bacteria are alive or dead. We are not aware of any way to do a viability assay of bacteria once they have been engulfed by cells.

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The green dye in the kit will label all the cells as it is a cell-permeant nucleic acid stain. The red dye is not cell permeant, and will only stain the cells with compromised membranes (dead cells). Therefore, any cells with red signal will be considered dead. It is possible that you will have some cells that are only red, some that are red and green, and some that are only green. Sometimes the red dye will displace the green dye. In any case, any red cells are dead.

Also, the green dye emission may bleed through into the red channel. Do a single-color staining and examine under both green and red filter sets to determine the level of bleedthrough. To avoid this bleedthrough, use a lower concentration of dye, and, if possible, use narrow bandpass filters.

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There are several options. We have two fluorescence based kits that are useful for bacterial counting: Live/Dead BacLight Bacterial Viability and Counting Kit, for flow cytometry (Cat. No. L34856) and Bacteria Counting kit, for flow cytometry (Cat. No. B7277). Another option is the Flow Cytometry Sub-micron Particle Size Reference Kit (Cat. No. F13839).

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