VIABILITY STAINING METHOD
The invention relates to a method of detecting viable cells in a cell sample, using a membrane permeable fluorescent label that permeates both viable and non-viable cells and a membrane impermeant quencher that selectively permeates non-viable cells.
1 . A method of detecting viable cells in a cell sample, the method comprising the steps of:
(a) exposing cells in the cell sample to (i) a membrane permeable fluorescent dye under conditions that permit the fluorescent dye to permeate both viable and non-viable cells, and (ii) a membrane impermeable fluorescence quencher capable of quenching fluorescence produced by the fluorescent dye under conditions to permit the quencher to selectively permeate non-viable cells but not viable cells;
(b) after step (a), exposing the cells to light having a wavelength capable of exciting the fluorescent dye to produce a fluorescent emission; and
(c) detecting the fluorescent emission, if any, from the cells, wherein the fluorescent dye within the non-viable cells emits substantially less fluorescence than the fluorescent dye within the viable cells, thereby to detect the viable cells in the cell sample.
2 . The method of claim 1 , wherein, in step (a), the cells are exposed to the fluorescent dye and then exposed to fluorescence quencher.
3 . The method of claim 1 , wherein, in step (a), the cells are exposed to the fluorescent dye and the fluorescence quencher at the same time.
4 . The method of claim 1 , wherein the fluorescent dye binds to a nucleic acid within the cell.
5 . The method of claim 1 , wherein the fluorescence quencher binds to a nucleic acid within the cell.
6 . The method of claim 4 , wherein the fluorescence quencher binds to a nucleic acid within the cell.
7 .- 14 . (canceled)
15 . The method of claim 1 , wherein the cells are captured on a porous membrane.
16 . (canceled)
17 . The method of claim 1 , wherein the cells are disposed upon a solid support.
18 . The method of claim 17 , wherein the solid support is a microscope slide.
19 . The method of claim 1 , wherein the cells are disposed within liquid.
20 . The method of claim 19 , wherein the cells are disposed within an optical cell.
21 .- 24 . (canceled)
25 . The method of claim 1 , wherein the cell sample is a liquid sample.
26 . The method of claim 1 further comprising, after step (c), culturing the cells under conditions that permit the growth and/or proliferation of the viable cells.
27 . The method of claim 1 , wherein the viable cells are microorganisms.
28 . (canceled)
29 . The method of claim 1 , wherein the cells are cultured under conditions to permit cell proliferation prior to step (a), during step (a), or prior to and during step (a).
30 . The method of claim 1 , wherein the cells are cultured under conditions to permit cell proliferation after step (a) but prior to step (b).