Viability staining method

Abstract

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).