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 with a detector, 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 . 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 with a detector configured to preferentially detect a detectable emission from the fluorescent dye rather than a detectable emission from the quencher so that the detectable emission from the quencher, if created, is no greater than 50% of the detectable emission from the fluorescent dye, wherein the non-viable cells emit substantially less fluorescence detected by the detector than the viable cells, thereby to detect the viable cells in the cell sample.   
     
     
         3 . The method of  claim 2 , wherein the detectable emission from the quencher, if created, is no greater than 30% of the detectable emission from the fluorescent dye. 
     
     
         4 . The method of  claim 1 , wherein the quencher, when exposed to the light in step (b), does not emit a fluorescent event that is detectable by the detector. 
     
     
         5 .- 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the fluorescent dye binds to a nucleic acid within the cell. 
     
     
         8 . The method of  claim 1 , wherein the fluorescence quencher binds to a nucleic acid within the cell. 
     
     
         9 . The method of  claim 1 , wherein the fluorescent dye and the fluorescence quencher bind one another within the cell. 
     
     
         10 .- 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the non-viable cells emit substantially no fluorescence detectable by the detector upon exposure to the light. 
     
     
         13 . The method of  claim 1 , wherein the non-viable cells emit substantially no fluorescence upon exposure to the light. 
     
     
         14 .- 17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein the cells are captured on a porous membrane. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the cells are disposed upon a solid support. 
     
     
         21 . The method of  claim 20 , wherein the solid support is a microscope slide. 
     
     
         22 . The method of  claim 1 , wherein the cells are disposed within liquid. 
     
     
         23 . The method of  claim 22 , wherein the cells are disposed within an optical cell. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1  further comprising the step of combining the cells in the sample with a plurality of fluorescent particles that emit a fluorescent signal upon activation by light having a wavelength in the range of from about 350 nm to about 1000 nm. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1  further comprising the step of determining the number of viable cells in at least a portion of the cell sample. 
     
     
         28 . The method of  claim 1 , wherein the cell sample is a liquid sample. 
     
     
         29 . 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. 
     
     
         30 . The method of  claim 1 , wherein the viable cells are microorganisms. 
     
     
         31 . (canceled) 
     
     
         32 . 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). 
     
     
         33 . The method of  claim 1 , wherein the cells are cultured under conditions to permit cell proliferation after step (a) but prior to step (b).