Optical method for detecting viable cells in a cell sample

Abstract

The invention relates to a method for determining the presence and/or amount of viable cells in a liquid sample. The method includes (a) labeling with a fluorescent label viable cells remaining on a porous membrane after passing the liquid sample therethrough, (b) scanning a portion of the porous membrane by rotating the porous membrane relative to a detection system, and (c) determining the presence and/or quantity of viable cells captured by the membrane.

What is claimed is: 
     
         1 . A method of detecting the presence and/or quantity of viable cells in a liquid sample, the method comprising the steps of:
 (a) labeling viable cells, if any, retained by at least a portion of a substantially planar porous membrane after passing the liquid sample therethrough with a fluorescent label;   (b) scanning the portion of the porous membrane by rotating the porous membrane relative to a detection system comprising,
 (i) a light source emitting a beam of light of a wavelength adapted to excite the fluorescent label to produce an emission event, and 
 (ii) at least one detector capable of detecting the emission event, 
 thereby to interrogate a plurality of regions of the planar porous membrane and to detect emission events produced by excitation of fluorescent label associated with any viable cells; and 
   (c) determining the presence and/or quantity of viable cells captured by the membrane based upon the emission events detected in step (b).   
     
     
         2 . The method of  claim 1 , wherein, in step (a), the cells are labeled using at least one of a viability stain and a viability staining system. 
     
     
         3 . The method of  claim 1 , wherein the beam of light source emits light having a wavelength in a range of from about to 350 nm to about 1000 nm. 
     
     
         4 . The method of  claim 1 , wherein the detector detects emitted light in a range of from about to 350 nm to about 1000 nm. 
     
     
         5 . The method of  claim 1 , wherein the porous membrane comprises a disc. 
     
     
         6 . The method of  claim 1 , wherein the porous membrane is substantially non-autofluorescent when exposed to light having a wavelength in the range from about 350 nm to about 1000 nm. 
     
     
         7 . The method of  claim 1 , wherein the porous membrane has a flatness tolerance of up to about 100 μm. 
     
     
         8 . The method of  claim 1 , wherein the porous membrane defines a plurality of pores having an average diameter less than about 1 μm so as to permit fluid to traverse the porous membrane while retaining cells thereon. 
     
     
         9 . The method of  claim 1 , wherein the porous membrane has a thickness in a range selected from the group consisting of from 1 μm to 3,000 μm; from 10 μm to 2,000 μm; and from 100 μm to 1,000 μm. 
     
     
         10 . The method of  claim 1 , wherein the porous membrane is disposed upon a fluid permeable support member. 
     
     
         11 . The method of  claim 10 , wherein the support member has a thickness in a range selected from the group consisting of from 0.1 mm to 10 mm; from 0.5 mm to 5 mm; and from 1 mm to 3 mm. 
     
     
         12 . The method of  claim 1  further comprising capturing on the porous membrane a plurality of fluorescent particles that emit a fluorescent event upon activation by light from the light source. 
     
     
         13 . The method of  claim 1  further comprising determining the quantity of viable cells in at least a portion of the liquid sample. 
     
     
         14 . The method of  claim 1  further comprising determining locations of the viable cells on the permeable membrane. 
     
     
         15 . The method of  claim 1  further comprising, after step (c), culturing the porous membrane under conditions that permit growth and/or proliferation of the viable cells captured by the porous membrane. 
     
     
         16 . The method of  claim 1 , wherein the viable cells are microorganisms. 
     
     
         17 . The method of  claim 1  further comprising identifying a genus and/or species of the viable cells. 
     
     
         18 . The method of  claim 1 , wherein the scanning step (b) comprises tracing at least one of a nested circular pattern and a spiral pattern on the porous membrane with the beam of light. 
     
     
         19 . The method of  claim 1 , wherein the viable cells are cultured under conditions to permit cell proliferation prior to step (a), during step (a), or prior to and during step (a). 
     
     
         20 . The method of  claim 1 , wherein the viable cells are cultured under conditions to permit cell proliferation after step (a) but prior to step (b).