Spectral intensity ratio (sir) analysis for rapid live microbial enumeration

Abstract

Single dye fluorescent staining (with a membrane—associated dye such as FM 1-43 or FM 4 -64) and the combination of differences in both intensity and spectral emission discriminate live from inactivated/dead bacteria and provides for rapid and accurate detection of live bacteria in mixed populations.

What is claimed is: 
     
         1 . A method for differentiating between live and inactivated bacteria in a treated sample comprising:
 staining the sample with a single membrane-associated dye, wherein the single membrane-associated dye stains both live and inactivated bacteria;   illuminating the sample with an incident light at an excitation wavelength, wherein, when stained with the single membrane-associated dye, an illuminated stained live bacteria exhibits a first maximum emission peak, and wherein an illuminated stained inactivated bacteria exhibits a second maximum emission peak that is shifted compared to the first maximum emission peak;   measuring, for each bacterial cell (i) the intensity I1 of emitted light from the single membrane-associated dye at wavelength 1; and (ii) the intensity 12 of emitted light from the single membrane-associated dye at wavelength 2, wherein I1 and I2 are from the same single membrane-associated dye;   calculating a ratio I2/I1; and   determining whether the bacterial cell is live or inactivated based on the calculated I2/I1 ratio.   
     
     
         2 . The method of  claim 1 , wherein the membrane-associated dye is N-(3-Triethylammoniumpropyl) -4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide. 
     
     
         3 . The method of  claim 1 , wherein the membrane-associated dye is N-(3-Triethylammoniumpropyl) -4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide. 
     
     
         4 . The method of  claim 1 , wherein the excitation wavelength is between 488 and 570 nm. 
     
     
         5 . The method of  claim 4 , wherein the excitation wavelength is 488 nm. 
     
     
         6 . The method of  claim 1 , wherein the emission wavelength range is between 520 and 850 nm. 
     
     
         7 . The method of  claim 1 , wherein wavelength 1 is 530 nm and wavelength 2 is 610 nm. 
     
     
         8 . The method of  claim 1 , wherein wavelength 1 is 670 and wavelength 2 is 780 nm. 
     
     
         9 . The method of  claim 1 , wherein live bacteria have a I2/I1<2.0, and inactivated bacteria have a I2/I1>2.0. 
     
     
         10 . The method of  claim 1 , wherein the step of determining whether the bacterial cell is live or inactivated based on the calculated I2/I1 ratio comprises calculating a second I2/I1 ratio of a control containing only live bacteria; and
 determining whether the bacterial cell is live or inactivated by comparing the I2/I1 ratio of the sample with the second I2/I1 ratio of the control containing only live bacteria.   
     
     
         11 . The method of  claim 10 , wherein the I2/I1 ratio of the sample is divided by the second I2/I1 ratio of the control containing only live bacteria to generate a non-viability parameter, wherein live bacteria have a non-viability parameter≤1.0, and inactivated bacteria have a non-viability parameter>1.0. 
     
     
         12 . A method for differentiating between live and inactivated bacteria in a whole sample comprising the steps of:
 staining the whole sample with a single membrane-associated dye;   illuminating the whole sample with an incident light at an excitation wavelength, measuring, for the stained whole sample, (i) the intensity I1 of emitted light at wavelength 1; and (ii) the intensity I2 of emitted light at wavelength 2, wherein the I1 and I2 are from the same single membrane-associated dye;   calculating a ratio I2/I1 of the whole sample and a non-viability parameter (NVP) by dividing the I2/I1 of the whole sample by I2/I1 of a control containing only live bacteria; and   comparing said NVP to a threshold value to determine if the bacteria are live or inactivated.   
     
     
         13 . The method of  claim 12 , wherein the single membrane-associated dye is a styryl dye or a cyanine dye. 
     
     
         14 . The method of  claim 12 , wherein the excitation wavelength is a wavelength selected between the range of 360 and 570 nm. 
     
     
         15 . The method of  claim 12 , wherein the excitation wavelength is a wavelength selected between the range of 488 and 570 nm. 
     
     
         16 . The method of  claim 12 , wherein the range of emitted light at wavelength 1 lies between 520 and 850 nm. 
     
     
         17 . The method of  claim 12 , wherein the range of emitted light at wavelength 1 lies between 530 and 750 nm. 
     
     
         18 . The method of  claim 12 , wherein the range of emitted light at wavelength 1 lies between 520 and 700 nm. 
     
     
         19 . The method of  claim 12 , wherein wavelength 1 is 530 nm and wavelength 2 is 610 nm. 
     
     
         20 . The method of  claim 12 , wherein wavelength 1 is 670 and wavelength 2 is 780 nm. 
     
     
         21 . The method of  claim 12 , further comprising treating live bacteria cells contained in the whole sample in an effort to inactivate the bacteria cells prior to the staining step. 
     
     
         22 . The method of  claim 12 , wherein the sample is a treated sample. 
     
     
         23 . The method of  claim 21 , further comprising the step of filtering the stained cells prior to the illuminating step and subsequent to the staining step. 
     
     
         24 . The method of  claim 21 , wherein the treating step comprises interacting the live bacteria with an antimicrobial drug. 
     
     
         25 . The method of  claim 21 , wherein the treating step comprises heating, adding an antibiotic drug, adding an antibacterial drug, adding ethanol, or ultraviolet (UV) irradiation. 
     
     
         26 . The method of  claim 12 , wherein the control containing only live bacteria has the same bacterial concentration as the sample. 
     
     
         27 . The method of  claim 12 , wherein the threshold value is 1.0, live bacteria have a non-viability parameter≤1.0, and inactivated bacteria have a non-viability parameter>1.0. 
     
     
         28 . A method for differentiating between live and inactivated bacteria in a whole sample and stained with a single membrane-associated dye, comprising the steps of:
 illuminating the stained whole sample with an incident light at an excitation wavelength;   measuring, for the stained whole sample, (i) the intensity I1 of emitted light at wavelength 1, and (ii) the intensity I2 of emitted light at wavelength 2, wherein the I1 and I2 are from the same single membrane-associated dye;   calculating a ratio I2/I1 of the whole sample and a non-viability parameter (NVP) by dividing the I2/I1 of the whole sample by I2/I1 of a control containing only live bacteria; and   comparing said NVP to a threshold value to determine if the bacteria are live or inactivated.   
     
     
         29 . The method of  claim 28 , further comprising treating live bacteria cells contained in the sample in an effort to inactivate the bacteria cells prior to the staining step. 
     
     
         30 . The method of  claim 28 , wherein the whole sample is a treated sample. 
     
     
         31 . The method of  claim 28 , further comprising the step of filtering the stained cells prior to the illuminating step and subsequent to the staining step. 
     
     
         32 . The method of  claim 29 , wherein the treating step comprises interacting the live bacteria with an antimicrobial drug. 
     
     
         33 . The method of  claim 29 , wherein the treating step comprises heating, adding an antibiotic drug, adding an antibacterial drug, adding ethanol, or ultraviolet (UV) irradiation.