Optical Method and Device for the Detection and Enumeration of Microorganisms

Abstract

A new device and method for detecting the presence of living microorganisms in test samples are described. The device comprises a container with at least one section transparent to light, a growth zone located in said container containing a mixture of growth media capable of supporting growth of the microorganisms, and at least one indicator substrate that changes its optical properties due to growth of the microorganisms. A detection zone is located in the container adjacent to the transparent section, and a barrier layer comprising porous solid material separates the two zones, allowing diffusion of molecules and ions of metabolic by-products of the organisms, while preventing microorganisms and particulate matter of the test sample from penetrating into the detection zone.

1 . A device for detecting the presence of living microorganisms in a test sample, comprising
 optical instrumentation, the optical instrumentation having a light source and at least one photodetector:   a container with at least one section transparent to light;   a growth zone located in the container, containing a mixture of growth media capable of supporting growth of the microorganisms, and at least one indicator substrate that changes optical properties due to growth of the microorganisms;   a detection zone located in the container adjacent to the transparent section and containing an identical mixture of the growth media and the indicator substrate; and   a barrier layer comprising porous material interposed between said growth and detection zones, and allowing diffusion of molecules of the growth media, the indicator substrate, and molecules and ions of metabolic by-products of the organisms, back and forth between the growth and detection zones, wherein the barrier layer prevents microorganisms and particulate matter of the test sample from penetrating into the detection zone; wherein barrier layer allows flow of liquids when a hydrostatic pressure gradient is present between its two surfaces;   wherein the light source is positioned exterior to the container and produces light passes through the detection zone prior to being detected by the photodetector.   
     
     
         2 . The device of  claim 1  wherein said barrier layer includes a membrane. 
     
     
         3 . The device of  claim 2  wherein said membrane is hydrophilic. 
     
     
         4 . The device of  claim 3  wherein said membrane allows the flow of liquids when a hydrostatic pressure gradient is present between its two surfaces 
     
     
         5 . The device of  claim 2  wherein the maximal pore size of said membrane is less than 1 micrometer. 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The device of  claim 1  wherein said porous material is a polymer. 
     
     
         9 . The device of  claim 1  wherein said indicator substrate is at least one of the following: is a visible dye capable of changing its color due to metabolic processes of the microorganisms; fluorescence dye capable of changing its fluorescent properties due to metabolic processes of the microorganisms 
     
     
         10 . (canceled) 
     
     
         11 . A method for detecting the presence of living microorganisms in a test sample, comprising the steps of:
 introducing the test sample into the growth zone of the device of  claim 1 ;   incubating the microorganisms in the growth media and allowing metabolic processes to generate by-products into the mixture of the media and the indicator substrate present in the growth zone, thereby changing the optical properties of the indicator substrate;   allowing the by-products to diffuse into the detection zone through the barrier layer that blocks the microorganisms and the particulate matter of the test sample from reaching the detection zone; and   observing the change in the optical properties of the indicator substrate in the detection zone through the transparent section.   
     
     
         12 . The method of  claim 11  further including the steps of:
 aiming in electromagnetic energy source at the transparent section; and 
 detecting the reactive electromagnetic energy resulting from the interaction of the electromagnetic energy with the indicator substrate present in the detection zone. 
 
     
     
         13 . The method of  claim 12  wherein said electromagnetic energy source is a visible light source and the indicator substrate is a visible dye. 
     
     
         14 . The method of  claim 13  wherein said electromagnetic energy source is a light-emitting diode. 
     
     
         15 . The method of  claim 12  wherein said electromagnetic energy source is an ultraviolet light source and the indicator substrate is a fluorescence dye generating visible reactive energy. 
     
     
         16 . The method of  claim 15  wherein said electromagnetic energy source is one of the following: an ultraviolet light-emitting diode; a gas discharge tube. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 12  wherein a photo detector is utilized for the detection of the reactive electromagnetic energy and wherein the photo detector is at least one of the following: at least one photo diode, at least one photon multiplying tube (PMT), at least one photo transistor. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 12  wherein said electromagnetic energy source comprises a multiple color light-emitting diode. 
     
     
         23 . The method of  claim 22  wherein said electromagnetic energy source further comprises at least one of the following: an ultraviolet light-emitting diode; an ultraviolet gas discharge tube. 
     
     
         24 . (canceled) 
     
     
         25 . A method of enumerating living microorganisms in a test sample, comprising the steps of:
 applying the steps described in  claim 12  and recording the detected reactive electromagnetic energy at predetermined time intervals as time sequence data;   analyzing the sequence data to determine the Detection Time, in which the difference between a predetermined number of consecutive data points in the sequence data changes to follow the growth pattern of the living microorganisms; and   evaluating the number of the living microorganisms in the samples by applying the equation:   
       
         
           
             
               CFU 
               = 
               
                 
                   log 
                   
                     - 
                     1 
                   
                 
                  
                 
                   ( 
                   
                     
                       log 
                        
                       
                         
                           
                             C 
                             si 
                           
                            
                           ln 
                            
                           
                               
                           
                            
                           2 
                         
                         
                           
                             K 
                             B 
                           
                            
                           
                             t 
                             g 
                           
                         
                       
                     
                     - 
                     
                       
                         
                           log 
                            
                           
                               
                           
                            
                           2 
                         
                         
                           t 
                           g 
                         
                       
                        
                       
                         ( 
                         
                           
                             t 
                             D 
                           
                           - 
                           
                             t 
                             L 
                           
                         
                         ) 
                       
                     
                   
                   ) 
                 
               
             
           
         
       
       wherein:
 CFU is the colony forming units; 
 log denotes the 10 base logarithmic function; 
 In denotes the natural logarithmic function; 
 C si  is the initial concentration of the dye substrate modifying reagents; 
 K B  is the bacterial activity; 
 t g  is the bacterial generation time; 
 t D  is said Detection Time; and 
 t L  is the time duration of the lag phase. 
 
     
     
         26 . The method of  claim 25  wherein the coefficients of said equation are determined empirically by performing a statistical best-fit linear regression analysis to derive from experimental data values A and B of the equation:
   Log(CFU)= A−B·t   D    
 
       wherein said experimental data consists of a multiplicity of tests for different test samples, each consisting of finding the CFU value using traditional plate counts methodology and the corresponding Detection Time t D . 
     
     
         27 . A method for testing the susceptibility of microorganisms to anti-microbial agents, comprising the steps of:
 dividing a sample of the microorganisms to a test sample and a reference sample with identical volumes and concentrations;   mixing the test sample with a target antimicrobial agent at a specific concentration to form a susceptibility test sample;   applying the steps described in  claim 12  to the susceptibility test sample and recording the detected reactive electromagnetic energy at predetermined time intervals as a sample time sequence data;   analyzing said sample sequence data to determine the sample Detection Time in which the slope calculated amongst a predetermined number of consecutive data points in said sample sequence data changes to follow the intrinsic slope of the logarithmic growth pattern of the microorganisms;   applying the steps described in  claim 12  to the reference sample and recording the detected reactive electromagnetic energy at predetermined time intervals as a reference time sequence data;   analyzing said sample sequence data to determine the reference Detection Time in which the slope calculated amongst a predetermined number of consecutive data points in said reference time sequence data changes to follow the intrinsic slope of the logarithmic growth pattern of the microorganisms; and   comparing the sample Detection Time to the reference Detection Time and providing criteria to determine the susceptibility of the microorganisms based upon said comparison.   
     
     
         28 . A method for testing the effectiveness of a preservative agent to reduce the growth of microorganisms in a sample, comprising the steps of:
 dividing a sample to a test sample and a reference sample with identical volumes and concentrations;   mixing the test sample with the preservative agent at a specific concentration ratio to form a challenge test sample;   applying the steps described in  claim 12  to the challenge test sample and recording the detected reactive electromagnetic energy at predetermined time intervals as a sample time sequence data;   analyzing said sample sequence data to determine the sample Detection Time in which the slope calculated amongst a predetermined number of consecutive data points in said sample time sequence data changes to follow the intrinsic slope of the logarithmic growth pattern of the microorganisms;   applying the steps described in  claim 12  to the reference sample and recording the detected reactive electromagnetic energy at predetermined time intervals as a reference time sequence data;   analyzing said sample sequence data to determine the reference Detection Time in which the slope calculated amongst a predetermined number of consecutive data points in said reference time sequence data changes to follow the intrinsic slope of the logarithmic growth pattern of the microorganisms; and   comparing the sample Detection Time to the reference Detection Time, and providing criteria to determine the effectiveness of the preservative based upon said comparison.   
     
     
         29 . An apparatus for detecting the presence of living microorganisms in a test sample, the apparatus comprising:
 optical instrumentation including a light source;   a container with at least one section transparent to light;   a growth zone located in the container, containing a mixture of growth media capable of supporting growth of the microorganisms, and at least one indicator substrate that changes optical properties due to growth of the microorganisms;   a detection zone located in the container adjacent to the transparent section and containing an identical mixture of the growth media and the indicator substrate; and   a barrier layer interposed between said growth and detection zones, the barrier layer comprising a polymeric material and allowing diffusion of molecules of the growth media, the indicator substrate, and molecules and ions of metabolic by-products of the organisms to pass from the growth zone to the detection zone, wherein the barrier layer prevents microorganisms and particulate matter of the test sample from penetrating into the detection zone; and   wherein said light source produces light that passes through said detection zone prior to being detected by said photodetector and wherein changes in the color of the dye indicator in the detection zone indicate presence of microorganisms growing in the growth zone.   
     
     
         30 . The apparatus of  claim 29  wherein said barrier layer comprises a membrane. 
     
     
         31 . The apparatus of  claim 30  wherein said membrane is hydrophilic. 
     
     
         32 . The apparatus of  claim 29  wherein said barrier layer allows the flow of liquids when a hydrostatic pressure gradient is present between its two surfaces. 
     
     
         33 . The apparatus of  claim 29  wherein said indicator substrate is at least one of the following: is a visible dye capable of changing its color due to metabolic processes of the microorganisms; fluorescence dye capable of changing its fluorescent properties due to metabolic processes of the microorganisms