Title of Invention

A METHOD FOR THE DETECTION FOR VIABLE MICROORGANISMS

Abstract

The proposed invention has incorporated two technologies that only combined can provide for a method for detection of viable microorganisms. These are: a. providing for a smooth surface on which the bacteria are placed so as not to need for continuously focusing on the slide as it scans the specimen. b. provide a system that allows for a very large view of vision. This has been accomplished by (a) using a sol-gel coated slide which has a smooth surface or any other smooth porous surface that allows for holding the enzyme detection chemicals ("markers") in place without washing the bacteria from the slide, and (b) using a 16 bit or higher CCD camera as outlined in illustration number 1. Another very important aspect that should be taken into account is that, by using ^ this concept the time for scanning the system can be shortened and two (or more) wavelengths for detecting various "markers" can be used simultaneously. This system can provide for preventing false positives.

Full Text

microorganisms, according to a preferred embodiment of the invention; Fig. 2 is a fluorescence picture of E. coli bacteria on a sol-gel surface at X400 magnification; and Fig. 3 is a chart-flow of a process according to a preferred embodiment of the invention. Detailed Description of the Invention The invention relates to a process of detection and enumeration of microorganisms such as bacteria in a relatively shorter period than any of the conventional methods and also to an apparatus for the performance of the said process. In one embodiment of the invention, the apparatus being used may be a stationery unit such as a laboratory unit or even can be a compact system such as a portable unit. The foregoing disclosure is a general way of describing the invention and does not construe to limit the scope of the invention. It may also be noted that the permutation and combination known to persons skilled in the art are included which may not be mentioned here. Accordingly, a process of detection and enumeration of viable micro-organisms such as bacteria in a shorter time period comprising the steps of (i) Providing a liquid composition comprising one or more marker(s) incorporated in a liquid sol-gel precursor or porous glass slide; or filter pad; (ii) Coating a transparent slide, porous surface e. g., a glass slide, with a thin uniform layer of said liquid sol-gel precursor composition; (iii) Filtering the said given sample such as a specimen liquid to be tested through a filter so as to capture the live bacteria on the filter; (iv) These captured bacteria that are on the filter are then exposed to a The size and shape of the light dots - light spots larger than 10 microns shall be considered not bacterial. Light glows and shadows are to be eliminated. The proposed invention can allow for preventing false positive results by incorporating several marker simultaneously. This allows for eliminating false positives that can be created by fluorescent artifacts or non target bacteria that could have broken down the marker. Such a case could be in the instance of conform detection when using derivatives of MUG. In this case not only coliform bacteria are detected by also aeromonas and psuedamonas can uptake the marker thereby causing a false positive. In order to overcome this the proposed invention has the capability of capturing several pictures of the specimen at various wavelengths thereby allowing for looking at various conditions of marker uptake. If for example, three markers e.g. Resorufin 1-D-galactopyranoside, SYBR Green and an oxidase fluorescent were mixed together by illuminating the specimen at different wavelengths a different information would arrive. The SYBR green would detect all microorganisms thereby eliminating non-organic fluorescent artifacts. Each light source would be given an exact x-y coordinate by the computer for reference to later readings. The resorufin would detect all viable bacteria that would be able to metabolize MUG derivatives (coliform, aeromonas and psuedomonas). Their exact position would be recorded so as to be analysed and compared to the previous picture taken by the CCD camera. The oxidase fluorescent would then determine which of the fluorescent bacteria are nonconform and again since the exact x-y coordinates are known these points would be subtracted from the light sources already known. Double Dye Approach: During this step bacteria are labeled in such a way to provide sufficient data for target bacteria discrimination. Dye chosen for bacteria labeling shall be accumulated and emit high intensity fluorescence light. Combining two differently fluorescing dyes targeted to different bacterium traits or organelles will provide us with target validation and confirmation. For the purpose of clarity, and as an aid in the understanding of the invention, as disclosed and claimed herein, the following abbreviations are defined below: CFU-colony forming unit. WE CLAIM 1. A process of detection and enumeration of viable micro-organisms such as bacteria in a shorter time period comprising the steps of: (i) Providing a liquid composition comprising one or more marker(s) incorporated in a liquid sol-gel precursor or porous glass slide; or filter pad. (ii) Coating a transparent and / or non-transparent slide, porous surface e. g., a glass slide, with a thin uniform layer of said liquid sol-gel precursor composition; (iii) Filtering the said given sample such as a specimen liquid to be tested through a filter so as to capture the viable micro-organisms such as live bacteria on the filter; (iv) These captured bacteria that are on the filter are then exposed to a biosensor that contains various fluorogenic substrates such as 3- carboxyumbelliferyl (3-D-galactopyranoside (CUG) or 4-chloromethyl-6, 8- difluoroumbelliferyl (3-D-galactopyranoside (CMDiFUG), resorufin 10-D- galactopyranoside or 6,8-difluoro-4-methylumbelliferyl 1-D- galactopyranoside (DiFMUG)for a period sufficient to their uptake into the metabolism of the live micro-organism cell and release of fluorogens due to the enzymetric break down of the fluorogenic substrates within the live cell; (v) Irradiating said sol-gel-coated slide or porous surface with an external energy source such as to generate detectable signals emitted from the marker(s) metabolized by the microorganisms; (vi) Detecting the micro-organisms by way of capturing the filtered emissions of light from fluorogens in the form of the specific light dots; (vii) Enumerating the specific light dots by way of comparing the different pictures and X-Y coordinates of the specific light dots only illuminating dots that have commonly shown up at least on two pictures. 2. An apparatus for detecting and enumerating the micro-organisms such as bacteria comprising a means of providing the excitation light to illuminate the biosensor, which is generally a slide on which the micro-organisms are placed on for detection and enumeration; a means of filtering system wherein the excited and emitted lights are filtered to prevent unwanted wavelengths; a lens system for focussing, adapting and magnification of light from the biosensor to the camera; a camera, preferably a 16bit CCD camera which shall be linked to a computer system through a direct ethernet cable to detect the weak light signals (luminescences) and coupling the CCD camera to an intensifier thereby creating a low light camera featuring a very high sensitivity along with a high resolution. 3. The process according to claim 1 wherein the microorganisms are bacteria. 4. The process according to claim 1 wherein the energy source is a visible light, fluorescent light, UV light or infrared light. 5. The process according to claim 1, wherein the microorganisms are isolated from water, milk, food, saliva, urine, throat swab tests, wounds, sputum, stomach content, or feces. 6. The process according to claim 1, wherein the marker is selected from 3-carboxyumbelliferyl (i-D-galactopyranoside 4-chloromethyl-6,8-difluoroumbelliferyl li-D-galactopyranoside, 4-methylumbelliferyl (i-D-galactopyranoside, fluorescein di (3-D-galactopyranoside, 6,8-difluoro-4-methylumbelliferyl (p-D-glucuronide, lithium sal, 2-dodecylresorufin, Elf-97, fluorescein [DI-B-glucuronide,] [5- (pentafluorobenzoylamino)] fluorescein [DI-fc-D-glucuronide,], 6,8-difluoro-4-methylumbelliferyl 1-D-galactopyranoside (DiFMUG), resorufin 10-D-galactopyranoside and [(3-trifluoromethylumbelliferyl (3-D-glucoronide.] 7. The process according to claim 1 wherein an antibiotic material is 34 incorporated into the sol-gel mixture for identifying bacterial antibiotic resistance. 8. The process according to claim 6, wherein the antibiotics are selected from Chloramphenicol, Erythromycin, Tetracycline, Streptomycin, Polymyxin, Nalidixic Acid, Novobyocin, Trimethoprin, Rifanapicin and Penicillin. 9. The process according to claim 1, wherein the markers are liposomes; films; multilayers; braided, lamellar, helical, tubular, and fiber-like shapes; solvated rods; solvated coils; and combinations thereof. 10. A method according to claim 1, wherein the number of microorganisms enumerated is less than 103 ml"1. 11. A method according to claim 1, wherein the slide and the filter are co-incubated for a period of time between 0.5 and 6 hours, at a temperature between 35 and 44°C. 12. The process according to claim 1, the liquid composition optimally comprises pyridine or potassium sulphate within the sol-gel for the detection of injured or stressed micro-organisms. 13. The process as claimed in claim 1 and claims 3 to 12 wherein two or more different excitation wavelengths coupled with the use of two or more fluorescent markers for providing different emission wavelengths from the viable micro-organisms to confirm that the emitted light is only from the viable micro-organisms. 14. The process as claimed in claim 1 and claims 3 to 13 wherein the emission wavelengths from the viable micro-organisms are compared from two pictures that have been taken at different wavelengths of the biosensor and eliminating those light sources that are not common. 35 15. The apparatus according to claim 2 wherein the incoming lamp light source is placed at an angle less than 90 degrees thereby preventing reflection of the artifact light into the receiving 16 bit or higher CCD camera. 16. The apparatus as claimed in claims 2 and 15 wherein the time allowed for taking a picture of the entire biosensor is a very short period i.e. few seconds so as to prevent photo bleaching of the bacteria. 17. The apparatus as claimed in claims 2,15 and 16 wherein a flat mirror is placed at 45 degree angle in between the lens system and the emission light filter for achieving a more compact design in the portable unit. 18. A process for the detection and enumeration of viable micro-organisms is substantially as herein described and exemplified with reference to the accompanying drawings. 19. An apparatus for the detection and enumeration of viable microorganisms is substantially as herein described with reference to the accompanying drawings. WE CLAIM J {\\) uoating a transparent ana / or non-transparent suae, porous sunace e. g., a glass slide, with a thin uniform layer of said liquid sol-gel precursor composition; (iii) Filtering the said given sample such as a specimen liquid to be tested through a filter so as to capture the viable micro-organisms such as live bacteria on the filter; (iv) These captured bacteria that are on the filter are then exposed to a biosensor that contains various fluorogenic substrates such as 3-carboxyumbelliferyl (3-D-galactopyranoside (CUG) or 4-chloromethyl-6, 8-difluoroumbelliferyl p-D-galactopyranoside (CMDiFUG), resorufin 10-D-galactopyranoside or 6,8-difluoro-4-methylumbelliferyl 1 -D-galactopyranoside (DiFMUG)for a period sufficient to their uptake into the metabolism of the live micro-organism cell and release of fluorogens due to the enzymetric break down of the fluorogenic substrates within the live cell; (v) Irradiating said sol-gel-coated slide or porous surface with an external energy source such as to generate detectable signals emitted from the marker(s) metabolized by the microorganisms; (vi) Detecting the micro-organisms by way of capturing the filtered emissions of light from fluorogens in the form of the specific light dots; (vii) Enumerating the specific light dots by way of comparing the different pictures and X-Y coordinates of the specific light dots only illuminating dots that have commonly shown up at least on two pictures. 2. An apparatus for detecting and enumerating the micro-organisms such as bacteria comprising a means of providing the excitation light to illuminate the biosensor, which is generally a slide on which the micro-organisms are placed on for detection and enumeration; a means of filtering system wherein the excited and emitted lights are filtered to prevent unwanted wavelengths; a lens system for focussing, adapting and magnification of light from the biosensor to the camera; a camera, preferably a 16bit CCD camera which shall be linked to a computer system through a direct ethernet cable to detect the weak light signals (luminescences) and coupling the CCD camera to an intensifier thereby creating a low light camera featuring a very high sensitivity along with a high resolution. 3. The process according to claim 1 wherein the microorganisms are bacteria. 7. The process according to claim 1 wherein the markers comprise antibiotic substances attached or coupled thereto. 8. The process according to claim 6, wherein the antibiotics are selected from Chloramphenicol, Erythromycin, Tetracycline, Streptomycin, Polymyxin, Nalidixic Acid, Novobyocin, Trimethoprin, Rifanapicin and Penicillin. 9. The process according to claim 1, wherein the markers are liposomes; films; multilayers; braided, lamellar, helical, tubular, and fiber-like shapes; solvated rods; solvated coils; and combinations thereof. 10. A method according to claim 1, wherein the number of microorganisms enumerated is less than 103 ml"1. 11. A method according to claim 1, wherein the slide and the filter are co-incubated for a period of time between 0.5 and 6 hours, at a temperature between 35 and 44°C. 12. The process according to claim 1, the liquid composition optimally comprises pyridine or potassium sulphate within the sol-gel for the detection of injured or stressed micro-organisms. 13. The process as claimed in claim 1 and claims 3 to 12 wherein two or more different excitation wavelengths coupled with the use of two or more fluorescent markers for providing different emission wavelengths from the viable micro-organisms to confirm that the emitted light is only from the viable micro-organisms. 14. The process as claimed in claim 1 and claims 3 to 13 wherein the emission wavelengths from the viable micro-organisms are compared from two pictures that have been taken at different wavelengths of the biosensor and eliminating those light sources that are not common. 15. The apparatus according to claim 2 wherein the incoming lamp light source is placed at an angle less than 90 degrees thereby preventing reflection of the artifact light into the receiving 16 bit or higher CCD camera. 16. The apparatus as claimed in claims 2 and 15 wherein the time allowed for taking a picture of the entire biosensor is a very short period i.e. few seconds so as to prevent photo bleaching of the bacteria. 17. The apparatus as claimed in claims 2,15 and 16 wherein a flat mirror is placed at 45degree angle in between the lens system and the emission light filter for achieving a more compact design in the portable unit 18. A process for the detection and enumeration of viable micro-organisms is substantially as herein described and exemplified with reference to the accompanying drawings. 19. An apparatus for the detection and enumeration of viable microorganisms is substantially as herein described with reference to the accompanying drawings.

Documents:

900-chenp-2003-abstract.pdf

900-chenp-2003-claims filed.pdf

900-chenp-2003-claims granted.pdf

900-chenp-2003-correspondnece-others.pdf

900-chenp-2003-correspondnece-po.pdf

900-chenp-2003-description(complete) filed.pdf

900-chenp-2003-description(complete) granted.pdf

900-chenp-2003-drawings.pdf

900-chenp-2003-form 1.pdf

900-chenp-2003-form 19.pdf

900-chenp-2003-form 26.pdf

900-chenp-2003-form 5.pdf

900-chenp-2003-pct.pdf