Method for the detection and enumeration of viable microorganisms. A liquid that comprises one or more markers incorporated in a liquid sol-gel precursor, is provided. A transparent slide is coated with a thin uniform layer of the liquid sol-gel precursor composition. The microorganisms are separate
Method for the detection and enumeration of viable microorganisms. A liquid that comprises one or more markers incorporated in a liquid sol-gel precursor, is provided. A transparent slide is coated with a thin uniform layer of the liquid sol-gel precursor composition. The microorganisms are separated from liquid sample to be analyzed by passing the sample through a filter, and then bringing the filter into close contact with the sol-gel coated slide. The filter is co-incubated with the sol-gel coated slide for a period of time and at a temperature suitable to promote uptake of the markers by the microorganisms. The gel-coated slide irradiated with an external energy source, so as to generate detectable signals emitted from the markers uptaken by the microorganisms. Image of the detectable signals emitted from the microorganisms are acquired, and analyzed using a computer system, in order to provide the identification and enumeration of the microorganisms.
대표청구항▼
The invention claimed is: 1. A method for detecting and enumerating viable microorganisms in a sample, providing a reliable direct count of individual viable microorganisms contained in the sample, comprising: providing a smooth transparent porous material composed of sol-gel and containing a marke
The invention claimed is: 1. A method for detecting and enumerating viable microorganisms in a sample, providing a reliable direct count of individual viable microorganisms contained in the sample, comprising: providing a smooth transparent porous material composed of sol-gel and containing a marker in pores thereof; separating the microorganisms from a sample to be analyzed and contacting the microorganisms with the smooth transparent porous material, thereby transferring the separated microorganisms to a surface of the smooth transparent porous sol-gel material; incubating the contacted microorganisms and smooth transparent porous marker containing sol-gel material for a time sufficient to update the marker from the pores, to promote uptake of the marker by viable microorganisms; irradiating the marker containing smooth transparent porous sol-gel material with an energy source to generate detectable signals emitted by the marker taken up by the viable microorganisms, and detecting the signals emitted and analyzing the signals to identify and enumerate the viable microorganisms. 2. A method according to claim 1 wherein the microorganisms are bacteria. 3. A method according to claim 1 wherein the energy source is selected from the group consisting of visible light, fluorescent light, UV light, infrared light, an electro-magnetic field, sonar, ultrasonic waves, radio waves, and short wave radiation. 4. A method according to claim 1, further comprising isolating the microorganisms from a sample selected from the group consisting of water, milk, food, saliva, urine, throat swab tests, wounds, sputum, stomach content, and feces. 5. The method according to claim 1, wherein the marker is selected from the group consisting of 3-carboxyumbelliferyl β-D-galactopyranoside, 4-chloromethyl-6,8-difluoroumbelliferyl β-D-galactopyranoside, 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di β-D-galactopyranoside, 6,8-difluoroumbelliferyl β-D-glucuronide, 5-(pentafluorobenzoylamino) fluorescein di-β-D-glucuronide, and β-trifluoromethylumbelliferyl β-D-glucuronide. 6. The method according to claim 1 further comprising attaching antibiotic substances to the marker. 7. The method according to claim 6, wherein the antibiotic substance is selected from the group consisting of Chloramphenicol, Lrythromycin, Tetracycline, Streptomycin, Polymyxin, Nalidixic Acid, Novobyocin, Trimethorprin, Rifanapicin and Penicillin. 8. The method according to claim 1, wherein the markers are provided in a form selected from the group consisting of liposomes; films; multilayers; braided; lamellar, helical, tubular, and fiber shapes; solvated rods; solvated coils; and combination thereof. 9. The method according to claim 1, farther comprising providing pyridine or K2SO4 in the smooth transparent porous material. 10. The method according to claim 1, further comprising detecting microorganisms present at less than 103 ml-1. 11. The method according to claim 1, wherein separating the microorganisms from a sample comprises filtering the sample to capture microorganisms on a filter media, and placing the filter media in contact with the smooth transparent porous material containing the marker. 12. The method of claim 11 further comprising co-incubating the filter media with the smooth transparent porous material. 13. The method of claim 12 wherein the co-incubating is conducted at 0.5 to 6 hours at a temperature of between 35 and 44째 C. 14. A system for identifying and enumerating viable microorganisms in a sample, the system being sufficiently sensitive to provide a reliable direct count of individual viable microorganisms contained in the sample comprising; a smooth transparent porous material composed of sol-gel and containing at least one marker in the pores therein; separation means for separating the microorganisms from a sample to be analyzed, the separated microorganisms transferred to a surface of the smooth transparent porous sol-gel material, such that the microorganisms contact the marker contained thereon; means for incubating the microorganisms with the marker-containing smooth transparent porous material such that the marker is uptaken from the pores, the viable microorganisms ingest the marker and metabolize the marker for emifting detectable signals from the viable microorganisms; means for irradiating the marker ingested by the viable microorganisms disposed on the marker-containing smooth transparent porous material for generating the detectable signals emitted by the marker; means for detecting the signals; and means for analyzing the detected signals to identify and enumerate the viable microorganisms. 15. The system of claim 14, wherein the irradiating means comprise an energy source selected from the group consisting of visible light, fluorescent light, UV light, infrared light, an electrochemical field, sonar, ultrasonic waves, radio waves and short wave radiation. 16. The system of claim 14, wherein the detecting means comprise a frame grabber, a CCD array camera, a microscope, and an autofocus system. 17. The system of claim 14, further comprising a mechanical x-y table. 18. The system of claim 16, wherein the frame grabber has a rate of 30 frames per second. 19. The system according to claim 14 wherein the microorganisms are bacteria. 20. The system according to claim 14 wherein the sample is selected from the group consisting of water, milk, food, saliva, urine, throat swab tests, wounds, sputum, stomach content, and feces. 21. The system according to claim 14 wherein the marker is selected from the group consisting of 3-carboxyumbelliferyl β-D-galactopyranoside, 4-chloromethyl-6,8-difluoroumbelliferyl β-D-galactopyranoside, 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di β-D-galactopyranoside, 6,8-difluoroumbelliferyl β-D-glucuronide, 5-(pentafluorobenzoylamino) fluorescein di-β-D-glucuronide, and β-trifluoromethylumbelliferyl β-D-glucuronide. 22. The system according to claim 14 wherein the marker has at least one antibiotic substance attached thereto. 23. The system according to claim 22, wherein the antibiotic substance is selected from the group consisting of Chloramphenicol, Lrythromycin, Tetracycline, Streptomycin, Polymyxin, Nalidixic Acid, Novobyocin, Trimethorprin, Rifanapicin and Penicillin. 24. The System according to claim 14, wherein the marker is in a form selected from the group consisting of liposomes; films; multilayers; braided; lamellar, helical, tubular, and fiber shapes; solvated rods; solvated coils; and combination thereof. 25. The system according to claim 14 wherein the smooth transparent porous material contains pyridine or K2SO4. 26. The system according to claim 14 wherein the system detects microorganisms present at less than 103 ml-1. 27. The system according to claim 14 wherein the separation means comprise a filter for filtering the sample to capture microorganisms on a filter media, the filter media contacted with the smooth transparent porous material containing the marker. 28. The system of claim 27 wherein the filter media is co-incubated with the smooth transparent porous material. 29. The system of claim 28 wherein the filter media is co-incubated with the smooth transparent porous material for 0.5 to 6 hours at a temperature of between 35 and 44째 C. 30. The system of claim 14 wherein the smooth transparent marker containing sol-gel material is a smooth transparent marker containing sol-gel coating supported on a slide.
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이 특허에 인용된 특허 (6)
Brocklehurst Timothy F.,GB3 ; Mackie Alan R.,GB3 ; Steer David C.,GB3 ; Wilson David R.,GB3, Detection of microbial growth.
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