US5164301A
Expired / Lapsed
US
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Process and kit for detecting microbial metabolism
Assignee: Difco Laboratories Inc
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Filing: 1990-06-22
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Granted: 1992-11-17
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Expires: 2010-06-22
Abstract
Abstract A process and kit for detecting microbial metabolism is disclosed, the process including the steps of combining medium, a metabolic dye having an absorbance spectrum which in the presence of microorganisms changes and responds to metabolism of the microorganism and a second analytical dye having an excitation or emission spectrum which overlaps with one of the changed or unchanged absorbance spectra of the first dye. The medium contains a sample solution to be analyzed for containing metabolizing microorganisms. The user observes either no change in the fluorescence emissions of the second analytical dye indicating the absence of metabolizing microorganisms in the sample or a change indicating the presence of metabolizing microorganisms in the sample.
Inventors
- 👤 Thomas E. Thompson
- 👤 Ruth F. Eden
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 23:43
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/02; C12Q1/04; C12Q2304/00; C12Q2304/20; Y; Y10; Y10S; Y10S436/00; Y10S436/80
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
138 words
A process for detecting microbial metabolism, said process including the steps of: combining a sample solution to be analyzed for metabolizing microorganisms, microbial growth medium, a nontoxic concentration of a first dye having an absorbance spectrum which in the presence of growing microorganisms changes in response to by products of metabolism released by the microorganism into the medium, and a nontoxic concentration of a second dye having an excitation or emission spectrum which overlaps with one of the changed or unchanged absorbance spectra of the first dye, neither dye being metabolized directly by the microorganisms; and observing either no change in the intensity of fluroescence emission of the second dye indicating the absence of metabolizing microorganisms in the sample or a change in the intensity of the fluorescence emission indicating the presence of metabolizing microorganisms in the sample.
Claim 2
Dependent Claim
138 words
A process for detecting microbial metabolism, said process including the steps of: combining a sample solution to be analyzed for metabolizing microorganisms, microbial growth medium, a nontoxic concentration of a first dye having an absorbance spectrum which in the presence of growing microorganisms changes in response to by products of metabolism released by the microorganism into the medium, and a nontoxic concentration of a second dye having an excitation or emission spectrum which overlaps with one of the changed or unchanged absorbance spectra of the first dye, neither dye being metabolized directly by the microorganisms; and observing either no change in the intensity of fluroescence emission of the second dye indicating the absence of metabolizing microorganisms in the sample or a change in the intensity of the fluorescence emission indicating the presence of metabolizing microorganisms in the sample.
Claim 3
Dependent Claim
76 words
A process as set forth in claim 1 wherein said combining step is further defined as changing the physical-chemical environment of the first dye as the microorganisms metabolize nutrients from the medium, and shifting the absorbance spectrum of the first dye, thus quenching or unquenching the emission spectrum of the second dye, said observing step being further defined as observing the quenching or unquenching of the second dye indicating the presence or absence of metabolizing microorganism.
Claim 4
Dependent Claim
79 words
A process as set forth in claim 2 wherein the growth medium contains specific nutrients which change the physical-chemical properties of the medium when metabolized by specific microorganisms, said changing step being further defined as changing the physical-chemical environment of the first dye as only a specific type of microorganism metabolizes the specific nutrient in the medium and observing the change in emission of the second dye as an identification of the presence of the specific type of microorganism.
Claim 5
Dependent Claim
54 words
A process as set forth in claim 3 further including the steps of repeating said combining step for several different media containing different specific nutrients, observing the changes or absence of changes of the emission spectrum of the second dye, and constructing an identification finger print of specific microorganisms based on the several observations.
Claim 6
Dependent Claim
41 words
A process as set forth in claim 2 wherein said changing step is further defined as changing the pH of the solution, the first dye having an absorbance spectrum which changes in response to the change in pH of the environment.
Claim 7
Dependent Claim
36 words
A process as set forth in claim 5 wherein said changing step is further defined as adding specific carbohydrates which are fermented to acidic end products by certain microorganisms thus changing the pH of the medium
Claim 8
Dependent Claim
41 words
A process as set forth in claim 5 wherein said changing step is further defined as adding urea which is enzymatically transformed by microorganisms containing urease and cleaved to ammonia and carbon dioxide and changing thus the pH of the environment.
Claim 9
Dependent Claim
43 words
A process as set forth in claim 2 wherein said changing step is further defined as growing the microorganisms and changing the reduction-oxidation potential of the environment, the first dye having an absorbance spectrum which changes in response to changes in reduction-oxidation potentials.
Claim 10
Dependent Claim
15 words
A process as set forth in claims 5 or 8 wherein the environment is intracellular.
Claim 11
Dependent Claim
15 words
A process as set forth in claims 5 or 8 wherein the environment is extracellular.
Claim 12
Dependent Claim
66 words
A process as set forth in claim 1 wherein said combining step is further defined as adding different antibiotics and/or growth inhibitor to different samples of the medium containing the microorganisms, said observing step being further defined observing which antibiotics and/or inhibitors prevent a change in the fluorescence emission of the second dye as indicating sensitivity or resistance of a microorganism to different antibiotics and/or inhibitors.
Claim 13
Dependent Claim
35 words
A process as set forth in claim 1 further including the step of incubating the combined medium and dyes at 30° to 35° C. for 2 to 8 hours while monitoring the changes in fluorescence.
Claim 14
Dependent Claim
36 words
A process as set forth in claim 1 wherein said combining step is further defined as adding 5 to 30 micromolar concentrations of the first dye and 2 to 20 micromolar concentrations of the second dye.
Claim 15
Dependent Claim
106 words
A kit for detecting microbial metabolism, said kit comprising: microbial growth medium; a nontoxic concentration of a first dye having an absorbance spectrum which in the presence of microorganisms changes in response to metabolism of the microorganism; a nontoxic concentration of a second dye having an exciation or emission spectrum which overlaps with one of the unchanged or changed absorbance spectrum of the first dye whereby addition of the first and second dyes to the medium without metabolizing microorganisms causes no change in the observed fluorescence emission of said second dye and with metabolizing microorganisms causes changes in the observed fluroescence emission of said second dye.
Claim 16
Dependent Claim
106 words
A kit for detecting microbial metabolism, said kit comprising: microbial growth medium; a nontoxic concentration of a first dye having an absorbance spectrum which in the presence of microorganisms changes in response to metabolism of the microorganism; a nontoxic concentration of a second dye having an exciation or emission spectrum which overlaps with one of the unchanged or changed absorbance spectrum of the first dye whereby addition of the first and second dyes to the medium without metabolizing microorganisms causes no change in the observed fluorescence emission of said second dye and with metabolizing microorganisms causes changes in the observed fluroescence emission of said second dye.
Claim 17
Dependent Claim
26 words
A kit as set forth in claim 14 wherein said first dye has an absorbance spectrum that changes in response to physical-chemical changes in the environment.
Claim 18
Dependent Claim
27 words
A kit as set forth in claim 15 wherein said first dye has an absorbance spectrum that changes in response to changes in pH of the environment.
Claim 19
Dependent Claim
26 words
A kit as set forth in claim 15 wherein said first dye has an absorbance spectrum that changes in response to reduction-oxidation potential of the environment.
Claim 20
Dependent Claim
27 words
A kit as set forth in claim 14 wherein said first dye has an absorbance spectrum, that changes when said first dye is metabolized by specific microorganisms.
References Cited (Backward Citations)
US3564588A
Cited
US3676679A
Cited
US3957583A
Cited
US4025393A
Cited
US4116775A
Cited
US4118280A
Cited
US4049499A
Cited
US4448534A
Cited
US4311794A
Cited
US4221867A
Cited
US4591554A
Cited
US4250266A
Cited
EP0070685A2
Cited
US4495293A
Cited
US4495293B1
Cited
US4556636A
Cited
US4639421A
Cited
US4665024A
Cited
US4743561A
Cited
US4861709A
Cited
US4916060A
Cited
US4859584A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-11-17 | 1996-05-18 | 1996-11-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-11-17 | 2000-05-18 | 2000-11-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-11-17 | 2004-05-18 | 2004-11-17 | $7700.00 | Window Closed / Lapsed |