Process and kit for detecting microbial metabolism

• US 5,164,301 A
• Filed: 06/22/1990
• Issued: 11/17/1992
• Est. Priority Date: 06/22/1990
• Status: Expired due to Fees

• Current Assignee: Difco Laboratories Ann Arbor MI
• Original Assignee: Difco Laboratories, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Eden, Ruth F., Thompson, Thomas E.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): WILLIAMS, JANE
• Application Number:
  US07/542,115
• Time in Patent Office: 879 Days
• Field of Search: 435/34, 435/29, 435/4, 436/800, 436/172, 250/461.2, 250/461.1
• US Class Current: 435/29
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 2304/20 Redox indicators
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

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.

Claims

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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

7. 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.

8. 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.

9. A process as set forth in claims 5 or 8 wherein the environment is intracellular.

10. A process as set forth in claims 5 or 8 wherein the environment is extracellular.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.