Microbial monitoring device

• US 5,567,598 A
• Filed: 03/03/1993
• Issued: 10/22/1996
• Est. Priority Date: 04/18/1991
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Burrell, Gregory J., Hu, Kwok-Yu, Sapitowicz, Robert, Monthony, James F., Stitt, David T.
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): Cano, Milton I.
• Application Number:
  US08/025,899
• Time in Patent Office: 1,329 Days
• Field of Search: 436/800, 436/172, 435/29, 435/33, 435/32, 435/301, 435/968, 435/39, 435/7.32, 435/288.4
• 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 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 2304/44 Oxygen
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

This invention presents methods for detection and evaluation of metabolic activity of microorganisms based upon their ability to consume dissolved oxygen. The methods utilize a fluorescence detection system which makes use of the sensitivity of the fluorescent emission of certain compounds to the presence of oxygen, which quenches (diminishes) the compound'"'"'s fluorescent emission in a concentration dependent manner. Respiring microorganisms will affect the oxygen concentration of a liquid medium in which they are immersed. Thus, this invention provides a convenient system to gather information on the presence, identification and metabolic activity of microorganisms by determining their effect on the oxygen concentration of the media in which they are present.

Claims

1. A method for detecting the presence of respiring microorganisms in a fluid comprising:

(i) contacting said fluid with a sensor composition which comprises a fluorescent compound that exhibits a reduction in fluorescent intensity, when irradiated with light containing wavelengths which cause said compound to fluoresce, upon exposure to oxygen, wherein the presence of the sensor composition is non-destructive to the microorganism;

(ii) irradiating said sensor composition with light containing wavelengths which cause said fluorescent compound to fluoresce;

(iii) measuring or visually observing the fluorescent light intensity from said fluorescent compound while irradiating said sensor compound with said light;

(iv) comparing said measurement to that of a control not containing a respiring microorganism, wherein said control is selected from the group consisting of;

a reagent control not in contact with respiring microorganisms and a calculated threshold, wherein a change in fluorescent intensity relative to the fluorescent intensity of the control is indicative of the presence of respiring microorganisms; and

(v) in the event that no such increase is measured or observed, repeat steps (ii), (iii), and (iv) as needed, to detect the presence of respiring microorganisms in said fluid.

2. The method of claim 1 where said matrix is a rubber or plastic matrix.

3. The method of claim 1 wherein said matrix is a silicone rubber matrix.

4. The method of claim 1 wherein said fluorescent compound is adsorbed on solid silica particles.

5. The method of claim 1 wherein said fluorescent compound is a tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) salt.

6. The method of claim 5 wherein said fluorescent compound is tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) chloride.

7. The method of claim 1 wherein said fluorescent compound is a tris-2, 2'"'"'-bipyridyl ruthenium (II) salt.

8. The method of claim 7 wherein said fluorescent compound is tris-2,2'"'"'-bipyridyl ruthenium (II) chloride hexahydrate.

9. The method of claim 1 wherein said fluorescent compound is 9, 10-diphenyl anthracene.

10. The method of claim 1 wherein said fluid is isolated from atmospheric oxygen wherein said fluid is contained in a closed system.

11. The method of claim 1 wherein said fluid is exposed to atmospheric oxygen.

12. A method for determining the effects of an antibiotic or antimicrobial composition on a respiring microorganism comprising:

(i) preparing a broth of said microorganism;

(ii) contacting said broth with a sensor composition which comprises a fluorescent compound that exhibits a reduction in fluorescent intensity, when irradiated with light containing wavelengths which cause said compound to fluoresce, upon exposure to oxygen, wherein the presence of the sensor composition is non-destructive to the microorganism;

(iii) admixing with said broth, a quantity of said antibiotic or antimicrobial composition;

(iv) irradiating said sensor composition with light containing wavelengths which cause said fluorescent compound to fluoresce;

(v) measuring or visually observing the fluorescent light intensity from said fluorescent compound while irradiating said sensor compound with said light; and

(vi) comparing said measurement to that of a control wherein said control is selected from the group consisting of;

a reagent control not in contact with a respiring microorganism or the antibiotic or antimicrobial composition, a reagent control in contact with a respiring microorganism but not in contact with the antibiotic or antimicrobial composition and a calculated threshold, wherein a change in fluorescent intensity relative to the control is indicative of the susceptibility or resistivity of the microorganisms to the quantity of said antibiotic or antimicrobial composition; and

(vii) in the event that no such change is measured or observed, repeat steps (iv), (v) and (vi), as needed, to determine the effects of the antibiotic or antimicrobial on the respiring microorganisms.

13. The method of claim 12 where said matrix is a rubber or plastic matrix.

14. The method of claim 12 wherein said rubber or plastic matrix is a silicone rubber matrix.

15. The method of claim 12 wherein said fluorescent compound is adsorbed on solid silica particles.

16. The method of claim 12 wherein said fluorescent compound is a tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) salt.

17. The method of claim 16 wherein said fluorescent compound is tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) chloride.

18. The method of claim 12 wherein said fluorescent compound is a tris-2, 2'"'"'-bipyridyl ruthenium (II) salt.

19. The method of claim 18 wherein said fluorescent compound is tris-2,2'"'"'-bipyridyl ruthenium (II) chloride hexahydrate.

20. The method of claim 12 wherein said fluorescent compound is 9, 10-diphenyl anthracene.

21. The method of claim 12 wherein said broth is isolated from atmospheric oxygen.

22. The method of claim 12 wherein said broth is exposed to atmospheric oxygen.

23. (amended) A method for detecting the presence of respiring microorganisms in a fluid comprising:

(i) contacting said fluid with a fluorescent sensor composition which comprises a fluorescent compound selected from the group consisting of tris-4,7-diphenyl-1,10-phenanthroline-ruthenium (II) chloride and tris-2,2'"'"'-bipyridyl ruthenium (II) chloride hexahydrate, wherein the presence of the sensor composition is non-destructive to the microorganism;

(ii) irradiating said sensor composition with light containing wavelengths which cause said fluorescent compound to fluoresce;

(iii) measuring or visually observing the fluorescent light intensity from said fluorescent compound while irradiating said sensor compound with said light;

(iv) comparing said measurement to that of a control, wherein said control is selected from the group consisting of;

a reagent control not in contact with respiring microorganisms and a calculated threshold, wherein a change in fluorescent intensity relative to the fluorescent intensity of the control is indicative of the presence of respiring microorganisms; and

(v) in the event that no such increase is measured or observed, repeat steps (ii), (iii), and (iv), as needed, to detect the presence of respiring microorganisms in said fluid.

24. The method of claim 23 where said matrix is a rubber or plastic matrix.

25. The method of claim 23 wherein said matrix is a silicone rubber matrix.

26. The method of claim 23 wherein said fluorescent compound is adsorbed on solid silica particles.

27. The method of claim 23 wherein said fluid compound is isolated from atmospheric oxygen.

28. The method of claim 23 wherein said fluid compound is exposed to atmospheric oxygen.

34. The method of claim 23 wherein said fluid compound is exposed to atmospheric oxygen.

29. A method of determining the effect of an antibiotic or antimicrobial composition on a respiring microorganism comprising:

(i) preparing a broth of said microorganism;

(ii) contacting said broth with a sensor composition which comprises a fluorescent compound selected from the group consisting of tris-4,7-diphenyl-1,10-phenanthroline-ruthenium (II) chloride and tris-2,2'"'"'-bipyridyl ruthenium (II) chloride hexahydrate, wherein the presence of the sensor composition is non-destructive to the microorganism;

(iii) admixing with said broth a quantity of said antibiotic or antimicrobial composition;

(iv) irradiating said sensor composition with light containing wavelengths which cause said fluorescent compound to fluoresce;

(iv) measuring or visually observing the fluorescent light intensity from said fluorescent compound while irradiating said sensor compound with said light;

(v) comparing said measurement to that of a control, wherein said control is selected from the group consisting of;

a reagent control not in contact with a respiring microorganism or the antibiotic or antimicrobial composition, a reagent control in contact with a respiring microorganism but not in contact with the antibiotic or antimicrobial composition and a calculated threshold, wherein a change in fluorescent intensity relative to the control is indicative of the susceptibility or resistivity of the microorganisms to quantity of the antibiotic or antimicrobial composition; and

(vi) in the event that no such change is measured or observed, repeat steps (iii), (iv), and (v), as needed, to detect the presence of respiring microorganisms in said fluid.

30. The method of claim 29 where said matrix is a rubber or plastic matrix.

31. The method of claim 29 wherein said matrix is a silicone rubber matrix.

32. The method of claim 29 wherein said fluorescent compound is adsorbed on solid silica particles.

33. The method of claim 29 wherein said broth compound is isolated from atmospheric oxygen wherein said fluid is contained in a closed system.