Microbial growth detector

• US 9,850,457 B2
• Filed: 12/23/2010
• Issued: 12/26/2017
• Est. Priority Date: 05/05/2010
• Status: Active Grant

• Current Assignee: Neogen Corporation
• Original Assignee: Neogen Corporation
• Inventors: Sarver, Jr., Ronald Waldo, Kariagin, Alexandr Y., Cooper, Christine Claire, McDougal, Susan Teruko
• Primary Examiner(s): Beisner, William H
• Assistant Examiner(s): HENKEL, DANIELLE B
• Application Number:
  US12/928,960
• Publication Number:
  US 20110275112A1
• Time in Patent Office: 2,560 Days
• Field of Search: 435 29, 4352872, 435 34, 4352875, 427 211
• CPC Class Codes: C12M 23/22 Transparent or translucent parts glassware for laboratory use B01L3/00
• CPC Class Codes: C12M 23/24 Gas permeable parts
• CPC Class Codes: C12M 41/26 of pH
• CPC Class Codes: C12M 41/34 of gas
• CPC Class Codes: G01N 21/80 Indicating pH value
• CPC Class Codes: G01N 31/22 using chemical indicators G01N31/02 takes precedence
• CPC Class Codes: G01N 33/02 Food

Abstract

The disclosure generally relates to a test device that detects microorganism growth by detecting a gas metabolite (e.g., carbon dioxide) produced during the growth of bacteria or other microorganism in a tested sample. The test device can contain a culture growth media separated from a detection area by a gas-permeable membrane. The gas-permeable membrane permits carbon dioxide to permeate into the detection area. The detection area includes a solidified mixture of pH indicators and a gelling agent in the form of a semi-permeable matrix. The optical properties, including the absorbance of light at various wavelengths, of the detection solution change with alterations in carbon dioxide concentration. This test device can then be placed in an incubation and optical detection instrument to monitor changes in optical properties of the detection are induced during microorganism growth in the culture medium.

Claims

1. An apparatus for detecting carbon dioxide, the apparatus comprising:

(a) a vessel comprising a wall, the wall defining (i) a detection region in the vessel and (ii) a growth region in the vessel;

(b) a semi-permeable matrix disposed in the detection region of the vessel, the matrix comprising a pH indicator distributed throughout the matrix;

(c) a gas-permeable membrane comprising a silicone polymer selected from the group consisting of room-temperature-vulcanized silicone, high-temperature-vulcanized silicone, ultraviolet-vulcanized silicone, and combinations thereof disposed inside the vessel, the gas-permeable membrane covering the semi-permeable matrix and defining a boundary between the detection region and the growth region of the vessel; and

(d) optionally, a culture medium disposed in the growth region of the vessel, the culture medium being capable of supporting the growth of a microorganism;

wherein;

(i) the gas-permeable membrane and the semi-permeable matrix are permeable to carbon dioxide, thereby permitting diffusive transport of carbon dioxide present in the growth region to the detection region;

(ii) the gas-permeable membrane is impermeable to liquid and solid materials present in the growth region;

(iii) the gas-permeable membrane is substantially free of any pH indicators present in the semi-permeable matrix;

(iv) the wall of the vessel is at least partially transparent in the detection region; and

(v) the semi-permeable matrix is permeable to liquids, but the presence of the gas-permeable membrane prevents the passage of liquids from the growth region to the detection region of the vessel.

2. The apparatus of claim 1, wherein the silicone polymer comprises ultraviolet-vulcanized silicone.

3. The apparatus of claim 1, wherein the gas-permeable membrane is attached to the wall of the vessel and forms a barrier isolating the detection region from the growth region.

4. The apparatus of claim 1, wherein the gas-permeable membrane has a thickness ranging from 10 pm to 2000 pm.

5. The apparatus of claim 1, wherein the gas-permeable membrane has a permeability ranging from 1×

10<sup>−

11</sup> cm²/(sec·

Pa) to 1×

10<sup>−

9</sup> cm²/(sec·

Pa) for carbon dioxide.

6. The apparatus of claim 1, wherein the semi-permeable matrix is in the form of a semi-solid or gel.

7. The apparatus of claim 1, wherein the semi-permeable matrix is in the form of a gel and comprises a gelling agent selected from the group consisting of agar, gelatin, carageenan, pectin, and combinations thereof.

8. The apparatus of claim 1, wherein the semi-permeable matrix is adhered to the wall of the vessel.

9. The apparatus of claim 1, wherein the pH indicator is evenly distributed throughout the semi-permeable matrix.

10. The apparatus of claim 1, wherein:

(i) the pH indicator comprises a first indicator and a second indicator; and

(ii) semi-permeable matrix comprises the first indicator and the second indicator in amounts and at a pH such that (A) the semi-permeable matrix has a first absorbance at a first wavelength, (B) the semi-permeable matrix has a second absorbance at a second wavelength, and (C) a ratio of the first absorbance to the second absorbance ranges from 0.2 to 4.

11. The apparatus of claim 1, wherein the pH indicator exhibits a color change at a pH value ranging from 6 to 10.

12. The apparatus of claim 1, wherein the pH indicator is selected from the group consisting of bromothymol blue, xylenol blue, methyl orange, α

-naphtholphthalein, fluorescein, coumarin, phenolphthalein, thymolphthalein, thymol blue, xylenol blue, and α

-naphtholbenzein, and combinations thereof.

13. The apparatus of claim 1, wherein the gas-permeable membrane, the growth region of the vessel, and the culture medium, when present, are free of any pH indicators present in the semi-permeable matrix.

14. The apparatus of claim 1, wherein:

(i) the pH indicator comprises bromothymol blue and xylenol blue; and

(ii) semi-permeable matrix comprises the bromothymol blue and the xylenol blue in amounts and at a pH such that (A) the semi-permeable matrix has a first absorbance at a first wavelength of about 615 nm, (B) the semi-permeable matrix has a second absorbance at a second wavelength of about 420 nm, and (C) a ratio of the first absorbance to the second absorbance ranges from 0.8 to 2.

15. The apparatus of claim 1, wherein the culture medium is present in the growth region of the vessel.

16. The apparatus of claim 15, wherein the culture medium comprises a Tryptic Soy Broth mixture.

17. The apparatus of claim 1, further comprising:

(e) a support material disposed in the growth region of the vessel, the support material providing a growth substrate for the microorganism.

18. A method of making an apparatus for detecting carbon dioxide, the method comprising:

(a) providing a vessel comprising a wall, the wall defining (i) a detection region in the vessel and (ii) a growth region in the vessel, wherein the detection region of the vessel contains a semi-permeable matrix disposed in the detection region of the vessel, the matrix comprising a pH indicator distributed throughout the matrix;

(b) applying a gas-permeable membrane precursor comprising a mixture comprising (i) a silicone prepolymer, (ii) a silicone crosslinking agent, and (iii) a curing catalyst in liquid form to an exposed surface of the semi-permeable matrix; and

(c) curing the gas-permeable membrane precursor, thereby forming a gas-permeable membrane in the vessel, the gas-permeable membrane covering the semi-permeable matrix and defining an interface between the detection region and the growth region of the vessel;

wherein;

(i) the gas-permeable membrane and the semi-permeable matrix are permeable to carbon dioxide, thereby permitting diffusive transport of carbon dioxide present in the growth region to the detection region;

(ii) the gas-permeable membrane is impermeable to liquid and solid materials present in the growth region;

(iii) the wall of the vessel is at least partially transparent in the detection region; and

(iv) the semi-permeable matrix is permeable to liquids, but the presence of the gas-permeable membrane prevents the passage of liquids from the growth region to the detection region of the vessel.

19. The method of claim 18, wherein the semi-permeable matrix in part (a) is formed by a process comprising:

(i) providing a mixture comprising (A) a liquid medium, (B) a matrix-forming agent in the liquid medium, and (C) a pH indicator in the liquid medium, wherein the mixture is at a temperature sufficient to maintain the mixture in liquid form;

(ii) dispensing the mixture in liquid form into the detection region;

(iii) cooling the mixture for a time sufficient to allow the matrix-forming agent to solidify, thereby forming the semi-permeable matrix comprising the pH indicator distributed throughout the matrix.

20. The method of claim 19, wherein the mixture is a solution in which the matrix-forming agent and the pH indicator are dissolved in the liquid medium.

21. The method of claim 19, wherein the matrix-forming agent is selected from the group consisting of agar, gelatin, carageenan, pectin, and combinations thereof.

22. The method of claim 18, further comprising:

(d) dispensing a culture medium in liquid form into the growth region of the vessel, the culture medium being in contact with the gas-permeable membrane and being capable of supporting the growth of a microorganism;

(e) sealing the vessel; and

(f) optionally exposing the sealed vessel to an ambient source of environmental carbon dioxide for a time sufficient for the semi-permeable matrix to attain an equilibrium level of carbon dioxide.

23. The method of claim 22, wherein part (d) further comprises inserting a support material into the growth region of the vessel, the support material being in contact with the culture medium and providing a growth substrate for the microorganism.

24. The method of claim 18, comprising applying the gas-permeable membrane precursor in an amount sufficient to completely coat the exposed surface of the semi-permeable matrix and to contact the wall of the vessel.

25. The method of claim 24, wherein the semi-permeable matrix is adhered to the wall of the vessel.

26. The method of claim 18, wherein curing the gas-permeable membrane precursor comprises exposing the gas-permeable membrane precursor to ultraviolet light.

27. The method of claim 18, comprising performing parts (a)-(c) in a continuous process.

28. The method of claim 18, comprising performing parts (a)-(c) in a batch process.

29. A method of detecting carbon dioxide, comprising:

(a) providing an apparatus including (A) a vessel comprising a wall, the wall defining (i) a detection region in the vessel and (ii) a growth region in the vessel;

(B) a semi-permeable matrix disposed in the detection region of the vessel, the matrix comprising a pH indicator distributed throughout the matrix;

(C) a gas-permeable membrane comprising a silicone polymer selected from the group consisting of room-temperature-vulcanized silicone, high-temperature-vulcanized silicone, ultraviolet-vulcanized silicone, and combinations thereof disposed inside the vessel, the gas-permeable membrane covering the semi-permeable matrix and defining a boundary between the detection region and the growth region of the vessel; and

(D) optionally, a culture medium disposed in the growth region of the vessel, the culture medium being capable of supporting the growth of a microorganism;

wherein;

(1) the gas-permeable membrane and the semi-permeable matrix are permeable to carbon dioxide, thereby permitting diffusive transport of carbon dioxide present in the growth region to the detection region;

(ii) the gas-permeable membrane is impermeable to liquid and solid materials present in the growth region;

(iii) the gas-permeable membrane is substantially free of any pH indicators present in the semi-permeable matrix;

(iv) the wall of the vessel is at least partially transparent in the detection region; and

(v) the semi-permeable matrix is permeable to liquids, but the presence of the gas-permeable membrane prevents the passage of liquids from the growth region to the detection region of the vessel;

wherein the culture medium is present in the growth region of the vessel;

(b) inserting a sample to be tested into the culture medium at a first time (t₁);

(c) sealing the vessel with the inserted sample;

(d) monitoring the detection region at a second time (t₂>

t₁) to detect changes in color of the pH indicator in the semi-permeable matrix; and

(e) correlating a change in the color of the pH indicator between the first time and the second time with a presence of carbon dioxide in the detection region.

30. The method of claim 29, further comprising:

(f) correlating a change in the color of the pH indicator between the first time and the second time with a presence of microorganisms in the sample.

31. The method of claim 30, wherein the microorganisms are selected from the group consisting of bacteria, yeasts, molds, and combinations thereof.

32. The method of claim 30, wherein the microorganisms comprise aerobic bacteria producing carbon dioxide as a metabolite.

33. The method of claim 29, wherein monitoring the detection region comprises incubating the vessel at a controlled temperature between the first time and the second time.

34. The method of claim 29, wherein monitoring the detection region comprises visually inspecting the semi-permeable matrix in the detection region to detect the changes in color of the pH indicator.

35. The method of claim 29, wherein monitoring the detection region comprises performing a spectrophotometric detection at one or more wavelengths.

36. The method of claim 35, wherein the one or more wavelengths are in the visible spectrum.

37. An apparatus for detecting a metabolic product gas of a microorganism, the apparatus comprising:

(a) a vessel comprising a wall, the wall defining (i) a detection region in the vessel and (ii) a growth region in the vessel;

(b) a semi-permeable matrix disposed in the detection region of the vessel, the matrix comprising a gas indicator distributed throughout the matrix;

(c) a gas-permeable membrane comprising a silicone polymer selected from the group consisting of room-temperature-vulcanized silicone, high-temperature-vulcanized silicone, ultraviolet-vulcanized silicone, and combinations thereof disposed inside the vessel, the gas-permeable membrane covering the semi-permeable matrix and defining a boundary between the detection region and the growth region of the vessel; and

(d) optionally, a culture medium disposed in the growth region of the vessel, the culture medium being capable of supporting the growth of a microorganism;

wherein;

(i) the gas-permeable membrane and the semi-permeable matrix are permeable to a metabolic product gas of microorganism growth, thereby permitting diffusive transport of the gas present in the growth region to the detection region;

(ii) the gas-permeable membrane is impermeable to liquid and solid materials present in the growth region;

(iii) the wall of the vessel is at least partially transparent in the detection region; and

(iv) the semi-permeable matrix is permeable to liquids, but the presence of the gas-permeable membrane prevents the passage of liquids from the growth region to the detection region of the vessel.

38. A method of detecting a metabolic product gas of a microorganism, comprising:

(a) providing an apparatus (A) a vessel comprising a wall, the wall defining (i) a detection region in the vessel and (ii) a growth region in the vessel;

(B) a semi-permeable matrix disposed in the detection region of the vessel, the matrix comprising a gas indicator distributed throughout the matrix;

(C) a gas-permeable membrane comprising a silicone polymer selected from the group consisting of room-temperature-vulcanized silicone, high-temperature-vulcanized silicone, ultraviolet-vulcanized silicone, and combinations thereof disposed inside the vessel, the gas-permeable membrane covering the semi-permeable matrix and defining a boundary between the detection region and the growth region of the vessel; and

(D) optionally, a culture medium disposed in the growth region of the vessel, the culture medium being capable of supporting the growth of a microorganism;

wherein;

(i) the gas-permeable membrane and the semi-permeable matrix are permeable to a metabolic product gas of microorganism growth, thereby permitting diffusive transport of the gas present in the growth region to the detection region;

(ii) the gas-permeable membrane is impermeable to liquid and solid materials present in the growth region;

(iii) the wall of the vessel is at least partially transparent in the detection region; and

(iv) the semi-permeable matrix is permeable to liquids, but the presence of the gas-permeable membrane prevents the passage of liquids from the growth region to the detection region of the vessel;

(b) inserting a sample to be tested into the culture medium at a first time (t₁);

(c) sealing the vessel with the inserted sample;

(d) monitoring the detection region at a second time (t₂>

t₁) to detect changes in color of the gas indicator in the semi-permeable matrix; and

(e) correlating a change in the color of the gas indicator between the first time and the second time with a presence of a metabolic product gas of microorganism growth in the detection region.