Test media and quantiative or qualitative method for identification and differentiation of biological materials in a test sample

• US 20050196825A1
• Filed: 04/01/2005
• Published: 09/08/2005
• Est. Priority Date: 07/20/1999
• Status: Active Grant

• Current Assignee: Micrology, Inc.
• Original Assignee: Micrology Laboratories LLC
• Inventors: Roth, Geoffrey N., Roth, Jonathan N.
• Granted Patent: US 7,344,854 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• 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 1/045 Culture media therefor
• CPC Class Codes: C12Q 1/10 Enterobacteria
• CPC Class Codes: C12Q 2334/00 O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
• CPC Class Codes: C12Q 2334/50 Indoles
• CPC Class Codes: G01N 2333/245 Escherichia (G)
• CPC Class Codes: G01N 2333/25 Shigella (G)
• CPC Class Codes: G01N 2333/255 Salmonella (G)
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A test medium and method for detecting, quantifying, identifying and differentiating up to four (4) separate biological materials in a test sample. A test medium is disclosed which allows quantifying and differentiating under ambient light aggregates of biological entities producing specific enzymes, which might include general coliforms, E. coli, Aeromonas, and Salmonella in a single test medium. A new class of nonchromogenic substrates is disclosed which produce a substantially black, non-diffusible precipitate. This precipitate is not subject to interference from other chromogenic substrates present in the test medium. In one embodiment, the substrates are selected such that E. coli colonies present in the test medium show as substantially black, general coliforms colonies show in the test medium as a blue-violet color, Aeromonas colonies present in the test medium show as a generally red-pink color, and Salmonella colonies show as a generally teal-green color. Other microorganisms and color possibilities for detection and quantification thereof are also disclosed. An inhibitor and method for making a test medium incorporating the inhibitor are disclosed.

Claims

1. A test medium for detecting, quantifying or differentiating general coliforms, E. coli, Aeromonas and Salmonella, said test medium comprising:

a β

-D-glucuronide chromogenic or non chromogenic substrate, which forms a first product that is visible in ambient light in the presence of E. coli;

an α

-D-galactoside chromogenic substrate, which forms a second product that is visible in ambient light in the presence of the Salmonella, E. coli and other coliforms; and

a β

-D-galactoside chromogenic substrate, which forms a third product that is visible in ambient light in the presence of Aeromonas, E. coli and other coliforms, said products of said α

-D-galactoside and said β

-D-galactoside substrates forming a combined color in the presence of general coliforms and E. coli, and wherein general coliforms, E. coli, Aeromonas, and Salmonella are all distinguishable from one another; and

a fourth substrate being one of a β

-D-glucuronide substrate, an α

-D-galactoside substrate, or a β

-D-galactoside substrate, which forms a product that fluoresces under ultraviolet light in the presence of at least one of E. coli, general coliforms, Salmonella, or Aeromonas.

2. The test medium of claim 1, wherein said fourth substrate is a β

-D-glucuronide substrate and the product of the fourth substrate formed in the presence of E. coli fluoresces under an ultraviolet light.

3. The test medium as set forth in claim 1, wherein said fourth substrate is an α

-D-galactoside substrate, and the product of the fourth substrate formed in the presence of E. coli, general coliforms and/or Salmonella fluoresces under an ultraviolet light.

4. The test medium as set forth in claim 1, wherein said fourth substrate is a β

-D-galactoside substrate and the product formed in the presence of E. coli, general coliforms and/or Aeromonas fluoresces under an ultraviolet light.

5. The test medium as set forth in claim 1, wherein said β

-D-glucuronide chromogenic or nonchromogenic substrate produces color and one of either said α

-D-galactoside chromogenic substrate or said β

-D-galactoside chromogenic substrate includes the same color component as said chromogenic or nonchromogencic β

-D-glucuronide substrate.

6. The test medium as set forth in claim 5, wherein said β

-D-glucuronide chromogenic or nonchromogencic substrate and said α

-D-galactoside chromogenic substrate or said β

-D-galactoside chromogenic substrate are present in equal amounts.

7. The test medium of claim 5, wherein said β

-D-glucuronide substrate and said one of either said α

-D-galactoside chromogenic substrate or said β

-D-galactoside chromogenic substrate are present in different amounts.

8. The test medium of claim 7, wherein there is more of said β

-D-glucuronide substrate than said α

-D-galactoside chromogenic substrate or said β

-D-galactoside chromogenic substrate of the same color component.

9. The test medium of claim 1, wherein said fouth substrate includes the fluorescent component 4-methylumbelliferyl.

10. A test medium for detecting, identifying and qualifying or quantifying biologic entities, said medium comprising:

a nutrient-based medium;

a first substrate, which forms a first product in the presence of a first biological entity;

a second substrate, which forms a second product in the presence of a second biological entity; and

a third substrate, which forms a third product in the presence of a third biological entity, said second and third substrates also forming a combined product in the presence of a fourth biological entity, and said first biological entity also forming a combined product in the presence of said second and third substrates.

11. The test medium as set forth in claim 10, wherein said first and said second substrates include the same color forming component, and said first biological entity appears darker than said fourth biological entity as said first biological entity reacts to all of said substrates.

12. The test medium as set forth in claim 11, wherein said first substrate includes more of said color forming component than said second substrate.

13. The test medium as set forth in claim 10, wherein said first and said third substrates include the same color forming component, and said first biological entity appears darker than said fourth biological entity as said first biological entity reacts to all of said substrates.

14. The test medium as set forth in claim 11, wherein said first substrate includes more of said color forming component than said third substrate.

15. The test medium as set forth in claim 10, including a fourth substrate, which forms a product in the presence of one of said biological entities that fluoresces under an ultraviolet light.

16. The test medium as set forth in claim 15, wherein said first substrate is a β

-D-glucuronide chromogenic or nonchromogenic substrate and said first biological entity is E. coli;

said second substrate is an α

-D-galactoside chromogenic substrate and said second biological entity is Salmonella;

said third substrate is a β

-D-galactoside chromogenic substrate and said third biological entity is Aeromonas; and

said fourth biological entity is general coliforms.

17. A test medium for detecting, identifying, or quantifying a biological entity, said medium comprising:

a nutrient-based medium;

a first substrate, which is a chromogenic or nonchromogenic substrate, which forms a product that is visible in ambient light in the presence of a first biological entity; and

a second substrate, which produces a product that fluoresces under an ultraviolet light in the presence of said first biological entity.

18. The test medium as set forth in claim 17, wherein said first substrate is a β

-D-glucuronide substrate and said first biological entity is E coli.

19. The test medium as set forth in claim 18, wherein said second substrate is a 4-methylumbelliferyl β

-D-glucuronide substrate.

20. The test medium as set forth in claim 19, wherein said medium provides a dual verification of the presence of E coli, wherein a first verification is the fluorescence of E coli under ultraviolet light and the second verification is a visual identification under ambient light of a product from the chromogenic or nonchromogenic substrate.

21. The test medium as set forth in claim 20, wherein said verification of E coli as fluorescing under an ultraviolet light is detectable before the visible identification from the chromogenic or nonchromogenic substrate.

22. The test medium as set forth in claim 17, wherein said first substrate is an α

-D-galactopyranoside and said second substrate is a β

-D-galactopyranoside.

23. The test medium as set forth in claim 22, wherein said first biological entity is total coliforms and said second substrate also produces a product that fluoresces under an ultraviolet light in the presence of Aeromonas.

24. The test medium as set forth in claim 23, wherein said first substrate is 5-bromo-4-chloro-3-indolyl-α

-D-galactopyranoside, and the product formed in the presence of total coliforms is teal, and a teal product is also formed in the presence of Salmonella.

25. The test medium as set forth in claim 17, wherein said first substrate is a β

-D-galactopyranoside and said second substrate is a α

-D-galactopyranoside.

26. The test medium as set forth in claim 25, wherein said first biological entity is total coliforms and said second substrate also produces a product that fluoresces under an ultraviolet light in the presence of Salmonella.

27. The test medium as set forth in claim 26, wherein said first substrate is 5-bromo-4-chloro-3-indolyl-β

-D-galactopyranoside, and the product formed in the presence of total coliforms is teal, and a teal product is also formed in the presence of Aeromonas.