Method and device for detecting bacteriophage using contrast-coloring and precipitable dyes

• US 6,090,541 A
• Filed: 07/09/1999
• Issued: 07/18/2000
• Est. Priority Date: 04/18/1997
• Status: Expired due to Term

• Current Assignee: 3M Innovative Properties Company (3M Company)
• Original Assignee: 3M Innovative Properties Company (3M Company)
• Inventors: Williams, Michael G., Krejcarek, Gary E., Wicks, James H.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US09/350,978
• Time in Patent Office: 375 Days
• Field of Search: 435/5, 435/29, 435/34, 435/287.1, 435/288.4
• US Class Current: 435/5
• 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 2334/52 5-Bromo-4-chloro-3-indolyl, i.e. BCI

Abstract

The use of a precipitable dye and a contrast-coloring dye together enhance visualization of plaques in confluent lawns of bacteria in bacteriophage and bacteria assays. A test sample suspected of containing a bacteriophage is combined with bacteria capable of replicating the bacteriophage, and applied to a water-proof surface to form a support for bacterial growth. The support is provided with the contrast-coloring dye and precipitable dye, and nutrients and salts capable of supporting growth of the bacteria. A lawn of bacteria is formed on the support, and plaques detected on the lawn indicate presence of the bacteriophage. The plaques contain a precipitate formed by enzymatic cleavage of the precipitable dye by an enzyme of the bacterial lawn. A similar procedure is used for detecting bacteria, except that a test sample suspected of containing a bacteria is combined with bacteriophage capable of replicating in the bacteria, and plaques detected indicate presence of the bacteria. The bacteriophage and bacteria assays are carried out with a disposable device containing at least one well having a water-proof surface and a depth of about at least 5 millimeters. A hydratable material containing the contrast-coloring dye and precipitable dye is positioned on the surface. The well may contain substantially vertical sides with a removable cover resting on top of the sides.

Claims

1. A method for detecting bacteriophage comprising the steps of:

combining a test sample comprising a suspect bacteriophage with bacteria capable of supporting replication of the bacteriophage to form a liquid sample;

applying the liquid sample to a water-proof surface, wherein the water-proof surface with the liquid sample comprises a support for bacterial growth, and wherein the support includes at least one contrast-coloring dye, and at least one precipitable dye, and nutrients and salts capable of supporting growth of the bacteria

forming a lawn of bacteria on the support; and

detecting at least one plaque formed on the surface of the bacterial lawn, wherein a precipitate is formed in the at least one plaque and the precipitate is the product of an enzymatic cleavage of the precipitable dye by an enzyme from the bacteria of the bacterial lawn and wherein detection of at least one plaque on the bacterial lawn indicates the presence of the suspect bacteriophage in the test sample.

2. The method of claim 1, wherein the liquid sample comprises a solidifiable support in liquid form.

3. The method of claim 1, wherein the water-proof surface to which the liquid sample is applied comprises a hydratable solidifying support.

4. The method of claim 1, wherein the water-proof surface to which the liquid sample is applied comprises a semi-solid support.

5. The method of claim 1, wherein the support comprises agar.

6. The method of claim 1, wherein the bacteriophage is capable of replicating in bacteria selected from the group consisting of E. coli, Enterobacter, Salmonella, Staphylococci, Listeria and Mycobacterium.

7. The method of claim 1, wherein the liquid sample applied to the water-proof surface comprises nutrients and salts to permit the growth of a confluent lawn of bacteria.

8. The method of claim 1, wherein the test sample is a dilution of an original sample containing bacteriophage.

9. The method of claim 1, wherein the contrast-coloring dye is crystal violet.

10. The method of claim 1, wherein the precipitable dye is chromogenic.

11. The method of claim 10, wherein the precipitable dye produces a blue precipitate.

12. The method of claim 1, wherein the surface with the liquid sample further comprises a pH sensitive dye.

13. The method of claim 12, wherein the pH sensitive dye is neutral red.

14. The method of claim 1, wherein the surface with the liquid sample includes an inducer for the enzyme capable of enzymatically cleaving the precipitable dye.

15. The method of claim 14, wherein the inducer is selected from the group of 1-O-methyl-β

-D-glucuronide, isopropyl-β

-D-thioglucuronic acid, isopropyl-β

-D-thiogalactopyranoside, 3-O-methyl-α

-D-glucopyranoside and 1-O-methyl-β

-D-glucopyranoside.

16. The method of claim 1, wherein the liquid sample comprises the contrast-coloring dye, the precipitable dye, and nutrients and salts for bacteria growth.

17. The method of claim 4, wherein the semi-solid support comprises the contrast-coloring dye, the precipitable dye, and nutrients and salts for bacterial growth.

18. The method of claim 3, wherein the hydratable solidifying support comprises the contrast-coloring dye, the precipitable dye, and nutrients and salts for bacterial growth.

19. The method of claim 10, wherein the precipitable dye is 5-bromo-4-chloro-3-indolyl-β

-D-glucuronic acid.

20. The method of claim 1, wherein the liquid sample is poured onto the water-proof surface.

21. The method of claim 1, wherein the enzyme cleaving the dye is selected from the group consisting of a β

-D-glucuronidase, a β

-D-galactosidase, an alkaline phosphatase and an acid phosphatase.

22. A method for detecting bacteriophage comprising the steps of:

combining a test sample comprising a suspect bacteriophage and bacteria capable of supporting replication of the bacteriophage to form a liquid sample, wherein the concentration of bacteria is sufficient to form a bacterial lawn;

adding the liquid sample to a well, the well comprising a water-proof surface with a water-hydratable material dispersed thereon, wherein the water-hydratable material comprises a contrast-coloring dye and a precipitable dye and wherein the liquid sample in the well comprises a support suitable for bacterial growth and for the formation and visualization of bacteriophage-derived plaques;

forming a lawn of bacteria on the water-hydratable material; and

incubating the liquid sample until at least one discrete plaque is visible, wherein a precipitate is formed in the at least one plaque and the precipitate is the product of an enzymatic cleavage of the precipitable dye by an enzyme from the bacteria of the bacterial lawn and wherein detection of at least one plague on the bacterial lawn indicates the presence of the suspect bacteriophage in the test sample.

23. The method of claim 22, wherein the bacteriophage is capable of replicating in bacteria selected from the group consisting of E. coli, Enterobacter, Salmonella, Staphylococci, Listeria and Mycobacterium.

24. The method of claim 22, wherein the liquid sample applied to the water-proof surface comprises nutrients and salts to permit the growth of a confluent lawn of bacteria.

25. The method of claim 22, wherein the hydratable material further comprises nutrients and salts for bacterial growth.

26. The method of claim 22, wherein the contrast-coloring dye is crystal violet.

27. The method of claim 22, wherein the precipitable dye produces a blue precipitate.

28. The method of claim 22, wherein the hydratable material further comprises a pH sensitive dye.

29. The method of claim 28, wherein the pH sensitive dye is neutral red.

30. The method of claim 22, wherein the hydratable material further comprises an inducer for the enzyme capable of enzymatically cleaving the precipitable dye.

31. The method of claim 30, wherein the inducer is selected from the group of 1-O-methyl-β

-D-glucuronide, isopropyl-β

-D-thioglucuronic acid, isopropyl-β

-D-thiogalactopyranoside, 3-O-methyl-α

-D-glucopyranoside and 1-O-methyl-β

-D-glucopyranoside.

32. The method of claim 22, wherein the precipitable dye is 5-bromo-4-chloro-3-indolyl-β

-D-glucuronic acid.

33. A disposable device to detect bacteriophage comprising:

at least one well comprising a water-proof surface, the well having a depth of about at least 5 millimeters; and

a hydratable material positioned on the water-proof surface, the material comprising a precipitable dye and a contrast-coloring dye wherein the contrast-coloring dye is capable of coloring the hydratable material and where the precipitable dye is capable of being cleaved by at least one enzyme from a bacteria to form precipitate at the sight of a bacteriophage plague, wherein the bacteriophage is capable of replicating in the bacteria.

34. The device of claim 33, further comprising the at least one well having substantially vertical sides and a removable cover that is substantially impermeable to bacteria on top of the substantially vertical sides.

35. The device of claim 33, wherein the cover is transparent.

36. The device of claim 33, wherein the water-proof surface is opaque.

37. The device of claim 33, wherein the water-proof surface is white.