Method for detecting bacteria using bacteriophage, contrast-coloring dye and precipitable dye

• US 5,958,675 A
• Filed: 04/18/1997
• Issued: 09/28/1999
• 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:
  US08/844,145
• Time in Patent Office: 893 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

Bacteria are detected in a test sample by contacting the test sample with a bacteriophage that is capable of replicating in the bacteria, adding the resultant sample to a water-proof surface of a support for bacterial growth that contains a contrast-coloring dye and a precipitable dye, forming a bacterial lawn of a bacteria in which the bacteriophage can replicate on the support and detecting plaques on the bacterial lawn as an indication of the presence of the bacteria. The combination of precipitable dye and contrast-coloring dye improves visualization of plaques. A precipitate is formed in plaques by enzymatic cleavage of the precipitable dye by an enzyme of the bacterial lawn. A procedure for detecting bacteriophage is similar to that for detecting bacteria, except that a test sample suspected of containing bacteriophage is combined with bacteria in which the bacteriophage can replicate, and plaques detected indicate presence of the bacteriophage. The bacteria and bacteriophage detections are carried out with a disposable device containing at least one well having a water-proof surface and substantially vertical sides that extend at least 5 millimeters in height from the surface. A hydratable material containing the precipitable dye and the contrast-coloring dye is positioned on the water-proof surface. A removable cover rests on top of the sides of the well.

Claims

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

contacting a test sample suspected of containing a suspect bacteria with bacteriophage in a liquid to form a liquid sample, wherein the bacteriophage is capable of replicating in the suspect bacteria;

removing bacteriophage not in contact with the suspect bacteria in the liquid sample;

adding the liquid sample to a waterproof surface, wherein the water-proof surface including the liquid sample comprises a support for bacterial growth, and wherein the support includes a contrast-coloring dye and a precipitable dye;

forming a bacterial lawn of a bacteria on the support, wherein the bacteriophage is capable of replicating in the bacteria of the bacterial lawn; and

detecting at least one plaque, wherein a precipitate is formed in the plaques 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 bacteria in the test sample.

2. The method of claim 1 wherein the suspect bacteria of the contacting step is different from the bacteria of the bacterial lawn.

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

4. The method of claim 1, wherein the water-proof surface comprises a solidifiable support in liquid form.

5. The method of claim 4, wherein the support comprises a hydratable solidifying support.

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

7. The method of claim 1, wherein the precipitable dye is 5-bromo-4-chloro-3-indolyl-β

-D-glucuronic acid.

8. The method of claim 1, wherein the support further comprises neutral red.

9. The method of claim 1, wherein the surface with the liquid sample includes an inducer for the enzyme of the detecting step.

10. The method of claim 9, wherein the inducer is selected from the group consisting 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.

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