Method for detecting staphylococci

• US 5,635,367 A
• Filed: 05/15/1995
• Issued: 06/03/1997
• Est. Priority Date: 01/31/1994
• Status: Expired due to Term

• Current Assignee: Minnesota Mining and Manufacturing Co (3M Company)
• Original Assignee: Minnesota Mining and Manufacturing Co (3M Company)
• Inventors: Lund, Marlys E.
• Primary Examiner(s): Tsang, Cecilia J.
• Assistant Examiner(s): Mohamed, Abdel A.
• Application Number:
  US08/441,452
• Time in Patent Office: 750 Days
• Field of Search: 435/34, 435/14, 435/29, 435/30, 435/39, 435/243, 435/253.6, 435/261, 435/291, 435/299, 435/310, 435/805, 435/810, 435/882
• US Class Current: 435/34
• CPC Class Codes: C12M 25/06 Plates; Walls; Drawers; Multilayer plates
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• 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 1/14 Streptococcus; Staphylococcus
• CPC Class Codes: C12Q 2334/50 Indoles
• CPC Class Codes: C12Q 2334/52 5-Bromo-4-chloro-3-indolyl, i.e. BCI
• CPC Class Codes: G01N 2333/31 from Staphylococcus (G)
• CPC Class Codes: G01N 2333/315 from Streptococcus (G), e.g. Enterococci
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/882 Staphylococcus

Abstract

The present invention exploits the herein first reported, empirical observation that even though staphylococci produce the enzyme, beta-glucosidase, this genus of bacteria is not able to produce a metabolite that will enzymatically react with 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, a substrate commonly used to detect the presence of beta-glucosidase produced by other bacteria. In view of this unexpected observation, one embodiment of the present invention includes a selective medium containing inhibitors to enhance staphylococci growth as well as a first glucopyranoside substrate, such as 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, and a second phosphatase substrate, Such as 6-chloro-3-indolylphosphate or 5-bromo-6-chloro-3-indolylphosphate. In a particularly preferred embodiment of this invention, a thin film culture plate device, such as a PETRIFILM culture plate device, is prepared using a dry culture medium containing selected inhibitors, a first glucopyranoside substrate and a second phosphate substrate. When the thin film culture plate device is inoculated with a sample and then incubated for a sufficient period of time, staphylococci in the sample will produce colonies on the thin film device having a red color and other bacteria in the sample will produce colonies on the device having a blue color.

Claims

1. A thin film culture plate device for identifying and enumerating staphylococci in a sample containing more than one species of bacteria, comprising:

i) a self-supporting, waterproof substrate to which is adhered a rehydratable selective medium powder containing nutrients, inhibitors to promote the growth of staphylococci, and at least one gelling agent, and

ii) a transparent cover sheet having a layer of adhesive consisting essentially of a dual substrate system having an indolylglucopyranoside substrate which provides a visible first color change in the presence of beta-glucosidase and a phosphate substrate which provides a visible second color change in the presence of staphylococci and at least one gelling agent.

2. The device of claim 1 wherein the inhibitors are selected from the group consisting of colistin methanesulfonate, nalidixic acid and lithium chloride.

3. The device of claim 1 wherein the indolylglucopyranoside substrate is 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside.

4. The device of claim 1 wherein the phosphate is selected from the group consisting of 6-chloro-3-indolylphosphate and 5-bromo-6-chloro-3-indolylphosphate.

5. The device of claim 1 wherein the gelling agent is selected from group consisting of xanthan gum, locust bean gum, rhamsan gum and guar gum.