Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2002-03-18
2003-09-09
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S034000, C435S038000, C435S039000, C435S040000, C435S253600, C435S287900
Reexamination Certificate
active
06617149
ABSTRACT:
BACKGROUND OF THE INVENTION
Escherichia coli
0157:H7 has been recognized as an important human pathogen. Studies have shown that it is principally transmitted through food,
Escherichia coli
0157:H7: Epidemiology, Pathogenesis, and Methods for Detection in Food, Nisha V. Padhye and Michael P. Doyle—Journal of Food Protection, Vol. 55, No. 7, Pages 555-565 (July 1992). There is thus a need for a rapid diagnostic test for the presence of
Escherichia coli
0157:H7 in food in order to prevent the spread of
Escherichia coli
0157:H7 through the food supply.
Pradhye and Doyle, supra, survey methods of detection of
Escherichia coli
0157:H7. A stable characteristic of
Escherichia coli
0157:H7 is that it will not ferment sorbitol within 24 hours whereas other strains of
Escherichia coli
will produce fermentation in sorbitol under incubation temperatures within 24 hours, and this characteristic has been used in processes for the isolation of
Escherichia coli
0157:H7 from other enterics. Since there are microorganisms other than
Escherichia coli
0157:H7 that do not ferment sorbitol, including some strains of
Escherichia coli,
this characteristic is not sufficiently specific to serve as an identifying test for
Escherichia coli
0157:H7.
Anita J. Okrend, Bonnie E. Rose and Charles P. Lattuada describe an improved plating medium in Use of 5-Bromo-4-Chloro-3-Indoxyl-Beta-D-Glucuronide in MacConkey Sorbitol Agar in the Isolation of
Escherichia coli
0157:H7 from Ground Beef, Journal of Food Protection, Vol. 53, No.11, Pages 941-943 (November 1990). This article describes a plating medium in which 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide acid cyclohexylammonium salt was dissolved in ethanol and the solution added to MacConkey Sorbitol Agar. Since approximately 97% of all
Escherichia coli
are beta-glucuronidase positive, but
Escherichia coli
0157:H7 is beta-glucuronidase negative, this medium responds to the presence of
Escherichia coli
0157:H7 by isolating white colonies rather than isolating blue colonies resulting from beta-glucuronidase positive microorganisms.
The process of isolating and detecting the presence of
Escherichia coli
0157:H7 in a test sample by means of the processes described above, requires inoculation of the plating medium with the test sample, incubating the inoculated plating medium for a period of time, usually over night, and examining the surface of the plating medium to locate colonies of microorganisms in the incubated plating medium. Identification of
Escherichia coli
0157:H7 is determined by the shape of the colony, size of the colony and color of the colony in the plating medium.
The color of the colony in the plating medium is a characteristic of the particular medium. U.S. Pat. No. 5,464,755 of Barry Bochner entitled Microbiological Medium and Method of Assay for Bacteria describes a plating medium adapted to produce colonies in three different colors. The examination of incubated plating media under a microscope is an exacting and time consuming task, and in the plating methods of the prior art, a positive response results in a presumptive identification of
Escherichia coli
0157:H7 which must be verified by other testing methods. In short, an identification of
Escherichia coli
0157:H7 cannot be made by prior art methods in less than about one day and at substantial expense due to the labor required to analyze the plating medium and the cost of the plating materials.
In an article published in the Journal of Microbiology—Volume 39 (1993) at pages 133-158, by P. M. Zadik, P. A. Chapman and C. A. Siddons, entitled Use of Tellurite for the Selection of Verocytotoxigenic
Escherichia coli
0157:H7, experiments are described in which plating media containing mixtures of MacConkey sorbitol and potassium tellurite are subjected to mixed cultures of microorganisms. It was found that such plating media can be effective to reduce the growth of other strains of
Escherichia coli
than
Escherichia coli
0157:H7 without materially affecting the growth of
Escherichia coli
0157:H7. Further, such plating media were found to suppress the growth of other important enteric microorganisms, excepting Shigella.
STATEMENT OF INVENTION
It is an object of the present invention to provide a method of isolating and presumptively identifying
Escherichia coli
0157:H7 which utilizes solid plating media and is more reliable than plating media methods known to the art, that is, makes a positive presumptive identification of
Escherichia coli
0157:H7 and reduces the percentage of false positive determinations from that of prior art plating methods.
It is a further object of the present invention to provide a method of isolating and presumptively identifying
Escherichia coli
0157:H7 which utilizes a solid plating medium and achieves its results in a significantly shorter time than processes of the prior art.
It is a further object of the present invention to provide a method of isolating and presumptively identifying
Escherichia coli
0157:H7 which utilizes a solid plating medium and achieves its results at a significantly lower cost than processes of the prior art.
It is also an object of the present invention to provide plating media adapted for use in the methods described above.
The present invention comprises a solid plating medium which utilizes three mechanisms to produce an indication of the presence of
Escherichia coli
0157:H7 in a test sample. First, the plating medium contains one or more carbohydrates which are not fermented by
Escherichia coli
0157:H7 but may be fermented by other microorganisms including other strains of
Escherichia coli.
If a microorganism is present which ferments the carbohydrate, the medium is selected to change to a first color and indicates the presence of a microorganism other than
Escherichia coli
0157:H7. Second, the plating medium contains a chromogen responsive to the presence of beta-galactosidase, and third, the plating medium contains ingredients for restricting the growth of microorganisms other than
Escherichia coli
0157:H7.
More specifically, a solid plating medium for the presumptive identification of
Escherichia coli
0157:H7 according to the present invention comprises: (1) at least one ingredient for differentiating
Escherichia coli
cells under incubation which is not fermented by
Escherichia coli
0157:H7 but is fermented by other microorganisms including other strains of
Escherichia coli,
and if fermented results in a change in the pH of the medium; (2) a pH indicator dye which changes to a first color when the pH of the medium changes, (3) an ingredient for inhibiting the growth of gram positive microorganisms under incubation; (4) an ingredient for inhibiting the growth of Proteus sp. under incubation; (5) an ingredient for inhibiting the growth of strains of
Escherichia coli
other than
Escherichia coli
0157:H7 under incubation; and (6) a chromogenic substrate that upon reacting to beta-galactosidase forms a second color that can contrast with the first color and combine with the first color to form a third color which contrasts with the first and second colors, however
Escherichia coli
0157:H7 retains the second color.
The method of presumptive identification of
Escherichia coli
0157:H7 according to the present invention comprises inoculating a mass of the plating medium described above with the sample under test, thereafter incubating the mass of inoculated plating medium at a temperature between 30 degrees and 40 degrees Celsius for a sufficient period of time to produce microorganism colonies in the mass of plating medium, and thereafter examining the surface of the mass of plating medium for colonies of the second color. Emergence of the first or the third color, which is a blend of the first and second colors, indicates the presence of microorganisms other than
Escherichia coli
0157:H7.
REFERENCES:
patent: 5393662 (1995-02-01), Roth et al.
patent: 5418167 (1995-05-01), Matner et al.
patent: 5541082 (1996-07-01), Botchner
patent: 6087156 (2000-07-01), Restaino
Burmeister Marshall A.
R&F Laboratories, Inc.
Tate Christopher R.
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