Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1995-02-27
2004-03-02
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S018000, C435S029000, C435S034000, C435S252800
Reexamination Certificate
active
06699685
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method, test medium, and novel chromogenic compounds for quantitatively identifying and differentiating general coliforms and
Escherichia coli.
2. Description of the Related Art
Currently, in microbiology, the presence of indicator organisms is widely used to determine the quality of various products. For example, in the analysis of water, food and dairy products, the presence of members of the “coliform” group as well as the presence of the bacterial species
Escherichia coli
are considered very significant quality indicators. Therefore, test methods to effective identify and enumerate these bacterial types are needed, and there is a continuing search for better, more accurate and simpler test methods in this area.
Numerous methods for determining, identifying and enumerating coliforms and
E. coli
currently exist, with varying degrees of accuracy and facility. Some test methods only indicate the presence or absence (P/A) of the organisms while some methods attempt to quantify the organisms in the test materials. Following are some of the current methods.
Violet Red Bile Agar (VRBA): This medium incorporates bile salts to inhibit non-coliforms. It also contains lactose with the pH indicator neutral red. As coliforms (especially
E. coli
) grow in the medium, the lactose is fermented with acid production and the neutral red in the area of the bacterial colony becomes a brick red color. Therefore, any colonies growing as a red color in 24-48 hours are considered to be coliforms. This medium is not easy to interpret and for
E. coli
quantification needs to be followed up by confirming tests such as brilliant green lactose broth fermentation or streaking on Eosin Methylene Blue Agar (EMBA). In spite of these shortcomings, VRBA is an approved method for testing dairy products.
The Most Probable Number (MPN) method: This method utilizes various broth (liquid) media in tubes. Samples to be tested are added in varying amounts to the broth media and after incubation, the tubes are checked for growth and gas formation. Estimates of the numbers (populations) of bacteria are determined from pre-existing tables. The method is in general use, but the results are given in a general range and therefore are riot very precise.
The Membrane Filter (MF) method: This method utilizes micropore filters through which samples are passed so that the bacteria are retained on the surface of the filter. It is used most often when bacterial populations are very small and a large sample is needed to get adequate numbers. The filter is then placed on the surface of a chosen medium, incubated and the bacterial colonies are counted and evaluated. This method is widely used and gives good results in general if combined with proper reagents and media, but is expensive and time-consuming. The MF method can be used well in combination with the new method described herein.
The Presence/Absence (P/A) test: This test, which involves the reagents O-nitrophenyl-&bgr;-D-galactopyranoside (ONPG), a &bgr;-galactosidase substrate and 4-methyl-umbelliferyl-&bgr;-D-glucuronide (MUG), a &bgr;-glucuronidase substrate, results in the determination of the presence or absence of general coliforms and
E. coli
. The test relies on the fact that generally all coliforms produce &bgr;-galactosidase, but only
E. coli
strains produce &bgr;-glucuronidase. If any coliforms are present, the broth medium turns a yellow color due to the activity of galactosidase enzyme on the ONPG material causing the release of a diffusible yellow pigment. If
E. coli
is present, the broth medium will demonstrate a blue fluorescence when irradiated with ultraviolet rays due to the breakdown of the MUG reagent with the release of the fluorogenic dye caused by the production of the glucuronidase enzyme. These reactions are very specific and allow both general coliforms and
E. coli
to be identified in a single test in a single sample. But, since both reagents produce diffusible pigments, the test has the disadvantage of not being directly quantitative for either bacterial type.
The reagent 5-bromo-4-chloro-3-indolyl-&bgr;-D-galactopyranoside (X-gal) is a known test compound for identifying coliforms. When acted on by the &bgr;-galactosidase enzyme produced by coliforms, X-gal forms an insoluble indigo blue precipitate. X-gal can be incorporated into a nutrient medium such as an agar plate, and if a sample containing coliforms is present, the coliforms will grow as indigo blue colonies. X-gal has the advantage over the compound ONPG, described above, in that it forms an insoluble precipitate, rather than a diffusible compound, thereby allowing the quantitative determination of coliforms.
Recently, a similar compound, 5-bromo-4-chloro-3-indolyl-&bgr;-D-glucuronide (X-gluc) has been developed for the identification of
E. coli
. When acted on by the &bgr;-glucuronidase enzyme produced by
E. coli
, X-gluc forms an insoluble indigo blue precipitate. X-gluc has the advantages over the compound MUG, described above, in that it forms an insoluble precipitate, rather than a diffusible compound, thereby allowing the quantitative determination of
E. coli
. Further, it does not require the use of ultraviolet light. X-gluc and its use to identify
E. coli
are described in Watkins, et al, Appl. Environ. Microbiol. 54:1874-1875 (1988). A similar compound, indoxyl-&bgr;-D-glucuronide, which also produces sharp blue colonies of
E. coli
, was described in Ley, et al, Can. J. Microbiol. 34:690-693 (1987).
X-gal and X-gluc have the disadvantage that they each contain the exact same chromogen and therefore they cannot be used together to identify and distinguish between both
E. coli
and general coliforms in a single test with a single sample. Both X-gal and X-gluc cause the formation of identically hued indigo blue colonies. A person using both reagents together would be able to quantitatively identify the total number of coliforms, the same as if X-gal were used alone, but would not be able to tell which of the colonies were
E. coli
and which were other coliforms besides
E. coli.
SUMMARY OF THE INVENTION
A method has now been found for quantitatively identifying and differentiating microorganisms having &bgr;-galactosidase but not &bgr;-glucuronidase activity and microorganisms having &bgr;-glucuronidase activity, comprising the steps of combining a chromogenic &bgr;-galactosidase substrate capable of forming an insoluble precipitate of a first color upon reacting with &bgr;-galactosidase, a chromogenic &bgr;-glucuronidase substrate capable of forming an insoluble precipitate of a second color contrasting with the first color upon reacting with &bgr;-glucuronidase, and a nutrient base medium to form a test medium, inoculating the test medium with a sample to be tested for the presence of microorganisms, incubating the test medium, examining the test medium for the presence of colonies of the first color, such colonies being colonies of microorganisms having &bgr;-galactosidase but not &bgr;-glucuronidase activity, and the presence of colonies of the second color, such colonies being colonies of microorganisms having &bgr;-glucuronidase activity, and enumerating the microorganisms having &bgr;-galactosidase but not &bgr;-glucurosidase activity and the microorganisms having &bgr;-glucuronidase activity.
The advantages of the new method include the following. First, it allows the chromogenic differentiation between general coliforms, which have &bgr;-galactosidase activity, and
E. coli
, which additionally have &bgr;-glucuronidase activity, in the same test plate with the same sample. It is also quantitative so that exact counts of the numbers of viable organisms of each type are determined. This is much more meaningful than just a presence/absence test as levels of contamination can be determined. The new method does not require any special apparatus or equipment such as a UV light source or special filter apparatus. The new method is based on enzymatic reactions rather
Ferguson Wilfred J.
Roth Jonathan N.
Lankford , Jr. Leon B.
RCR Scientific, Inc.
Roylance Abrams Berdo & Goodman L.L.P.
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