Appeal No. 1999-1206 Page 5
Application No. 08/394,608
especially dairy products. According to the specification, A[n]umerous methods for
determining, identifying and enumerating coliforms and E. coli [are known],@ but none
can be used to both quantify, and distinguish between, general coliforms and E. coli, in
a single test on a single sample. Specification, page 2. For example (Id., pages 3-4)
The Presence/Absence (P/A) test . . . which involves the reagents O-
nitrophenyl-β-D-galactopyranoside (ONPG), a β-galactosidase substrate
and 4-methyl-umbelliferyl-β-D-glucuronide (MUG), a β-D-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 β-galactosidase, but only E. coli strains produce β-
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 . . . glucuronidase . . .
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.
[t]he reagent 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) is
a known test compound for identifying coliforms. When acted on by the β-
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
. . . ONPG . . . in that it forms an insoluble precipitate, rather than a
diffusible compound, thereby allowing the quantitative determination of
coliforms.
[a] similar compound, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc)
has been developed for the identification of E. coli. When acted on by the
β-glucuronidase enzyme produced by E. coli, X-gluc forms an insoluble
indigo blue precipitate. X-gluc has the advantages over . . . MUG . . . in
that it forms an insoluble precipitate, rather than a diffusible compound,
thereby allowing the quantitative determination of E. coli [and] it does not
require the use of ultraviolet light.
X-gal and X-gluc have the disadvantage that they each contain the exact
same chromogen and therefore cannot be used together to identify and
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