Appeal No. 1999-1206 Page 6
Application No. 08/394,608
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 . . .
The present specification describes a number of chromogenic substrates of β-
galactosidase3 and β-gucuronidase,4 each of which forms an insoluble magenta
precipitate upon enzymatic hydrolysis, readily distinguishable from the insoluble indigo
blue precipitate produced upon enzymatic hydrolysis of X-gal or X-gluc. Pages 6 and
10-16. Thus, colonies of E. coli (indigo blue) are easily differentiated from colonies of
general coliforms (magenta or purple) when both are grown on solidified culture
medium containing one of the magenta precipitate-forming β-galactosidase substrates,
e.g., 6-chloroindolyl-β-D-galactoside, and an indigo blue precipitate-forming β-
gucuronidase substrate, e.g., X-gluc. Similarly, colonies of E. coli will be magenta,
while colonies of general coliforms will be indigo blue, when grown on a medium
containing one of the magenta precipitate-forming β-glucuronidase substrates, e.g., 6-
chloroindolyl-β-D-glucuronide, and an indigo blue precipitate-forming β-galactosidase
3 E.g., 6-chloroindolyl-β-D-galactoside, 4,6-dichloroindolyl-β-D-galactoside, 6,7-
dichloroindolyl-β-D-galactoside and 4,6,7-trichloroindolyl-β-D-galactoside.
4 E.g., 6-chloroindolyl-β-D-glucuronide, 4,6-dichloroindolyl-β-D-glucuronide, 6,7-
dichloroindolyl-β-D-glucuronide and 4,6,7-trichloroindolyl-β-D-glucuronide.
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