Chemical, Biological, and Physical Methods
249
5. l-alanine- p-nitroanilide (LAPN),
6. 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (sodium or cyclohexylammonium salt—
variously BCIG, X-Gluc, X-GlcA),
7. 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal), and
8. indoxyl-β-d-glucuronide (IBDG).
These substrates are employed in various ways in plating media, MPN broths, and for membrane
filtration methods. MUG is the most widely used of the fluorogenic substrates, and it is hydrolyzed by
β-d-glucuronidase (GUD) to release the fluorescent 4-methylumbelliferyl moiety, which is detected
with long-wave ultraviolet light. The value of MUG is that E. coli is the primary producer of GUD,
and this substrate has found wide use as a differential agent in media and methods for this organism.
A few salmonellae and shigellae are also GUD positive, as are some corynebacteria.
The first to employ MUG for E. coli detection were Feng and Hartman,
57 who incorporated it
into lauryl tryptose broth (LTB) and other coliform-selective media and found that in LTB-MUG, one
E. coli cell could be detected in 20 hours. Because about 10
7 E. coli cells are needed to produce enough
GUD to provide detectable MUG results, the time for results depends on the initial number of cells.
Although most positive reactions occurred in 4 hours, some weak GUD-positive strains required up
to 16 hours for reaction. An important aspect of this method is the occurrence of fluorescence before
gas production from lactose. Employing the Feng–Hartman method, another group examined 1,020
specimens by a three-tube MPN and were able to detect more E. coli-positive samples than with a
conventional MPN.
213 The greater effectiveness of LTB-MUG resulted because some E. coli strains
are anaerogenic. No false-negative results were obtained. In an evaluation of MUG added to lauryl
sulfate broth (LSB), a 94.8% agreement was obtained on 270 product samples with 4.8% false positives
but no false negatives with LSB-MUG.
172 Oysters contain endogenous glucuronidase, but an E. coli
(EC) broth–MUG method was employed successfully in one study where 102 of 103 fluorescing tubes
were positive for E. coli.
113 A 20-minute tube procedure employing MUG was applied to 682 E. coli
cultures, and 630 (92.4%) were positive.
207 Of 188 0157 strains of E. coli, 166 were MUG negative and
all were positive for the vero toxin. By use of this 20-minute method, MUG-negative E. coli are very
likely to be verotoxigenic.
207 In a study of molluscan shellfish, EC-MUG broth with MUG employed
at 50 ppm, 95% of E. coli, were positive with 11% being false negative.
169 When compared to the
Association of Official Analytical Chemists (AOAC) method for E. coli, LST-MUG (lauryl sulfate
tryptose) was found to be equivalent for one product and better than AOAC for some others tested,
163
whereas in another, LST-MUG was found to be comparable to the AOAC MPN method.
164
MUGal has received limited study as a fluorogenic substrate, but in one study it was used to detect
fecal coliforms in water by use of a membrane filter method where as few as 1 cfu/100 ml of water
could be detected in 6 hours.
16 It has also been used to differentiate species of enterococci.
128 The
method employed dyed starch along with the substrate, both of which were added to a medium
selective for enterococci. By observing for starch hydrolysis and fluorescence, 86% of enterococci
from environmental samples were correctly differentiated. ONPG is a colorithmetric substrate that is
specific for coliforms. The substrate is hydrolyzed by β-galactosidase to produce a yellow color that
can be quantitated at 420 nm. To determine E. coli in water, the organisms are collected on a 0.45-µm
membrane and incubated in EC medium for 1 hour followed by the addition of filter-sterilized ONPG.
Incubation is continued at 45.5
◦ C until color develops that can be read at 420 nm.
218 The sensitivity
of ONPG is similar to that of MUG, with about 10
7 cells required to produce measurable hydrolysis.
ONPG is employed in a modification of the classical presence–absence method for coliforms in
water.
49 By this modified method, tubes that contain coliforms become yellow. To detect E. coli, each
yellow tube is viewed with a hand-held fluorescent lamp (366 nm); those that contain E. coli fluoresce
249
5. l-alanine- p-nitroanilide (LAPN),
6. 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (sodium or cyclohexylammonium salt—
variously BCIG, X-Gluc, X-GlcA),
7. 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal), and
8. indoxyl-β-d-glucuronide (IBDG).
These substrates are employed in various ways in plating media, MPN broths, and for membrane
filtration methods. MUG is the most widely used of the fluorogenic substrates, and it is hydrolyzed by
β-d-glucuronidase (GUD) to release the fluorescent 4-methylumbelliferyl moiety, which is detected
with long-wave ultraviolet light. The value of MUG is that E. coli is the primary producer of GUD,
and this substrate has found wide use as a differential agent in media and methods for this organism.
A few salmonellae and shigellae are also GUD positive, as are some corynebacteria.
The first to employ MUG for E. coli detection were Feng and Hartman,
57 who incorporated it
into lauryl tryptose broth (LTB) and other coliform-selective media and found that in LTB-MUG, one
E. coli cell could be detected in 20 hours. Because about 10
7 E. coli cells are needed to produce enough
GUD to provide detectable MUG results, the time for results depends on the initial number of cells.
Although most positive reactions occurred in 4 hours, some weak GUD-positive strains required up
to 16 hours for reaction. An important aspect of this method is the occurrence of fluorescence before
gas production from lactose. Employing the Feng–Hartman method, another group examined 1,020
specimens by a three-tube MPN and were able to detect more E. coli-positive samples than with a
conventional MPN.
213 The greater effectiveness of LTB-MUG resulted because some E. coli strains
are anaerogenic. No false-negative results were obtained. In an evaluation of MUG added to lauryl
sulfate broth (LSB), a 94.8% agreement was obtained on 270 product samples with 4.8% false positives
but no false negatives with LSB-MUG.
172 Oysters contain endogenous glucuronidase, but an E. coli
(EC) broth–MUG method was employed successfully in one study where 102 of 103 fluorescing tubes
were positive for E. coli.
113 A 20-minute tube procedure employing MUG was applied to 682 E. coli
cultures, and 630 (92.4%) were positive.
207 Of 188 0157 strains of E. coli, 166 were MUG negative and
all were positive for the vero toxin. By use of this 20-minute method, MUG-negative E. coli are very
likely to be verotoxigenic.
207 In a study of molluscan shellfish, EC-MUG broth with MUG employed
at 50 ppm, 95% of E. coli, were positive with 11% being false negative.
169 When compared to the
Association of Official Analytical Chemists (AOAC) method for E. coli, LST-MUG (lauryl sulfate
tryptose) was found to be equivalent for one product and better than AOAC for some others tested,
163
whereas in another, LST-MUG was found to be comparable to the AOAC MPN method.
164
MUGal has received limited study as a fluorogenic substrate, but in one study it was used to detect
fecal coliforms in water by use of a membrane filter method where as few as 1 cfu/100 ml of water
could be detected in 6 hours.
16 It has also been used to differentiate species of enterococci.
128 The
method employed dyed starch along with the substrate, both of which were added to a medium
selective for enterococci. By observing for starch hydrolysis and fluorescence, 86% of enterococci
from environmental samples were correctly differentiated. ONPG is a colorithmetric substrate that is
specific for coliforms. The substrate is hydrolyzed by β-galactosidase to produce a yellow color that
can be quantitated at 420 nm. To determine E. coli in water, the organisms are collected on a 0.45-µm
membrane and incubated in EC medium for 1 hour followed by the addition of filter-sterilized ONPG.
Incubation is continued at 45.5
◦ C until color develops that can be read at 420 nm.
218 The sensitivity
of ONPG is similar to that of MUG, with about 10
7 cells required to produce measurable hydrolysis.
ONPG is employed in a modification of the classical presence–absence method for coliforms in
water.
49 By this modified method, tubes that contain coliforms become yellow. To detect E. coli, each
yellow tube is viewed with a hand-held fluorescent lamp (366 nm); those that contain E. coli fluoresce