250 Modern Food Microbiology
brightly. The Colilert and ColiQuik systems employ both ONPG and MUG as sole nutrient substrates
where total coliforms are indicated by a yellow color; E. coli is indicated by MUG fluorescence.
BCIG or X-Gluc is employed in plating media for the detection of E. coli. When added at 500 ppm
to a peptone–Tergitol agar, E. coli produced a blue color in 24 hours that did not diffuse from colonies,
and did not require fluorescent light.
64 In another study, no differences were observed between results
from a three-tube standard MPN on 50 ground beef samples.
170 When used in lauryl tryptose agar at
a final concentration of 100 ppm, only 1% of 1,025 presumptively positive E. coli cultures did not
produce the blue color, whereas 5% of 583 non-E. coli colonies were false positive.
219 The plating
medium was incubated at 35
◦ C for 2 hours and then at 44.5
◦ C for 22–24 hours.
The LAPN substrate is specific for Gram-negative bacteria on the premise that aminopeptidase is
restricted to this group. The enzyme cleaves l-alanine- p-nitroanilide to yield p-nitroaniline, a yellow
compound that is read spectrophotometrically at 390 nm.
29 When used to determine Gram-negative
bacteria in meats, 10
4 –5 × 10
5 cfu was the minimum detectable number.
41 Numbers of 10
6 –10
7
cfu/cm
2 could be detected in 3 hours. The Limulus test has received more study for Gram-negative
bacteria, and because it gives results within 1 hour, the LAPN method cannot be considered to be
comparable.
The combination of MUP and ONPG has been used in HEPES buffer as a test for Clostridium
perfringens, and 164 of 333 presumptive isolates from TSC agar were positive compared to 153 by
standard identification methods, and results were obtained in 4 hours.
1 To determine the contamination
level of beef carcass swab samples, a luminescence-based phosphatase test kit was employed on 70 beef
carcass swab samples, and the results were highly correlated with APC and obtained in 10 minutes.
104
A chromogenic substrate (BCM; Biosynth, Inc.) available in a Listeria monocytogenes plating agar is
specific for this species and L. ivanovii based on its capacity to respond to phosphatidylinositol-specific
phospholipase C (PI-PLC). When using a combination of BCM with either Oxford or Palcam agars,
one group of investigators found Oxford-BCM to be 99.3; Palcam-BCM 99.2; and Oxford-Palcam
90.2% sensitive when tested on 2,000 food and environmental samples.
98
Since the botulinal neurotoxins (BoNTs) are metalloproteases that exhibit stringent substrate requirements relative to amino acid sequences, Schmidt and Stafford
180 developed fluorogenic protease
assays for types A, B, and F toxins. The synthetic peptide substrates consisted of having the P 1 and
P
3 residues substituted with 2,4-dinitrophenyl-lysine (for P 1 ) and S-(N -[4-methyl-7-dimethylaminocoumarin-3-yl]-carboxamidomethyl)-cystine (for P
3 ). When the BoNTs were added to these synthetic
substrates, fluorescence increased over time and results were obtained in 1 or 2 minutes with BoNT
concentrations of 60 ng/ml. The three BoNTs cleaved the substrates at the same locations, and the
substrates were selective.
IMMUNOLOGICAL METHODS
Serotyping
Serotyping is most widely applied to Gram-negative enteric bacterial pathogens such as Salmonella
and Escherichia. Among Gram positives, serotyping is important for the genus Listeria. The gist of a
typical serotyping scheme is the use of specific antibodies (antiserum) to identify homologous antigens.
In the case of many foodborne pathogens, the antigens are particulate, and agglutination methods are
employed. For soluble antigens such as toxins, methods such as gel diffusion may be used. The O and
H antigens of enteric bacteria are illustrated in Figure 11–2. The serologic classification of salmonellae
was begun by Kauffmann in the early 1940s.
106 He defined and numbered the first 20 O groups. This
brightly. The Colilert and ColiQuik systems employ both ONPG and MUG as sole nutrient substrates
where total coliforms are indicated by a yellow color; E. coli is indicated by MUG fluorescence.
BCIG or X-Gluc is employed in plating media for the detection of E. coli. When added at 500 ppm
to a peptone–Tergitol agar, E. coli produced a blue color in 24 hours that did not diffuse from colonies,
and did not require fluorescent light.
64 In another study, no differences were observed between results
from a three-tube standard MPN on 50 ground beef samples.
170 When used in lauryl tryptose agar at
a final concentration of 100 ppm, only 1% of 1,025 presumptively positive E. coli cultures did not
produce the blue color, whereas 5% of 583 non-E. coli colonies were false positive.
219 The plating
medium was incubated at 35
◦ C for 2 hours and then at 44.5
◦ C for 22–24 hours.
The LAPN substrate is specific for Gram-negative bacteria on the premise that aminopeptidase is
restricted to this group. The enzyme cleaves l-alanine- p-nitroanilide to yield p-nitroaniline, a yellow
compound that is read spectrophotometrically at 390 nm.
29 When used to determine Gram-negative
bacteria in meats, 10
4 –5 × 10
5 cfu was the minimum detectable number.
41 Numbers of 10
6 –10
7
cfu/cm
2 could be detected in 3 hours. The Limulus test has received more study for Gram-negative
bacteria, and because it gives results within 1 hour, the LAPN method cannot be considered to be
comparable.
The combination of MUP and ONPG has been used in HEPES buffer as a test for Clostridium
perfringens, and 164 of 333 presumptive isolates from TSC agar were positive compared to 153 by
standard identification methods, and results were obtained in 4 hours.
1 To determine the contamination
level of beef carcass swab samples, a luminescence-based phosphatase test kit was employed on 70 beef
carcass swab samples, and the results were highly correlated with APC and obtained in 10 minutes.
104
A chromogenic substrate (BCM; Biosynth, Inc.) available in a Listeria monocytogenes plating agar is
specific for this species and L. ivanovii based on its capacity to respond to phosphatidylinositol-specific
phospholipase C (PI-PLC). When using a combination of BCM with either Oxford or Palcam agars,
one group of investigators found Oxford-BCM to be 99.3; Palcam-BCM 99.2; and Oxford-Palcam
90.2% sensitive when tested on 2,000 food and environmental samples.
98
Since the botulinal neurotoxins (BoNTs) are metalloproteases that exhibit stringent substrate requirements relative to amino acid sequences, Schmidt and Stafford
180 developed fluorogenic protease
assays for types A, B, and F toxins. The synthetic peptide substrates consisted of having the P 1 and
P
3 residues substituted with 2,4-dinitrophenyl-lysine (for P 1 ) and S-(N -[4-methyl-7-dimethylaminocoumarin-3-yl]-carboxamidomethyl)-cystine (for P
3 ). When the BoNTs were added to these synthetic
substrates, fluorescence increased over time and results were obtained in 1 or 2 minutes with BoNT
concentrations of 60 ng/ml. The three BoNTs cleaved the substrates at the same locations, and the
substrates were selective.
IMMUNOLOGICAL METHODS
Serotyping
Serotyping is most widely applied to Gram-negative enteric bacterial pathogens such as Salmonella
and Escherichia. Among Gram positives, serotyping is important for the genus Listeria. The gist of a
typical serotyping scheme is the use of specific antibodies (antiserum) to identify homologous antigens.
In the case of many foodborne pathogens, the antigens are particulate, and agglutination methods are
employed. For soluble antigens such as toxins, methods such as gel diffusion may be used. The O and
H antigens of enteric bacteria are illustrated in Figure 11–2. The serologic classification of salmonellae
was begun by Kauffmann in the early 1940s.
106 He defined and numbered the first 20 O groups. This