The Relationship of Microorganisms to Sanitation 65closely associated with virulence. Using this
method, the virulence ofL. monocytogenes
may be determined.
Fraser Enrichment Broth/Modified
Oxford Agar
This method was developed for Listeria
detection using Fraser enrichment broth com-
bined with modified Oxford agar for motility
enrichment. The Listeria organisms are
enriched in Fraser broth and held at 30ºC for
24 hours, and 1 mL of the enrichment broth
is placed in the Fraser broth in the left arm of
a U-shaped tube. The Fraser broth selectively
isolates and promotes Listeriagrowth and
precludes the growth of nonmotile microor-
ganisms. The microbes migrate through the
modified Oxford agar and arrive as a pure
culture in the second branch of the Fraser
broth. This becomes the second enrichment
necessary for the identification ofListeria.An
easier indication that Listeriaorganisms are
present is the formation of a black precipitate
as the bacteria move through the modified
Oxford agar. When turbidity develops, a sam-
ple can be taken for DNA probe analysis to
confirm the presence ofListeria. The second
enrichment step requires 12 to 24 hours.
Crystal Violet Test
The retention of crystal violet by Y. ente-
rocoliticacorrelates with virulence. Most
Y. enterocoliticastrains isolated from meat
and poultry are avirulent. Thus, this rapid
test allows samples with virulent strains to
be identified and discarded quickly.
Methyl Umbelliferyl Glucuronide Test
Methyl umbelliferyl glucuronide (MUG) is
split by the enzyme glucuronidase produced
by most E. coliand other microbes, such as
Salmonella. When split, MUG becomes fluo-
rescent under ultraviolet illumination of a
specific wavelength and permits rapid identi-
fication in tubed media or on spread plates for
enumeration.Assay for E. coli
At the time of this writing, several tech-
niques are being evaluated for the rapid
identification of microorganisms. Many tech-
niques have not been available long enough to
establish track records for their efficacy or to
achieve AOAC approval. Although several
methods are available, most require 24 to 48
hours for incubation of the microorganisms
and may need additional testing to confirm
the presence of E. coli. Many commercial
assays for the detection ofE. coliincorpo-
rate membrane filtration technology, and oth-
ers employ a reagent/sample mixture that is
incubated for 24 to 48 hours to obtain a pres-
ence/absence result of total E. colicontami-
nation.
A new assay for a rapid, inexpensive deter-
mination ofE. coliconcentrations in aqueous
environments has been developed which is
called the IME.Test™-EC KOUNT Assayer.
This assayer uses a reagent mixture contain-
ing an indicator compound that provides a
colorimetric (bright blue) indication ofE. coli
concentration in a water-based sample, predi-
cated on cleavage by the beta galactosidase
enzyme specific to E. coli. This assay provides
a simple method for quantifying the concen-
tration of viable E. coliin an aqueous sample
in 2 to 10 hours.
The procedure involves filling a snapping
cup with a sample and introducing it to a
vacuum-sealed test ampoule by snapping off
the ampoule’s sealed tip in one of the holes
in the bottom of the cup. The ampoule auto-
matically fills with the aqueous sample. Then
the sample is incubated at 35ºC and moni-
tored for the production of a blue fluores-
cence resulting from enzymatic cleavage of
the indicator molecule, MUG. The time
required for the production of a bright blue