color, visualized under long-wave ultraviolet
light optically or via instrument, is propor-
tional to the total E. coli/mL in the sample.
Based on time to positive, a comparison
chart provides the corresponding E. coli
count for the sample. Concentration and
detection times are:
E. coliConcentration Detection Time
9.9× 106 CFU/mL 2 hours
100 CFU/mL 10 hours
Further incubation of samples that are neg-
ative at 12 hours provides a presence/absence
determination after 24 hours. This technique
permits sampling at a remote site and return
to a laboratory for analysis. The major limita-
tion appears to be that not all of the E. coli
bacteria react in the presence of MUG.
Micro ID and Minitek
Micro ID is a self-contained identification
unit containing reagent-impregnated paper
discs for biochemical testing for the differen-
tiation of Enterobacteriaceae in approxi-
mately 4 hours. This technique has provided
reliable results. The Minitek system is
another miniaturized test kit for the identifi-
cation of Enterobacteriaceae. This kit also
utilizes reagent-impregnated paper discs
requiring 24 hours of incubation. It is consid-
ered to be accurate and versatile. The Ana-
lytab Products, Inc. (API) strip is the most
commonly used identification unit.
DNA Hybridization and Colorimetric
Detection
This assay methodology combines DNA
hybridization technology with nonradioac-
tive labeling and colorimetric detection. With
the appropriate specific DNA probes, enrich-
ment, and sample preparation procedures for
a particular organism, this basic assay can be
applied to the analysis of a wide variety of
microbes. The assay can be completed in
approximately 2.5 to 3 hours after 2 days of
broth culture enrichment of the sample.
An application of this principle is a colori-
metric assay, which employs synthetic
oligonucleotide DNA probes against riboso-
mal RNA (rRNA) of the target organism.
This approach offers increased sensitivity
because rRNA, as an integral part of the
bacterial ribosome, is present in multiple
copies (1,000 to 10,000) per cell. The number
of ribosomes present per cell is dependent on
the growth state of the bacterial culture.Polymerase Chain Reaction (PCR)
This technique detects low levels of
pathogens found in food products. PCR
amplifies very low DNA levels (as low as one
molecule) or detectable levels of target DNA
(approximately 10^6 ) through a series of
DNA hybridization reactions and thermocy-
cling. PCR products are detected by various
methods, such as gel electrophoreses, calori-
metric, or chemiluminescent assays. Its util-
ity is limited in food analyses, due to the
complexity of the procedure and certain
equipment requirements (Phebus and Fung,
1994).Biosensors
Biosensors similar to pregnancy test kits
are being developed and evaluated for rapid,
reliable, and inexpensive identification and
quantification of pathogenic microorgan-
isms as well as for biosafety and biosecurity.
The bioanalytical microsystem, fabricated
using nanotechnology, contains a microflu-
idic biosensor with the desired characteris-
tics of the black box type of pathogen
sensor. Furthermore, lateral flow assays that
are being incorporated to detect pathogens
are based on antibodies that detect pathogens
with a 10- to 20-minute assay (Baeumner,
2004). Baeumner (2004) developed a lateral
flow universal biosensor that can be made