1. food and drink

883
REVIEW / SYNTHÈSE
A review of conventional detection and enumeration
methods for pathogenic bacteria in food
Kiev S. Gracias and John L. McKillip
Abstract: With continued development of novel molecular-based technologies for rapid, high-throughput detection of
foodborne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration,
selective isolation of bacteria on commercial media, and immunoassays seems tenuous. In fact, a number of unique
approaches and variations on existing techniques are currently on the market or are being implemented that offer ease
of use, reliability, and low cost compared with molecular tools. Approaches that enhance recovery of sublethally injured
bacteria, differentiation among species using fluorogenics or chromogenics, dry plate culturing, differentiation among
bacteria of interest using biochemical profiling, enumeration using impedence technology, techniques to confirm the
presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring
are summarized here and discussed in relation to their specific advantages or disadvantages when implemented in a
food microbiology setting.
Key words: food pathogen, detection, enumeration methods, food safety.
Résumé : Avec le développement continuel des nouvelles technologies moléculaires permettant la détection rapide et à
haut débit de bactéries alimentaires pathogènes, l’avenir des méthodes microbiologiques conventionnelles comme
l’énumération des cellules viables, l’isolation sélective de bactéries sur milieu commercial, et les essais immunologiques,
semble trouble. En fait, un certain nombre d’approches uniques et de variations de techniques existantes sont actuellement
sur le marché ou sont mises en application afin d’offrir une facilité d’usage, une fiabilité et un coût réduit par rapport
aux outils moléculaires. Cet article résume des approches augmentant la récupération de bactéries endommagées sous le
seuil de la létalité, qui distinguent les espèces d’intérêt à l’aide de profilage biochimique, qui permettent l’énumération
en utilisant la technologie d’impédance, des techniques qui confirment la présence de pathogènes à l’aide de méthodes
immunologiques et des applications de la bioluminescence pour le suivi hygiénique. Nous discutons des avantages et
des désavantages spécifiques à chacune des approches lorsque appliquées dans un contexte de microbiologie alimentaire.
Mots clés : pathogènes alimentaires, détection, méthodes d’énumération, sécurité des produits alimentaires.
[Traduit par la Rédaction]
Gracias and McKillip
Introduction
Of the more than 200 known diseases transmitted through
food (in the United States) annually, approximately 8000
deaths are recorded from over 75 million reported cases.
Surveillance and detection of food-associated pathogenic
bacteria are complicated by emerging strains not routinely
encountered and the unclear route of transmission of many
bacteria (Mead et al. 1999). With regard to hygiene monitoring, Hazard Analysis Critical Control Point (HACCP) programs are designed to monitor critical control points for
potential contamination during food processing. Rigorous
adherence to sanitary practices in a food-processing environReceived 8 March 2004. Revision received 11 June 2004.
Accepted 30 June 2004. Published on the NRC Research
Press Web site at http://cjm.nrc.ca on 6 January 2005.
K.S. Gracias and J.L. McKillip.1 Department of Biology,
Ball State University, Muncie, IN 47306, USA.
1
Corresponding author (e-mail: [email protected]).
Can. J. Microbiol. 50: 883–890 (2004)
890
ment necessitates rapid assay results (Marriott 1999). High
throughput screening of an increasingly diverse array of
both fresh and processed foods requires that pre- and
postharvest food safety practices be dynamic, sensitive, specific, as well as versatile and cost-effective for large sample
numbers. No single assay or method will address all of these
criteria optimally, particularly culture-based techniques.
However, these methods do offer certain advantages for
monitoring microbiological quality of foods, including costeffectiveness, ease of use, and familiarity among users, as
described herein.
The last several years have revealed a new generation of
innovative methods and technologies for pathogen enumeration
and detection in contaminated foods. Molecular approaches
that entail near-time or real-time bacterial detection are rapid,
sensitive, and specific for the target pathogen (Smith et al.
2000; Feng 2001; Rijpens and Herman 2002) or one of the
varied virulence determinants of that pathogen (Fig. 1). The
reliability, cost, and novelty of some of these methods may
still limit their adoption and scale-up practicality in a food
doi: 10.1139/W04-080
© 2004 NRC Canada
884
Fig. 1. Targets or features of interest on a general foodassociated pathogenic or spoilage bacterium that are exploited
for selective isolation, differential media design, or
immunoassays. A few representative virulence determinants are
indicated.
processing scenario, obligating many laboratories to rely more
on traditional microbiological methods for quality control/quality
assurance of foods (Jaykus 2003). These methods are essentially designed around the recovery and (or) enumeration of
viable bacteria in the food matrix. Familiarity and acceptance among food processors with methods such as the standard plate count or selective and differential media for
bacterial isolation and detection as well as commercially
available biochemical profiling systems for identification of
specific food isolates continue to serve as the basis in many
food microbiology laboratories that lack the necessary resources to utilize some of the emerging molecular-based
technologies. Novel means of detecting and enumerating
bacteria of interest are continually being reported, and although the majority entail molecular biological approaches,
many fall into the category of conventional techniques. This
review will present a comparative overview of many conventional approaches in food microbiology for enumeration
and (or) detection of bacteria in foods.
Background on traditional microbiological
methods in food
Standard/aerobic plate counts and relevant variations
Detection of viable bacteria is traditionally performed by
implementing a means of culturing/measuring growth of individual microorganisms. Hundreds of commonly used bacteriological media in the food industry are met with unique
ways of applying them to best monitor for spoilage and (or)
pathogenic bacteria in food (Harrigan 1998). The use of routine nonselective media such as trypticase soy agar or stan-
Can. J. Microbiol. Vol. 50, 2004
Fig. 2. Agar overlay schematic that allows for recovery of
sublethally injured bacteria from food samples using a
nonselective basal layer (pour plated). Incubation/recovery in this
medium for a few hours followed by overlay with the selective
medium of choice increases the sensitivity of a viable cell count
compared with plating directly onto the selective medium.
dard methods agar, known as the aerobic plate count (APC)
or standard plate count (SPC), is worthy of discussion in
terms of the many offshoot methods that have been designed
to improve upon it.
Increased sensitivity of SPC/APC has been achieved using
a selective agar overlay approach designed to recover a
larger proportion of bacteria from food matrices (compared
with straight plating onto selective media) following
sublethal stressors experienced during processing steps, such
as heat, cold, acid, or osmotic shock (Harrigan 1998; Speck
et al. 1975). Detecting these sublethally damaged bacteria is
of vital significance in the food industry, since the selective
agar overlay technique aids in the resuscitation of bacterial
cells from food, which may still be viable and pose a threat
to human health as a result. In this approach, the inoculum is
pour-plated with a basal layer of trypticase soy agar, or comparable nonselective medium, and incubated for 1–4 h. During
this time, sublethally injured bacteria are given a chance to recover and begin growing prior to an overlay with the appropriate selective medium (Hurst 1977; Ray 1986) (Fig. 2). This
approach has been demonstrated predominately with enteric
bacteria such as Escherichia coli and Salmonella typhimurium,
although it has shown promise when modified to enumerate
Gram-positive pathogens such as Listeria monocytogenes,
Bacillus cereus, and enterotoxigenic Staphylococcus aureus
(Golden et al. 1988; Kang and Siragusa 1999, 2001; Kang
and Fung 1999, 2000; Hara-Kudo et al. 2000; Hajmeer et al.
2001; McKillip 2001; Wu et al. 2001; Sandel et al. 2003).
Slight variations on the resuscitation technique have been
employed, including a membrane or solid-support-based
transfer of E. coli O157:H7 from the trypticase soy agar recovery medium onto sorbitol MacConkey agar, which has
been shown to improve recovery of sublethally injured cells
by a factor of 3 log10 (Blackburn and McCarthy 2000).
Pre-enrichment of the suspect food sample in a
nonselective or selective broth culture is another strategy to
increase numbers of injured but viable target bacteria to
dectectable levels (Zhao and Doyle 2001), although depending on the food product being analyzed, the enrichment
step(s) may require an additional 8–24 h before enumeration
or detection can be completed. In the above study applied to
heat-injured pathogens, enrichment in the appropriate selective media was essential to obtain densities of at least 4 log10
to confirm the presence of the pathogen using immunoassay.
© 2004 NRC Canada
Gracias and McKillip
Therefore, despite the advantages of these familiar culture
methods in detecting viable bacteria, such as ease of use and
low cost, assay sensitivity is still relatively low compared
with alternative methods (e.g., molecular-based approaches
such as PCR). In PCR, the enrichment step is crucial in increasing bacterial cell numbers, prior to nucleic acid extraction
(thereby obtaining higher yield and good quality DNA) and
primer-specific amplification, in the highly sensitive and
quick detection of bacterial contaminants in food matrices. It
is important to note that an SPC is done both before and after enrichment to enumerate the bacteria in the food sample
prior to PCR (McKillip et al. 2002; Nakano et al. 2004).
Therefore, the time it takes to obtain data prevents the SPC/APC
technique from being considered among the “rapid methods”.
In many cases, the food microbiologist may find it necessary to implement a bacterial concentration or immobilization step immediately prior to plating to sequester cells
within an otherwise polluted and heterogenous food matrix.
Several general approaches have been described for selective
removal of cells from a fluid food system, and each offers
the potential to greatly increase detection and differentiation
of target bacteria prior to enumeration (Sharpe 1997). Immunomagnetic separation (IMS) is one such technique that employs antibodies linked to magnetic beads that are placed in
the food slurry and allowed to interact with specific epitopes
on the bacterial cell surface (Fig. 1). The material is then
exposed to a magnetic field that essentially pulls the bacteria
out of suspension for plating or molecular-based (e.g., PCR)
detection/enumeration (Jinneman et al. 1995; Tomoyasu
1998). One such commercial application using IMS,
DynabeadsTM (Dynal Botech, Oslo, Norway), has proven effective for isolation of a variety of food pathogens, including L. monocytogenes, E. coli O157:H7, and Salmonella spp.
(Hsih and Tsen 2001; Hudson et al. 2001; Ogden et al. 2001;
Chandler et al. 2001).
An effective alternative to IMS is the use of metal hydroxide
based bacterial concentration, which may be used either bound
to a solid support through which the food slurry passes or
applied directly to a liquid food matrix or dilution in floc
form. Titanous, hafnium, or zirconium hydroxide suspensions interact with the opposing charge of the bacterial cell
(whether viable or dead) and may be separated using low-speed
centrifugation, resuspended, and directly plated (or used for
molecular-based downstream detection assays). Like IMS,
metal hydroxide based concentration has been demonstrated
successfully for many common food-associated bacterial pathogens and is useful for removal and detection of spores as
well (Lucore et al. 2000; McKillip et al. 2000; Cullison and
Jaykus 2002; Jaykus 2003).
For selective isolation and differentiation of food-associated
spoilage and pathogenic bacteria of interest, a variety of
chromogenic and fluorogenic culture media are available. Incorporation of enzyme substrates into selective media may
expedite (or eliminate) followup biochemical confirmation
of bacterial identity. Fluorogenic enzyme substrates consist
of a sugar or amino acid conjugated to a fluorogen (Manafi
1996, 2000). A commonly used example, methylumbelliferyl,
has been employed in a wide variety of media and other detection formats for coliforms (e.g., E. coli O157:H7). When
cleaved by enzymes produced from specific target species, a
blue fluorescence is observed when suspect colonies are ex-
885
posed to long-wave ultraviolet light (Alonso et al. 1998,
1999; Berg and Fiksdal 1988). Enterohemorrhagic E. coli
O157:H7 is unique in that it is methylumbelliferyl-β-Dglucoronidase negative, whereas virtually all other coliforms
are positive for this enzyme (Hartman 1989). This important
distinction is useful for confirming enterohemorrhagic
E. coli O157:H7 presence in water or food samples (although additional steps are also necessary) (Bettelheim
1998) as growth on solid agar or liquid broth media (Manafi
2000).
Chromogenic substrates such as 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (XGAL) are useful for coliforms such as E.
coli as well and produce a distinctive blue colony when this
compound is cleaved by β-galactosidase produced during cell
growth, particularly if 1-isopropyl-β-D-thiogalactopyranoside is
added to the medium to enhance activity of this enzyme.
Other fluorogenic or chromogenic media for coliforms are
available under various trade names, including Rainbow
agar, BCM O157:H7 (and other coliforms), CHROMagar,
and Colilert, all of which have been applied in the clinical or
food industry to distinguish key species based on coloration
of suspect colonies.
For Gram-positive pathogens, fluorogenic and chromogenic
media are also readily available with similar substrates and
indicator systems. Staphylococcus aureus CHROMagar may
be supplemented with antibiotics to cross-check for methicillinresistant S. aureus, for example, in clinical applications.
Bacillus cereus, as a food-associated pathogen of growing
concern, may produce a phosphatidylinositol-specific phospholipase C, the key enzyme reacting with 5-bromo-4chloro-3-indoxyl-myoinositol-1-phosphate, giving distinctive
turquoise colonies. Analogous indicator systems are
commercially available for Clostridium perfringens, L.
monocytogenes, and Bifidobacterium spp. (Baumgart et al.
1990; Restaino et al. 1999; Chevalier et al. 1991).
Dry plate culturing (e.g., 3M Petrifilm or similar) is yet
another widely used means of assessing microbiological
quality of a wide range of foods for coliforms, aerobic
mesophilic bacteria, psychrotrophs, and staphylococci
(Blackburn 1996; Linton 1997; Silbernagel and Lindberg
2001; Ellis and Meldrum 2002). This system consists of
multiple layers of plastic film encasing a dehydrated disk of
the appropriate medium. Typically, a single sheet of plastic
is aseptically peeled back and 1 mL of inoculum is used to
rehydrate the medium while the film is reapplied and
pressed flat. In some types of rehydratable films, gas production (e.g., from Enterobacteriaceae) may be detected as
bubbles in the film following incubation. Dry media culture
plates have been applied in settings ranging from predicting
shelf life and monitoring the microbiological quality of milk
(Hughes and Sutherland 1987; Phillips and Griffiths 1990)
to assessing surface contamination of meat and poultry
(Guthrie et al. 1994; Park et al. 2001; Erdmann et al. 2002)
and constitute an AOAC-approved method in food microbiology for quality control. Owing to their small size,
Petrifilm plates are convenient for larger sample numbers
common in quality control laboratories and require less
space in a typical incubator but possess the same limitations
as standard plate counting in terms of poor sensitivity and
the likelihood of encountering false-negative results from
bacteria that may be sublethally injured, precluding accurate
© 2004 NRC Canada
886
enumeration from suspect foods. This and other methods for
food-associated pathogen detection are summarized in Feng
(1998).
Other means of measuring cell growth
Impedance microbiology
The use of the impedance allows for detection of microbes
directly through metabolite accumulation in surrounding
substrate or indirectly by carbon dioxide liberation (Silley
and Forsythe 1996; Marshall 1999). Direct impedance detection is measured by the increase in the conductivity of the
medium as a function of continually accumulating organic
acids, ammonium ions, and other compounds that are produced from growth of a pure isolate. Whether direct or indirect measurements are used, the time required for detectable
changes to occur depends on inoculum size, although detection limits here are theoretically a single cell, if conditions
are permissible for growth. Conductance assays are widely
used in food microbiology for prediction of shelf life and a
variety of bacterial growth modeling studies for specific
pathogens, including E. coli (Madden and Gilmour 1995;
Edmiston 1998, 2000) and Campylobacter spp. (Moore and
Madden 2002). This technique requires specialized instrumentation with significant up-front costs, however, and necessitates the establishment of stringently reproducible
growth curve profiles of isolated strains under each temperature or culture condition of interest (Duran and Marshall
2002). Variability may ensue from different food matrices
containing inconsistent levels of interfering organic material
and, as with the SPC, stressed bacteria may introduce an additional element of variability into experimental runs, making reproducibility difficult to achieve. Once impedance
curves are established for specific isolates under standard
sample preparation regimes, high throughput is possible with
relative ease.
Miniaturized assays
Frequently, bacteria isolated from contaminated food are
identified to the species level after growth on (or in) the appropriate selective medium. Selective enrichment steps in
broth are sometimes employed to increase the density of a
target microbe or population suspected to be present. As
mentioned above, this regime is both time- and laborintensive and may not directly correlate with the presence of
pathogenic strains of interest if one simply relies on matching the biochemical profile of the food isolate with a data set
from a type strain. Since bacterial virulence factors may be
shared among a wide variety of species, or conversely may
be absent in an otherwise correctly identified strain, the notion of using species identification as a hallmark of obligatory pathogenicity is an obsolescent design strategy. For
example, the flagship food-associated pathogen within
Bacillaceae, B. cereus, frequently produces enterotoxins that
elicit toxicoinfection-related illness (Granum and Lund
1997; McKillip 2000). Of course, not all B. cereus strains
are enterotoxigenic, and many species outside the B. cereus
phylogenetic group may harbor enterotoxin genes and express
them in varied growth environments (Hoult and Tuxford
1991; Damgaard 1995; Beattie and Williams 1999; Pruß et
al. 1999; Phelps and McKillip 2002). Food microbiologists
Can. J. Microbiol. Vol. 50, 2004
must operate under the assumption that such observations are
likely the rule rather than the exception and design and implement detection methodologies with this in mind. Regardless,
the myriad of commercially available kit/strip-based biochemical systems allow for confirmation of isolated bacteria
from food, particularly during the development of faster and
more sensitive molecular assays. The utility and evolution of
many of these miniaturized biochemical systems have been
reported (Cox et al. 1987; Dziezak 1987; Fung et al. 1988;
Feng 1996, 1997, 1998; Shi and Fung 2000).
As is common with identification strategies, many of the
rapid kit-based systems commercially available were first introduced in the clinical arena and were then eventually
adapted for use in food microbiology (Stager and Davis
1992). The majority of the biochemical test kits currently
sold assess fermentation of a battery of carbohydrates via
pH indicators and (or) specific amino acid or alternative substrate utilization. Most are designed for the differentiation
and putative identification of Gram-negative bacteria, although
a few are sold for use on Gram-positives such as staphylococci and Bacillaceae, although with greater variability
(Feng 2001; Murtiningsih 1997).
Each of these systems requires a pure culture for inoculation and 16–24 h for incubation to obtain results. If used
with a standardized inoculum, accuracy of identification
may exceed 95%, particularly if one uses some of the latest
kits on the market that are compatible with instruments designed to facilitate data acquisition and quantitation (Feng
1996, 1998). However, the higher sample throughput afforded by this automatability increases cost significantly.
Commercially available miniaturized assays are overviewed
in Russell et al. (1997) and in tabular format in Feng (1998).
Molecular-based semi-automated detection
formats
Immunoassays
Among the widely accepted molecular methods in use for
a battery of food pathogens is the enzyme-linked immunosorbent assay (ELISA), which potentially offers much greater
specificity compared with SPC owing to the antibody–target
interaction (although contaminants and food matrix debris
may interfere). This process essentially consists of the addition of the suspect antigen (food slurry or diluted extract) to
the sample wells in a microtiter plate containing a (primary)
antibody with specificity to the target molecule. This molecule may be a component of the pathogen, such as a cell or
flagellar antigen, or a product of the bacteria (e.g., enterotoxin)
(Notermans and Wernars 1991). Following an incubation step,
unbound material is rinsed away and the secondary anitibody
is added to form a “sandwich” of the antigen between the 2
antibody molecules. After a second rinse, the assay is developed per the conjugate or tag bound to the secondary antibody (Fig. 3). If serial dilutions of the suspect antigen (e.g.,
food sample) are made, one may glean relative quantitative,
high-throughput data if a spectrophotometer with microtiter
plate capabilities is employed for data collection. ELISA has
been successfully used to detect either whole-cell antigen
targets or products (e.g., virulence determinants) of pathogens
such as Salmonella spp., E. coli O157:H7, Campylobacter
spp., B. cereus, and L. monocytogenes, among others (Peplow
© 2004 NRC Canada
Gracias and McKillip
Fig. 3. Schematic of ELISA for high-throughput screening of
food pathogens using any one of a number of target antigens. A
bound capture antibody may be used to immobilize or remove
the target microbe or molecule from a heterogeneous matrix such
as a food slurry; following a rinse step, a labeled secondary antibody is added, and detection ensues via an enzyme–substrate
(e.g., colorimetric) reaction. Alternatively, the secondary antibody
may be conjugated to a fluorophore and read as fluorescence following excitation.
et al. 1999; Chen et al. 2001; Valdivieso-Garcia et al. 2001;
Daly et al. 2002; de Paula et al. 2002; Yeh et al. 2002;
Bolton et al. 2002), in a more user-friendly format than reverse passive latex agglutination assays (explained below).
ELISA is automatable, however, and is convenient for large
sample numbers with relative ease. Despite these advantages, ELISA methods may still suffer in terms of desired
sensitivity, with a typical detection limit of 104 CFU/mL,
depending on the food being analyzed (Cox et al. 1987;
Hartman et al. 1992).
A less quantitative means of confirming the presence of a
pure culture of a specific pathogen following isolation could
entail the use of reverse passive latex agglutination. This
rapid test is performed by mixing a suspension of test cells
(usually following enrichment) with sensitized latex beads
containing antibodies specific for the target pathogen under
study. After several minutes, visible clumping is observed in
a positive reaction compared with control samples. This procedure is typically performed from components sold commercially in kit form from a large number of clinical/food
microbiology supply companies.
ATP bioluminescence
If a food microbiology laboratory is predominately interested in general monitoring of processing plant sanitation
without the necessary isolation, recovery, and subsequent
identification of specific bacteria, bioluminescence assays
may suffice. In particular, bioluminescence assays that detect
bacterial adenosine triphosphate (ATP) are a good indicator
of the presence of microbial contamination in foods ranging
from meat and poultry to dairy products and the processing
surfaces in the vicinity (Griffiths 1993, 1996). It is important
to note that in addition to detecting microbial contamination,
ATP bioluminescence detects the presence of food residues
and therefore gives an indication of total surface contamination, a capability exploited within the realms of surface hygiene monitoring/assessment. Since ATP is present within all
actively metabolizing cells, in order for ATP bioluminescence
to solely detect microorganisms, the assay procedure must
887
include steps to either segregate microbial from somatic
ATP (filtration step in the protocol) or destroy the somatic
ATP so that all that remains in the sample is that that is associated with microbial contaminants, although these steps
extend assay time and decrease sensitivity.
In the ATP bioluminescence approach, luciferase is added
to a sample, along with luciferin, in a magnesium-containing
buffer. As the luciferin is oxidized to oxyluciferin, the photons of light emitted may be measured using a luminometer.
Once a standard curve is established with dilutions of bacteria at known densities, the level of contamination may be assessed in the food sample or on the contact surface (Poulis et
al. 1993; de Boer and Beumer 1999). Although ATP assays
do not require an enrichment step, the sensitivity is typically
on the order of 104 CFU/mL. Bioluminescence lends itself
well to rapid spot monitoring, as the results are obtained in
less than 15 min (Feng 2001). This technique has been used
on a diverse array of foods, including raw and pasteurized
dairy products (Webster et al. 1988; Kyriakides 1992;
Brovko et al. 1999; Niza-Ribeiro et al. 2000; Samkutty et al.
2001), meat and poultry products (Bautista et al. 1994, 1995;
Siragusa and Cutter 1995; Chen 2000), beer (Dowhanick
and Sobczak 1994), and fruit (Ukuku et al. 2001).
Perspectives
In order for any conventional or molecular-based detection format to be a feasible tool in the food industry for hygiene monitoring or quality control, it must demonstrate
reproducible sensitivity (ability to detect target cells or molecules at very low levels), marked specificity (ability to exhibit positive results in a high background of nontarget
molecules or cells), speed in obtaining results, low cost per
assay, acceptability and ease of use by the scientific community and food microbiology laboratory staff, and standardized protocols and data interpretation (Fung 2002; Malorny
et al. 2003). Since no single approach satisfies all or even
most of these criteria, the user must prioritize the features of
each available format against the needs of the facility and
implement the most practical method(s) accordingly.
References
Alonso, J.L., Soriano, K., Amoros, I., and Ferrus, M.A. 1998.
Quantitative determination of E. coli and fecal coliforms in water
using a chromogenic medium. J. Environ. Sci. Health, 33: 1229–
1248.
Alonso, J.L., Soriano, A., Carbajo, O., Amoros, I., and Garelick, H.
1999. Comparison and recovery of Escherichia coli and thermotolerant coliforms in water with a chromogenic medium incubated
at 41 and 44.5 °C. Appl. Environ. Microbiol. 65: 3746–3749.
Baumgart, J., Baum, O., and Lippett, S. 1990. Schneller and direkter
Nachweis von Clostridium perfringens. Fleischwirtschaft, 70:
1010–1014.
Bautista, D.A., Vaillancourt, J.P., Clarke, R.A., Renwick, S., and
Griffiths, M.W. 1994. Adenosine triphosphate bioluminescence
as a method to determine microbial levels in scald and chill
tanks at a poultry abattoir. Poult. Sci. 73: 1673–1678.
Bautista, D.A., Vaillancourt, J.P., Clarke, R.A., Renwick, S., and
Griffiths, M.W. 1995. Rapid assessment of the microbiological
quality of poultry carcasses using ATP bioluminescence. J. Food
Prot. 58: 551–554.
© 2004 NRC Canada
888
Beattie, S.H., and Williams, A.G. 1999. Detection of toxigenic
strains of Bacillus cereus with an improved cytotoxicity assay.
Lett. Appl. Microbiol. 28: 221–225.
Berg, J.D., and Fiksdal, L. 1988. Rapid detection of total and fecal
coliforms in water by enzymatic hydrolysis of 4-methylumbelliferone-β-D-galactoside. Appl. Environ. Microbiol. 54:
2118–2122.
Bettelheim, K.A. 1998. Reliability of CHROMagar O157 for the
detection of enterohaemorrhagic E. coli EHEC O157 but not
EHEC belonging to other serogroups. J. Appl. Microbiol. 85:
425–428.
Blackburn, C. de W. 1996. Evaluation of Petrifilm methods for
enumeration of aerobic flora and coliforms in a wide range of
foods. Lett. Appl. Microbiol. 22: 137–140.
Blackburn, C., and McCarthy, J.D. 2000. Modifications to methods
for the enumeration and detection of injured Escherichia coli
O157:H7 in foods. Int. J. Food Microbiol. 55: 285–290.
Bolton, F.J., Sails, A.D., Fox, A.J., Wareing, D.R.A., and
Greenway, D.L.A. 2002. Detection of Campylobacter jejuni in
foods by enrichment culture and polymerase chain reaction
enzyme-linked immunosorbent assay. J. Food Prot. 65: 760–767.
Brovko, L., Froundjian, V.G., Babunova, V.S., and Ugarova, N.N.
1999. Quantitative assessment of bacterial contamination of raw
milk using bioluminescence. J. Dairy Res. 66: 627–631.
Chandler, D.P., Brown, J., Call, D.R., Wunschel, S., Grate, J.W.,
Holman, D.A., Olson, L., Stottlemyre, M.S., and Bruckner-Lea, C.J.
2001. Automated immunomagnetic separation and microarray
detection of E. coli O157:H7 from poultry carcass rinse. Int. J.
Food Microbiol. 70: 143–154.
Chen, C.H., Ding, H.C., and Chang, T.C. 2001. Rapid identification
of Bacillus cereus based on the detection of a 28.5-kilodalton
cell surface antigen. J. Food Prot. 64: 348–354.
Chen, J. 2000. ATP bioluminescence: a rapid indicator for environmental hygiene and microbial quality of meats. Dairy Food Environ. Sanit. 20: 617–620.
Chevalier, P., Roy, D., and Savoie, L. 1991. X-α-Gal-based medium for simultaneous enumeration of bifidobacteria and lactic
acid bacteria in milk. J. Microbiol. Methods, 13: 75–83.
Cox, N.A., Fung, D.Y.C., Bailey, J.S., Hartman, P.A., and
Vasavada, P.C. 1987. Miniaturized kits, immunoassays and DNA
hybridization for recognition and identification of foodborne
bacteria. Dairy Food Environ. Sanit. 7: 628–631.
Cullison, M.A., and Jaykus, L. 2002. Magnetized carbonyl iron
and insoluble zirconium hydroxide mixture facilitates bacterial
concentration and separation from nonfat dry milk. J. Food Prot.
65: 1806–1810.
Daly, P., Collier, T., and Doyle, S. 2002. PCR–ELISA detection
of Escherichia coli in milk. Lett. Appl. Microbiol. 34: 222–
226.
Damgaard, P.H. 1995. Diarrhoel enterotoxin production by strains
of Bacillus thuringiensis isolated from commercial Bacillus
thuringiensis-based insecticides. FEMS Immunol. Med. Microbiol.
12: 245–250.
de Boer, E., and Beumer, R.R. 1999. Methodology for detection
and typing of foodborne microorganisms. Int. J. Food Microbiol.
50: 119–130.
de Paula, A.M.R., Gelli, D.S., Landgraf, M., Destro, M.T., and
Franco, B. 2002. Detection of Salmonella in foods using Tecra
Salmonella VIA and Tecra Salmonella UNIQUE rapid immunoassays and a cultural procedure. J. Food Prot. 65: 552–555.
Dowhanick, T.M., and Sobczak, J. 1994. ATP bioluminescence
procedure for viability testing of potential beer spoilage microorganisms. J. Am. Soc. Brew. Chem. 52: 19–23.
Can. J. Microbiol. Vol. 50, 2004
Duran, G.M., and Marshall, D.L. 2002. Rapid determination of
sanitizer concentration using impedance-based methods. J. Food
Prot. 65: 1422–1427.
Dziezak, J.D. 1987. Rapid methods for microbiological analysis of
food. Food Technol. 41: 56–73.
Edmiston, A.L., and Russell, S.M. 1998. A rapid microbiological
method for enumerating Escherichia coli from broiler chicken
carcasses. J. Food Prot. 61: 1375–1377.
Edmiston, A.L., and Russell, S.M. 2000. Specificity of a conductance assay for enumeration of Escherichia coli from broiler
carcass rinse samples containing genetically similar species. J.
Food Prot. 63: 264–267.
Ellis, P., and Meldrum, R. 2002. Comparison of the compact dry
TC and 3M Petrifilm ACP dry sheet media methods with the
spiral plate method for the examination of randomly selected
foods for obtaining aerobic colony counts. J. Food Prot. 65:
423–425.
Erdmann, J.J., Dickson, J.S., and Grant, M.A. 2002. A new technique for Escherichia coli testing of beef and pork carcasses. J.
Food Prot. 65: 192–195.
Feng, P. 1996. Emergence of rapid methods for identifying microbial pathogens in foods. J. AOAC Int. 79: 809–812.
Feng, P. 1997. Impact of molecular biology on the detection of
foodborne pathogens. Mol. Biotechnol. 7: 267–278.
Feng, P. 1998. Rapid methods for detecting foodborne pathogens.
In FDA bacteriological analytical manual. 8A ed. AOAC International, Gaithersburg, Md. pp. App. 1.01 – App. 1.16.
Feng, P. 2001. Development and impact of rapid methods for
detection of food-borne pathogens. In Food microbiology: fundamentals and frontiers. Edited by M.P. Doyle, L.R., Beuchat,
and T.J. Montville. ASM Press, Washington, DC. pp. 775–796.
Fung, D.Y.C. 2002. Predictions for rapid methods and automation
in food microbiology. J. AOAC Int. 85: 1000–1002.
Fung, D.Y.C., Cox, N.A., and Bailey, J.S. 1988. Rapid methods
and automation in the microbiological examination of foods.
Dairy Food Environ. Sanit. 8: 292–296.
Golden, D.A., Beuchat, L.R., and Brackett, R.E. 1988. Evaluation
of selective direct plating media for their suitability to recover
uninjured, heat-injured, and freeze-injured Listeria monocytogenes
from foods. Appl. Environ. Microbiol. 54: 1451–1456.
Granum, P.E., and Lund, T. 1997. Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Lett. 157: 223–228.
Griffiths, M.W. 1993. Applications of bioluminescence in the dairy
industry. J. Dairy Sci. 76: 3118–3125.
Griffiths, M.W. 1996. The role of ATP bioluminescence in the food
industry: new light on old problems. Food Technol. 50: 62–72.
Guthrie, J.A., Dunlop, J.J., and Saunders, G.A. 1994. Use of
Petrifilm 3M to assess coliform numbers on lamb carcasses. J.
Food Prot. 57: 924–927.
Hajmeer, M.N., Fung, D.C., Marsden, J.L., and Milliken, G.A.
2001. Effects of preparation method, age, and plating technique
of thin agar layer media on recovery of Escherichia coli
O157:H7 injured by sodium chloride. J. Microbiol. Methods,
47: 49–253.
Hara-Kudo, Y., Ikedo, M., Kodaka, H., Nakagawa, H., Goto, K.,
Masuda, T., Konuma, H., Kojima, T., and Kumagai, S. 2000.
Selective enrichment with a resuscitation step for isolation of
freeze-injured Escherichia coli O157:H7 from foods. Appl.
Environ. Microbiol. 66: 2866–2872.
Harrigan, W. 1998. Laboratory methods in food microbiology. 3rd
ed. Academic Press, San Diego, Calif. pp. 43–70.
Hartman, P.A. 1989. The MUG glucoronidase test for E. coli food
and water. In Rapid methods and automation in microbiology
© 2004 NRC Canada
Gracias and McKillip
and immunology. Edited by A. Turano. Brixia Academics Press,
Brescia, Italy. pp. 290–308.
Hartman, P.A., Swaminathan, B., Curiale, M.S., Firstenberg-Eden,
R., Sharpe, A.N., Cox, N.A., Fung, D.Y.C., and Goldschmidt,
M.C. 1992. Rapid methods and automation. In Compendium of
methods for the microbiological examination of foods. Edited by
C. Vanderzant and D.F. Splittstoesser. American Public Health
Association, Washington, DC. pp. 665–746.
Hoult, B., and Tuxford, A.F. 1991. Toxin production by Bacillus
pumilus. J. Clin. Pathol. 44: 455–458.
Hsih, H.Y., and Tsen, H.Y. 2001. Combination of immunomagnetic
separation and polymerase chain reaction for the simultaneous
detection of Listeria monocytogenes and Salmonella spp. in food
samples. J. Food Prot. 64: 1744–1750.
Hudson, J.A., Lake, R.J., Savill, M.G., Scholes, P., and McCormick,
R.E. 2001. Rapid detection of Listeria monocytogenes in ham
samples using immunomagnetic separation followed by polymerase chain reaction. J. Appl. Microbiol. 90: 614–621.
Hughes, D., and Sutherland, P.S. 1987. Evaluation of Petrifilm SM
for determining bacteriological counts on raw milk by comparison
with the standard plate count and micropipet count techniques.
Aust. J. Dairy Technol. 47: 59–61.
Hurst, A. 1977. Bacterial injury: a review. Can. J. Microbiol. 23:
936–944.
Jaykus, L.A. 2003. Challenges to developing real-time methods to
detect pathogens in foods. ASM News, 69: 341–347.
Jinneman, K.C., Trost, P.A., Hill, W.E., et al. 1995. Comparison of
template preparation methods from froods for amplification of
Escherichia coli O157 shiga-like toxins type I and II DNA by
multiplex polymerase chain reaction. J. Food Prot. 58: 722–726.
Kang, D.H., and Fung, D.Y.C. 1999. Thin agar layer method for
recovery of heat-injured Listeria monocytogenes. J. Food Prot.
62: 1346–1349.
Kang, D., and Fung, D. 2000. Application of thin agar layer method
for recovery of injured Salmonella typhimurium. Int. J. Food
Microbiol. 54: 127–132.
Kang, D.H., and Siragusa, G.R. 1999. Agar underlay method for
recovery of sublethally heat-injured bacteria. Appl. Environ.
Microbiol. 65: 5334–5337.
Kang, D.H., and Siragusa, G.R. 2001. A rapid twofold dilution
method for microbial enumeration and resuscitation of uninjured
and sublethally injured bacteria. Lett. Appl. Microbiol. 33: 232–
236.
Kyriakides, A. 1992. ATP bioluminescence applications for microbiological quality control in the dairy industry. J. Soc. Dairy
Technol. 45: 91–93.
Linton, R.H. 1997. Comparison of conventional plating methods
and Petrifilm for the recovery of microorganisms in a ground
beef processing facility. J. Food Prot. 60: 1084–1088.
Lucore, L., Cullison, M.A., and Jaykus, L. 2000. Immobilization
with metal hydroxides as a means to concentrate food-borne
bacteria for detection by cultural and molecular methods. Appl.
Environ. Microbiol. 66: 1769–1776.
Madden, R.H., and Gilmour, A. 1995. Impedance as an alternative
to MPN enumeration of coliforms in pasteurized milks. Lett.
Appl. Microbiol. 21: 387–388.
Malorny, B., Tassios, P.T., Radstrom, P., Cook, N., Wagner, M.,
and Hoorfar, J. 2003. Standardization of diagnostic PCR for the
detection of foodborne pathogens. Int. J. Food Microbiol. 83:
39–48.
Manafi, M. 1996. Fluorogenic and chromogenic substrates in culture
media and identification tests. Int. J. Food Microbiol. 31: 45–58.
Manafi, M. 2000. New developments in chromogenic and fluorogenic
culture media. Int. J. Food Microbiol. 60: 205–218.
889
Marriott, N.G. 1999. Principles of food sanitation. 4th ed. Aspen
Publishers, Gaithersburg, Md. pp. 75–89.
Marshall, D.L. 1999. Rapid impedance methods for seafood. In
Handbook for rapid methods and automation in microbiology
workshop. Sect. C. Chap. 11. Edited by D.Y.C. Fung. Kansas
State University, Manhattan, Kans. pp. 109–121.
McKillip, J.L. 2000. Prevalence and expression of enterotoxins in
Bacillus cereus and other Bacillus spp., a literature review. Antonie
Leeuwenhoek, 77: 393–399.
McKillip, J. 2001. Recovery of sublethally injured bacteria using
selective agar overlays. Am. Biol. Teach. 63: 184–188.
McKillip, J.L., Jaykus, L.A., and Drake, M.A. 2000. A comparison
of methods for the detection of Escherichia coli O157:H7 from
artificially contaminated dairy products using PCR. J. Appl.
Microbiol. 89: 49–55.
McKillip, J.L., Jaykus, L.A., and Drake, M.A. 2002. Influence of
growth in a food medium on the detection of Escherichia coli
O157:H7 by polymerase chain reaction. J. Food Prot. 65: 1775–
1779.
Mead, P.S., L. Slutsker, V., Dietz, L.F., McCaig, J.S., Bresee, C.,
Shapiro, P.M., Griffin, and Tauxe, R.V. 1999. Food-related illness
and death in the United States. Emerg. Infect. Dis. 5: 607–625.
Moore, J.E., and Madden, R.H. 2002. Impediometric detection of
Campylobacter coli. J. Food Prot. 65: 1660–1662.
Murtiningsih, S., and Cox, J.M. 1997. Evaluation of the Serobact
and Microbact systems for the detection and identification of Listeria spp. Food Control, 8: 205–210.
Nakano, S., Kobayashi, T., Funabiki, K., Matsumura, A., Nagao,
Y., and Yamada, T. 2004. PCR detection of Bacillus and Staphylococcus in various foods. J. Food Prot. 67: 1271–1277.
Niza-Ribeiro, J., Louza, A.C., Santos, P., and Lima, M. 2000.
Monitoring the microbiological quality of raw milk through the
use of an ATP bioluminescence method. Food Control, 11: 209–
216.
Notermans, S., and Wernars, K. 1991. Immunological methods for
detection of foodborne pathogens and their toxins. Int. J. Food
Microbiol. 12: 91–102.
Ogden, I.D., Hepburn, N.F., and MacRae, M. 2001. The optimization
of isolation media used in immunomagnetic separation methods
for the detection of Escherichia coli O157:H7 in foods. J. Appl.
Microbiol. 91: 373–379.
Park, Y.H., Seo, K.S., Ahn, J.S., Yoo, H.S., and Kim, S.P. 2001.
Evaluation of the Petrifilm plate method for the enumeration of
aerobic microorganisms and coliforms in retailed meat samples.
J. Food Prot. 64: 1841–1843.
Peplow, M.O., Correa-Prisant, M., Stebbins, M.E., Jones, F., and
Davies, P. 1999. Sensitivity, specificity, and predictive values of
three Salmonella rapid detection kits using fresh and frozen
poultry environmental samples versus those of standard plating.
Appl. Environ. Microbiol. 65: 1055–1060.
Phelps, R.J., and McKillip, J.L. 2002. Enterotoxin production in
natural isolates of Bacillaceae outside the Bacillus cereus group.
Appl. Environ. Microbiol. 68: 3147–3151.
Phillips, J.D., and Griffiths, M.W. 1990. Prediction of the shelf-life
of pasteurized milk using dry medium culture plates (3M Petrifilm,
Hygicult and Millipore sampler). J. Soc. Dairy Technol. 43: 45–49.
Poulis, J.A., Pijper, M. de., and Mossel, D.A.A. 1993. Assessment
of cleaning and disinfection in the food industry with the rapid
ATP-bioluminescence technique combined with the tissue fluid
contamination test and a conventional microbiological method.
Int. J. Food Microbiol. 20: 109–116.
Pruß, B.M., Dietrich, R., Nibler, B., Martlbauer, E., and Scherer, S.
1999. The hemolytic enterotoxin HBL is broadly distributed
© 2004 NRC Canada
890
among species of the Bacillus cereus group. Appl. Environ.
Microbiol. 65: 5436–5442.
Ray, B. 1986. Impact of bacterial injury and repair in food microbiology: its past, present, and future. J. Food Prot. 49: 651–655.
Restaino, L., Frampton, E.W., Irbe, R.M., Schabert, G., and Spitz, H.
1999. Isolation and detection of Listeria monocytogenes using
fluorogenic and chromogenic substrates for phosphatidylinositolspecific phospholipase C. J. Food Prot. 62: 244–251.
Rijpens, N.P., and Herman, L.M.F. 2002. Molecular methods for
identification and detection of bacterial food pathogens. J. AOAC
Int. 85: 984–995.
Russell, S.M., Cox, N.A., Bailey, J.S., and Fung, D.Y.C. 1997.
Miniaturized biochemical procedures for identification of bacteria.
J. Rapid Methods Autom. Microbiol. 5: 169–177.
Samkutty, P.J., Gough, R.H., Adkinson, R.W., and McGrew, P.
2001. Rapid assessment of bacteriological quality of raw milk
using ATP bioluminescence. J. Food Prot. 64: 208–212.
Sandel, M.K., Wu, Y.-F.G., and McKillip, J.L. 2003. Detection and
recovery of sublethally-injured enterotoxigenic Staphylococcus
aureus. J. Appl. Microbiol. 94: 90–94.
Sharpe, A.N. 1997. Separation and concentration of pathogens from
foods. In Food microbiological analysis: new technologies. Edited
by M.L. Tortorello and S.M. Gendel. Marcel Dekker, Inc., New
York. pp. 27–44.
Shi, X., and Fung, D.Y.C. 2000. Control of foodborne pathogens
during sufu fermentation and aging. Crit. Rev. Food Sci. Nutr.
40: 399–425.
Silbernagel, K.M., and Lindberg, K.G. 2001. PetrifilmTM rapid S.
aureus count plate method for rapid enumeration of Staphylococcus aureus in selected foods: collabaorative study. J. AOAC
Int. 84: 1431–1443.
Silley, P., and Forsythe, S. 1996. Impedance microbiology — a
rapid change for microbiologists (a review). J. Appl. Bacteriol.
80: 233–243.
Siragusa, G.R., and Cutter, C.N. 1995. Microbial ATP bio-
Can. J. Microbiol. Vol. 50, 2004
luminescence as a means to detect contamination on artificially
contaminated beef carcass tissue. J. Food Prot. 58: 764–769.
Smith, T.J., O’Connor, L., Glennon, M., and Maher, M. 2000.
Molecular diagnostics in food safety: rapid detection of foodborne pathogens. Ir. J. Agric. Food Res. 39: 309–319.
Speck, M., Ray, B., and Read, R., Jr. 1975. Repair and enumeration
of injured coliforms by a plating procedure. Appl. Microbiol.
29: 549–550.
Stager, C.E., and Davis, J.R. 1992. Automated systems for identification of microorganisms. Clin. Microbiol. Rev. 5: 302–327.
Tomoyasu, T. 1998. Improvement of the immunomagnetic separation
method selective for Escherichia coli O157 strains. Appl. Environ.
Microbiol. 64: 376–382.
Ukuku, D.O., Pilizota, V., and Sapers, G.M. 2001. Bioluminescence
ATP assay for estimating total plate counts of surface microflora
of whole cantaloupe and determining efficacy of washing treatments. J. Food Prot. 64: 813–819.
Valdivieso-Garcia, A., Riche, E., Abubakar, O., Waddell, T.E., and
Brooks, B.W. 2001. A double antibody sandwich enzyme-linked
immunosorbent assay for the detection of Salmonella using
biotinylated monoclonal antibodies. J. Food Prot. 64: 1166–1171.
Webster, J.A.J., Hall, M.S., Rich, C.N., Gilliland, S.E., Ford, S.R.,
and Leach, F.R. 1988. Improved sensitivity of the bioluminescent
determination of numbers of bacteria in milk samples. J. Food
Prot. 51: 949–954.
Wu, V.C.H., Fung, D.Y.C., and Kang, D.H. 2001. Evaluation of
thin agar layer method for recovery of cold-injured foodborne
pathogens. J. Rapid Methods Autom. Microbiol. 9: 11–26.
Yeh, K.S., Tsai, C.E., Chen, S.P., and Liao, C.W. 2002. Comparison
between VIDAS automatic enzyme-linked fluorescent immunoassay and culture method for Salmonella recovery from pork
carcass sponge samples. J. Food Prot. 65: 1656–1659.
Zhao, T., and Doyle, M.P. 2001. Evaluation of universal preenrichment broth for growth of heat-injured pathogens. J. Food
Prot. 64: 1751–1755.
© 2004 NRC Canada