FOOD BIOLOGICAL CONTAMINANTS

Elevated-Temperature, Colorimetric, Monoclonal,

Enzyme-Linked Immunosorbent Assay for Rapid Screening of
Salmonella in Foods: Collaborative Study
ECKNER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 77, NO. 2, 1994
KARL F. ECKNER, WENDY A. DUSTMAN, MICHAEL S. CURIALE, and RUSSELL S. FLOWERS
Silliker Laboratories Group, Inc., 1304 Halsted St, Chicago Heights, IL 60411
BARBARA J. ROBISON
Organon Teknika Corp., 100 Akzo Ave., Durham, NC 27704
Collaborators: M. Befus; S. Bell; J. Benson; D. Blank; J. Bozzuffi; D. Bryant; M. Buchanan; T. Burns; M. Cirigliano; D.C.
Clifford; M. Cook; A.E. Cruse; M. Davis; C. Dittman; W. Dustman; R. Dyer; K. Eckner; K. Faucher; J. Fechner; E.W.
Frampton; J. Fuls; K. Givich; R. Hamilton; L. Harris; D. Higgins; A. Holder; O. Imami; K. Jost-Keating; C. Kennett; R.
Koeritzer; J. Kornacki; C. Lin; M.M. Lister; R. Lubbat; E. McHugh; R. McKenna; G. Miklovic; C. Monaghan; C. OBryan;
C.U. Okolo; P. Perez; M. Peterson; D. Potel; M. Pratt; U. Purvis; W. Reid; L. Restaino; N. Richmond; B. Robison; C. Roderick;
B. Rodriguez; L. Saunders; K. Scheider; R. Schnitzer; M. Schulke; V. Smith; R. Sparano; L. Stoltzner; J. Stone; P. Trost; K.
Vought; C. Wilch

A collaborative study was performed by 30 laboratories in 3 sets of trials to validate a modified colorimetric monoclonal enzyme-linked immunosorbent assay (ELISA) method for Salmonella
detection. The modifications to the current methodology included incubation of enrichments and postenrichments at an elevated temperature, addition
of novobiocin to the M-broth post-enrichment, and
elimination of the centrifugation and agitation
steps. Five artificially contaminated foods (nonfat
dry milk, milk chocolate, dried egg, ground black
pepper, and soy flour) and 1 naturally contaminated food (raw ground turkey) were analyzed. The
artificially contaminated foods were inoculated
with individual Salmonella serotypes at a high (10
50 cells/25 g) and low (15 cells/25 g) contamination level. Results from the modified ELISA method
were compared to the Bacteriological Analytical
Manual (BAM)/AOAC culture method. In 2 of the
food products, milk chocolate and pepper, a number of laboratories isolated Salmonella from uninoculated control samples, thus invalidating their
data. As a result, there were too few laboratories remaining with valid data, and these foods were repeated. In the completed study, there were 11 false
negative results obtained by the modified ELISA
Submitted for publication October 8, 1992.
The recommendation was approved by the Committee on Microbiology
and Extraneous Materials and was adopted by the Official Methods Board
of the Association. See AOAC International Official Methods Board
News (1993) J. AOAC Int. 76, 33A, and Methods Adopted First Action
(1993) The Referee, 17, March issue.

method, while there were 28 false negatives produced by the BAM/AOAC procedure. There were 11
ELISA positive assays which could not be confirmed by culture methods. Statistically, there were
no differences between the modified, colorimetric,
monoclonal ELISA and the reference culture
method in all foods except raw turkey, where the
ELISA method was more productive. The colorimetric monoclonal enzyme immunoassay (SalmonellaTek) method for detecting Salmonella in all foods
has been adopted first action by AOAC INTERNATIONAL.

monoclonal antibody-based colorimetric enzyme immunoassay (Salmonella Bio-EnzaBead) was demonstrated to be an effective assay in rapidly screening
foods and ingredients for Salmonella (13). This ELISA
method was adopted official first action by AOAC in 1986 (4)
and was given final action status in 1988 (986.25 [5]). Subsequently, the bead assay was discontinued and replaced by a
microtiter plate format (Salmonella-Tek), which was shown to
be equivalent to the bead assay in a comparison study (6).
Several modifications were proposed to reduce the incidence of false-positive reactions in the ELISA method and to
simplify the assay. These modifications to the current ELISA
methodology included: (a) incubation of the tetrathionate selective broth and post-enrichment broth at an elevated temperature of 42C to inhibit growth of competing bacteria; (b) addition of novobiocin containing 10 cells/mL to the M-broth
post-enrichment (MN broth); (c) elimination of agitation of the

microtiter plates during incubation steps; and (d) elimination of

the centrifugation of the M-broth post-enrichment broths.
Use of elevated temperature during incubation of enrichments to enhance recovery of Salmonella is well documented
in the literature. A review of the utilization of elevated temperature to recover Salmonella by standard culture methods has recently been completed (7). Recovery of Salmonella from
tetrathionate enrichments incubated at 4143C has been demonstrated to be equivalent to, or better than, recovery from enrichments incubated at 35C (717). Novobiocin was added
into the M-broth post-enrichment medium primarily to restrict
growth of Proteus sp. Plate agitation and centrifugation steps
were eliminated to make the assay more convenient.
The modified ELISA procedure was validated in-house using 20 different food types. Based on those results, a collaborative study was performed to further evaluate the second-generation, modified ELISA method as compared to the
Bacteriological Analytical Manual (BAM)/AOAC culture
methods.
Collaborative Study
The selection of the food types (nonfat dry milk, dried egg,
milk chocolate, soy flour, finely ground black pepper, and raw
ground turkey) was based on previous collaborative studies (1
4). The products tested reflected a wide variety of foods incriminated in food-borne disease outbreaks. Furthermore, several different pre-enrichment protocols were required. The use
of the same products as those tested in previous studies also
allows performance comparisons among methods.

Preparation of Salmonella Serotypes

The Salmonella serotypes used in the study were inoculated
into individual 100 mL volumes of trypticase soy broth (TSB)
from frozen glycerol stock cultures held at 70C. The TSB
cultures were incubated 24 h at 35C. Cells were subsequently
centrifuged at 4000 g for 10 min. The pellet was resuspended
in 10 mL of sterile 10% (w/v) reconstituted nonfat dry milk in
a Nalgene 250 mL container. The cell suspension was frozen in
a 20C freezer for 30 min and lyophilized for 25 h in a VirTis
Model 10-117 freeze-drying chamber (Gardiner, NY).

Inoculation of Samples
Dry, powdered products (nonfat dry milk, soy flour, dried
egg, and ground pepper) were inoculated in the same manner.
The lyophilized Salmonella culture was ground in a mortar and
pestle with a small quantity of test product. The inoculated
product was added to a sterile plastic bag containing ca 1000 g
of test product, and well mixed by manual agitation. This larger
quantity of inoculated product (seeded product) was held for
2 weeks at room temperature (20C) to stabilize the level of
viable, lyophilized Salmonella cells in the product. After the
stabilization period, the product was sampled, serially diluted,
and plated to xylose lysine deoxycholate agar. The estimated
cell counts were used to determine the product dilution factors
to obtain the high and low level inoculation target values, 10
50 cells/25 g and 12 cells/25 g, respectively. The appropriate

portions of inoculated product were mixed manually or with a

Stephan VMC-12 mixer into 25003000 g of noninoculated
product to obtain target levels. After blending, the product was
tested by Salmonella most probable number (MPN) procedure
to determine if the levels were appropriate (Tables 1 and 2).
The milk chocolate seed was prepared by inoculating the
Salmonella culture into tempered chocolate. The sample was
agitated, and then held at room temperature for 3 weeks. The
milk chocolate was tempered at 55C and blended with the inoculated sample in a Stephan VCM-12 blender to achieve the
high and low contamination levels desired. The inoculated
chocolate was poured onto a chilled aluminum tray lined and
covered with aluminum foil. The warm, tempered milk chocolate was placed into a 4C incubator for 24 h to harden. The
hardened chocolate was cut into squares of appropriate weight
and placed into Whirl-Pak bags. Since these heating and cooling steps resulted in visual differences between inoculated and
control samples, all chocolate samples, including the MPB
samples, were warmed for 1 h at 45C to obtain a uniform appearance, and then cooled at 4C and held overnight prior to
shipping.
The raw, ground turkey was naturally contaminated. Two
batches of turkey containing high and low contamination levels
were tested. The turkey was stored at 20C before preparation,
and then thawed to 4C and thoroughly mixed for 30 min in a
Hobart blender (Model A-200, Troy, OH). Samples of about
8090 g were weighed out and frozen at 20C prior to shipment. Control samples were prepared by steaming a sub-sample of the turkey which had the lower contamination level. This
heat treatment (10 min at 100C) was performed in an effort to
prevent Salmonella-positive control samples, but not completely destroy the natural microflora of the turkey.

Sample Shipment
Food samples of ca 3035 g each were prepared except for
turkey (8090 g because of protocol requirement 325 g sample/assay). Dry and low moisture foods were shipped at ambient temperature in insulated styrofoam containers via overnight
courier service. The turkey samples were frozen at 20C
overnight and shipped frozen with ice packs in insulated Styrofoam containers by overnight courier service.

Analysis of Test Products

Five 25 g samples inoculated at high level and five 25 g
samples inoculated at a low level were analyzed by the AOAC
culture (5) and by the ELISA (Figures 1 and 2) methods. In
addition, 5 uninoculated control samples of each food group
were analyzed. For the purposes of this study, all broth cultures
(tetrathionate, selenite cystine, and MN broths) were streaked
on Hektoen enteric (HE) agar, xylose lysine deoxycholate
(XLD) agar, and bismuth sulphite (BS) agar to determine if the
salmonellae were present, regardless of the ELISA assay result.
Each food product was scheduled for analysis on a different
week. Salmonella MPN determinations were performed by the
initiating laboratory on the same day that the comparative
analyses were started. Sample sizes employed in triplicate for
MPN analysis were 100 g, 10 g, and 1 g. MPN analyses were

done using the reference culture method only. The number of

Salmonella-positive and -negative samples were recorded, and
the levels of Salmonella determined from the MPN table.
Thirty laboratories participated in the study. Each collaborator received a set of samples of the food to be analyzed, Salmonella-Tek ELISA kits, and Micro-ID strips for biochemical
identification. Some collaborators chose to use other AOACapproved biochemical identification procedures. All other materials were provided by the collaborators. Analyses were initiated by the collaborators on the same day. Except for the raw
turkey, both methods utilized a common pre-enrichment.
993.08 Salmonella in FoodsColorimetric,
Monoclonal, Enzyme Immunoassay Method
(Salmonella-Tek)
First Action 1993
Method is procedure for presence of Salmonella in all foods;
test is not confirmatory because monoclonal antibodies used in
test may cross-react with small percentage of non-Salmonella.
Enrichment broths and M-broth (with novobiocin) from
samples positive by this test must be streaked on selective media as in 967.26B and typical or suspicious colonies must be
identified as in 967.26C, 967.27 (alternatively 978.24 or
989.12), and 967.28.
Determination of positive results is objective and must be
performed using filter photometer with 450 nm filter. Positive
result is valid only when negative and positive controls possess
acceptable optical density readings.
Method Performance:
See Table 993.08 for method performance data.

A. Principle
Detection of Salmonella antigens is based on enzyme immunoassay (EIA) using specific monoclonal antibodies. Polystyrene microtiter wells are coated with monoclonal antibodies
to Salmonella antigens. Samples and controls are placed into
wells. Salmonella antigens present in sample will attach to specific antibodies adsorbed on well. Wells are washed and conjugate is added, binding to Salmonella antigens attached to adsorbed antibody on well surface. Wells are washed to remove
unbound conjugate, and enzyme substrate is added. A blue
color is produced which turns yellow when reaction is stopped
with stop solution. Presence of Salmonella antigen in sample
depends on intensity of this color development.

B. Apparatus
(a) Microtiter strip plate holder.Polystyrene frame to
hold 18 microtiter strips.
(b) Plate sealers.Adhesive sheets to cover microtiter
plate.
(c) Clamp and rod.Closure for foil pack.
(d) Package insert.
(e) Incubator.Capable of maintaining 35-37 1.0.
(f) Water baths.(1) Capable of maintaining 42 0.5,
and 35 1.0. (2) Capable of maintaining 100 1.0. (Note:

Autoclave set at 100C or generators of flowing steam are acceptable alternatives.)

(g) Manual or automated EIA wash system.System designed to wash coated wells in 12 8 microwell plate, consists
of vacuum pump which aspirates well contents and dispensing
system which fills wells with wash solution.
(h) EIA plate reader.Photometer with 450 nm filter, capable of reading mictotiter plates.
(i) Micropipets.Capable of accurately delivering in range
50200 L, both single and multichannel. Disposable micropipet tips.
(j) Reservoir troughs.Reagent reservoir used with multichannel pipetters to hold EIA reagents for dispensing to microtiter plate wells.

C. Reagents
(a) Antibody coated microtiter strip plates.Polystyrene
microtiter strip plates, each containing 12 wells coated with
monoclonal antibodies to Salmonella species. Store at 28.
(b) Control antigens.(1) Positive control.Heat-killed,
lyophilized S. san diego (reacts with antibodies to Salmonella).
(2) Negative control.Lyophilized 1% nonfat dry milk
(non-reactive with antibodies to Salmonella).
Both controls preserved with 0.1 mg/mL gentamicin sulfate.
(c) Conjugate diluent.Phosphate-buffered saline containing 1% fetal bovine serum, 0.05% Tween 20
(polyoxethylene 20 sorbitan monolaurate), 0.1 mg/mL gentamicin sulfate, 0.16 mg/mL cinnamaldehyde, and
0.025 mg/mL FD&C Red No. 2.
(d) Conjugate.Peroxidase-conjugated antibodies to Salmonella species (lyophilized).
(e) TMB peroxidase substrate (Solution A).3,3,5,5Tetramethylbenzidine, 0.4 g/L, in proprietary organic base.
(f) TMB peroxidase substrate (Solution B).Citric acid
buffer containing 0.02% hydrogen peroxide.
(g) Wash concentrate, 50.50% glycerol and 2.5%
Tween 20 in H2O.
(h) Stop solution.2N sulfuric acid. (Caution: Corrosive,
avoid contact with skin. If contact occurs, wash area thoroughly with water.)
(i) MN broth (M-broth with 10 g/mL novobiocin).5.0 g
yeast extract, 12.5 g tryptone, 2.0 g D-mannose, 5.0 g Na citrate, 5.0 g NaCl, 5.0 g K2HPO4, 0.14 g MnCl2, 0.8 g MgSO4,
0.04 g FeSO4, and 0.75 g Tween 80. Suspend ingredients in
1 L H2O, and heat to boiling and boil 12 min. Dispense 9 mL
portions into 16 125 mm screw-cap tubes. Cap tubes loosely
and autoclave 15 min at 121. Prepare novobiocin stock solution (100 g/mL in H2O) and filter-sterilize. Add 1.0 mL novobiocin solution aseptically to each sterile MN broth tube.
Novobiocin stock solution may be stored at 70, but do not
refreeze after thawing.
(j) Diagnostic reagents.See 967.25B (for culture confirmation of presumptive positive EIA tests).
Items B(a)(d) and C(a)(h) are available as SalmonellaTek ELISA test system (Organon Teknika Corp., 100 Akzo
Ave, Durham, NC 27704).

D. General Instructions
All kit components used in an assay must be same kit lot
number. Do not use materials after expiration date.
Bring foil pack to room temperature (2025) before opening to prevent condensation on strips. After foil pack has been
opened, strips are stable for 3 months at 28 if foil pack is
resealed with clamp and rod provided and silica gel bag is not
removed.
It is not necessary to perform immunoassay under sterile
conditions.
Bring components and test samples to room temperature
(2025) before testing begins. Include 1 positive control and
2 negative controls with each group of test samples. All controls must function properly for test to be valid. Use separate
pipet tips for each sample and kit reagent to avoid cross contamination. Keep plastic troughs well separated, if used to dispense conjugate and substrate. Store all components at 28
when not in use.
Do not reuse microtiter strips.

E. Preparation of Sample
(1) Pre-enrichment.Pre-enrich product in noninhibitory
broth to initiate growth of Salmonella. Methods used may vary
with product, and should be performed as in 967.26A, or in
current edition of the Bacteriological Analytical Manual with
the following exceptions:
Raw or highly contaminated products.Aseptically weigh
ca 25 g sample into sterile blender jar. Add 225 mL sterile lactose broth. Blend 2 min at high speed (ca 20 000 rpm). Cap jar
securely and let stand 60 min at room temperature. Mix well
by shaking and determine pH with test paper. Adjust pH to 6.8
0.2, if necessary, using sterile 1N NaOH or HCl, capping jar
securely and mixing well before determining final pH. Aseptically transfer contents of each jar to sterile wide-mouth screwcap 500 mL jar. Loosen jar cap 14 turn and incubate 24 2 h
at 35.
Processed food products.Incubate pre-enrichments 18
24 h at 35.
(2) Selective enrichment.Transfer 1 mL incubated preenrichment mixture to selenite cystine broth and 1 mL to
tetrathionate broth as in 967.26B(a), except prewarm
tetrathionate broth to 42 and incubate in 42 0.5 water bath.
For all foods other than raw or highly contaminated products,
incubate 68 h. Selective enrichments of raw or highly contaminated products must be incubated 1824 h.
(3) Post-enrichment.Remove selective broths from incubation and hand mix or use vortex mixer. Remove 1 mL from
tetrathionate broth tube and transfer to 10 mL tube of sterile
MN broth prewarmed to 42 0.5. Remove 1 mL from selenite cystine broth tube and transfer to separate tube of prewarmed MN broth. For all foods other than raw or highly contaminated products, incubate MN broths 1418 h in 42 0.5 water
bath and incubate selective enrichments an additional 1418 h
at 42 0.5 (tetrathionate broth) or 35 (selenite cystine broth).
For raw or highly contaminated products, incubate MN broth
tubes 46 h at 42 0.5 and incubate selective enrichment

broths an additional 6 h at their respective incubation temperatures.

(4) Preparation of sample for EIA analysis.Remove MN
broth tubes from incubation and hand mix or use vortex mixer.
Combine 0.5 mL from each MN broth tube in clean screw-cap
tube and heat in boiling water bath or in flowing steam 20 min.
Store remaining tubes of MN, tetrathionate, and selenite cystine broths from (3) at 28 for culture confirmation of any
positive samples. Cool heated MN broths to 2537 prior to
EIA analysis.

F. Enzyme Immunoassay
Prepare following reagents prior to commencing assay:
(1) Prepare 1 wash solution by diluting 10 mL 50 wash
concentrate, C(g), with 490 mL H2O.
(2) Reconstitute control antigens by adding 2 mL sterile
H2O to negative control vial and 1 mL sterile H2O to positive
control vial. Mix well. Reconstituted antigens are stable
60 days stored at 28.
(3) Reconstitute conjugate by adding 6 mL conjugate diluent, C(c), to vial of lyophilized conjugate, C(d). Mix gently
by inverting bottle several times. Reconstituted conjugate is
stable 30 days stored at 28.
Remove needed number of strips from foil pouch (1 well per
food sample and 3 wells for controls). Secure strips in holder.
Transfer 0.1 mL each heated MN broth sample to single well.
Transfer 0.1 mL negative control solution, (2), into 2 wells and
0.1 mL positive control solution, (2), into 1 well.
Cover plate with plate sealer and incubate 30 min at 37.
After incubation, aspirate contents from wells and add 0.2
0.3 mL 1 wash solution, (1), to each well. Repeat this step 2
times. Aspirate final wash.
Pipet 0.1 mL reconstituted enzyme conjugate, (3), into each
well, cover, and incubate 30 min at 37.
Aspirate and wash each well 6 times with 0.20.3 mL 1
wash solution. Prior to aspiration of final wash, prepare TMB
substrate by mixing equal amounts TMB solution A, C(e), and
TMB solution B, C(f), in glass tube. Pipet 0.1 mL TMB substrate into each well. Incubate 30 min at room temperature
(2025). Add 0.1 mL stop solution, C(h), to each well. Read
plate within 1015 min as in G.

G. Reading
Select 450 nm wavelength on plate reader. Zero reader on
air (without stripholder and strips) and read absorbance, A, of
solution in each well. Calculate mean A for negative control
wells (should be <0.30). A for positive control must be >0.70.
A for controls must be within these limits for test to be valid.
Cutoff value is calculated by adding 0.25 to mean A for negative controls. Test sample is presumed positive if sample A is
greater than or equal to cutoff value. Test sample is negative if
sample A is less than cutoff value.

H. Confirmation of Positive EIA Samples

Positive EIA reading indicates Salmonella may be present.
Since antibodies may cross-react with a few other organisms,
culture confirmations should be performed by streaking Hek-

toen enteric (HE), xylose lysine deoxycholate (XLD), and bismuth sulfite (BS) agar plates from tetrathionate broth, selenite
cystine broth, and associated MN broth tubes as in 967.26B.
Identify typical or suspicious colonies as in 967.26C, 967.27
(alternatively, 978.24 or 989.12), and 967.28.
Ref.: JAOAC 77, 374 (1994)
Results and Discussion
Certain data sets (Apendixes 1-9) were deleted from the statistical analyses due to method deviations. Most data exclusions occurred because Salmonella was isolated by 1 or both
methods from uninoculated control samples (e.g., collaborator
4, Appendix 2). In addition, 1 collaborator (laboratory 16)
failed to perform biochemical identification procedures properly on 3 foods. One collaborator made the M-broth with the
incorrect concentration of novobiocin (collaborator 1, Appendix 1).

Nonfat Dry Milk, Milk Chocolate (Trial 1), Soy Flour,

Dried Egg, Black Pepper (Trial 1)
The following number of data were available for statistical
analysis: 11 for dried egg, 10 for nonfat dry milk, 10 for soy
flour, 8 for chocolate, and 7 for black pepper. In the nonfat dry
milk and the chocolate samples, Salmonella was detected in all
high and low contamination level samples by both methods by
all of the collaborators. In the dried egg samples, there were 21
high and 3 low contamination level samples found to contain
Salmonella by both methods by all collaborators, and in the
black pepper there were 35 high and 25 low contamination
level samples positive by both methods for all collaborators.
There was perfect agreement between the methods for these 4
foods. The soy flour data contained 10 high contamination
level samples which were positive for Salmonella by the
ELISA method and 11 positive by the culture, while there were
2 low contamination level positives by the ELISA and only 1
by culture method. Agreement between the methods in the soy
flour methods was 98% for both the high and low level inoculated samples. Since AOAC requires valid data from 10 laboratories in a qualitative collaborative study, the chocolate (8
valid data sets) and pepper (7 valid data sets) were repeated.

Ground Black Pepper (Trial 2)

There were 13 valid data sets for black pepper after eliminating data from 5 collaborators who isolated Salmonella from
control samples. All collaborators found 62 high and 22 low
contamination level samples positive using both methods. Two
Salmonella-Tek positive assay results could not be confirmed
using culture method.

Milk Chocolate (Trials 2 and 3)

Very few positive results were found by collaborators testing milk chocolate in Trial 2. MPN values were 0.004 cell/g for
high and <0.003 cell/g for low contamination level samples indicating the levels were too low for adequate recovery and relevant statistical analysis. There were 14 samples positive by the
ELISA and 16 by AOAC culture method. There were 2 Salmo-

nella-Tek positive assays, which could not be confirmed by the

reference culture method, and 4 false negative ELISA results.
To ensure adequate recovery, the third trial used 4 inoculation levels. There was perfect agreement between the methods
for the very high, high, and low contamination level (55 positives/level). There was 1 BAM/AOAC false negative result in
the very low contamination level samples and 3 SalmonellaTek positive assays which were not able to be confirmed by
culture methods.

Raw Ground Turkey

Naturally contaminated turkey meat was used for this study.
As a result, all data were considered to be valid, even if salmonellae were isolated from the steamed control samples.
Eleven laboratories evaluated the turkey samples. There were
69 samples positive by both methods, 26 detected by the
ELISA method alone, 6 samples positive by culture only, and
64 negatives by both methods.
The turkey data indicate that the ELISA method has greater
sensitivity (Table 3) than the reference culture method. This
difference may be due to the use of a pre-enrichment step for
the ELISA method, and also may be attributed to the use of
elevated temperature for incubation of the selective and postenrichment broths. A direct comparison of each individual sample cannot be made since the individual turkey samples were
incubated in separate and different pre-enrichments and enrichments. Since the results were not obtained from a common preenrichment as with the other test food samples, some variability
can be expected in results obtained. However, a general trend
of superior ELISA performance against the AOAC culture
method (Tables 4 and 5) can be observed in the raw turkey
samples.
The Salmonella-Tek ELISA method was equivalent to the
BAM/AOAC culture method in all foods tested with the exception of the raw ground turkey, where the ELISA method was
more productive than culture method.
Recommendation
On the basis of the results of this study, it is recommended
that Salmonella detection in all foods by colorimetric monoclonal enzyme immunoassay (Salmonella-Tek) method be
adopted first action.
Acknowledgments
The authors thank the following:
M. Befus, R. Koeritzer, L. Saunders, and K. Vought, Land
OLakes, Arden Hills, MN
S. Bell, J. Fechner, L. Harris, C. Kennett, M. Schulke, V.
Smith and L. Stoltzner, Kraft General Foods, Glenview, IL
J. Benson, Silliker Laboratories of Georgia, Stone Mountain, GA
D. Blank, C. Monaghan, and M. Pratt, USDA/FSIS, Midwestern Laboratory, St. Louis, MO
J. Bozzuffi, B. Rodriguez, K. Schneider, and R. Sparano,
M&M Mars, Hackettstown, NJ

D. Bryant, D. Higgins, and B. Robison, Organon Teknika

Corp., Durham, NC
M. Buchanan, M. Cirigliano, R. McKenna and J. Stone, Lipton, Englewood, NJ
T. Burns and P. Perez, Quality Sausage, Dallas, TX
D.C. Clifford and R. Dyer, Nestle USA, Inc., Dublin, OH
M. Cook, Tyson Food, Springdale, AR
A.E. Cruse, FDA-NY, Brooklyn, NY
M. Davis and J. Kornacki, Silliker Laboratories of Pennsylvania, Sinking Spring, PA
C. Dittman, O. Imami, N. Richmond and R. Schnitzer, SaniPure, Saddlebrook, NJ
W. Dustman and K. Eckner, Silliker Laboratories Research,
Chicago Heights, IL
K. Faucher, Sandoz Nutrition, Minneapolis, MN
E.W. Frampton and L. Restaino, R & F Laboratories/Gycor
International, Bridgeview, IL
J. Fuls and K. Jost-Keating, Silliker Laboratories of New
Jersey, Garwood, NJ
K. Givich, Guittard Chocolate, Burlingame, CA
R. Hamilton and E. McHugh, Quaker Oats, Barrington, IL
A. Holder and M. Peterson, Standard Process Laboratories,
Palmyra, WI
C. Lin, Silliker of California, Carson, CA
M.M. Lister and C.N. Roderick, FDA-Dallas, Dallas, TX
R. Lubbat, Chem-Bio Consultants and Laboratories, Chicago, IL
G. Miklovic and D. Potel, Stouffer Frozen Foods, Solon, OH
C. OBryan, Hudson Food Laboratory, Rogers, AR
C.U. Okolo, Con-Agra Frozen Food Co., Batesville, AR
U. Purvis, Health Protection Branch, Health and Welfare
Canada, Scarborough, Ontario, Canada
W. Reid, Silliker Laboratories of Illinois, Chicago Heights, IL
P. Trost, Seafood Products Research Center, Bothell, WA
C. Wilch, IBP, Dakota City, NE

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B. Ray (Ed.), CRC Press, Inc., Boca Raton, FL, pp. 55113
(9) DAoust, J.-Y. (1984) J. Food Prot. 47, 588590
(10) DAoust, J.-Y., Daley, E., & Sewell, A.M. (1990) J. Food
Prot. 53, 841845
(11) Kafel, S., & Bryan, F.L. (1977) Appl. Environ. Microbiol. 34,
285291
(12) Miller, M.L., & Koburger, J.A. (1984) J. Food Prot. 47, 267
269
(13) Morris, G.K., & Dunn, C.G. (1970) Appl. Microbiol. 20,
192195
(14) Rengel, A., & Mendoza, S. (1984) J. Food Prot. 47, 213216
(15) Silliker, J.H., & Gabis, D.A. (1974) Can. J. Microbiol. 20,
813816
(16) Yde, M., & Ghysels, G. (1984) J. Food Prot. 47, 217219
(17) Bailey, J.S., Cox, N.A., & Blankenship, L.C. (1991) J. Food
Prot. 54, 354356
(18) McClure, F.D. (1990) J. Assoc. Off. Anal. Chem. 73, 953960

Table 993.08A Method performance (specificity rate) for colorimetric, monoclonal, enzyme-linked immunosorbent
assay for Salmonella in foods
ELISA method
a

Food

Nonfat dry milk

Milk chocolate 1
Milk chocolate 2
Milk chocolate 3
Soy flour
Dried egg
Black pepper 1
Black pepper 2
a
b
c
d

Reference method

Known negativeb

Test negativec

Specificity rated

Test negativec

Specificity rated

50
40
95
55
50
55
35
65

46
40
95
52
49
55
35
64

0.92
1.00
1.00
0.94
0.98
1.00
1.00
0.98

50
40
95
55
50
55
35
65

1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00

All samples were uninoculated, Salmonellanegative, control samples.

Known negative (McClure, 1990) represents total number of expected negative samples.
Test negative is number of known negative samples which tested negative.
Specificity is test negative to known negative ratio (McClure, 1990).

Table 993.08B Method performance (sensitivity rate) for colorimetric, monoclonal, enzyme-linked immunosorbent
assay for Salmonella in foods
ELISA method

Reference method

Food

MPN level

Known positivea

Test positiveb

Sensitivity ratec

Test positiveb

Sensitivity ratec

Nonfat dry milk

High
Low
High
Low
High
Low
Very High
High
Low
Very Low
High
Low
High
Low
High
Low
High
Low
High
Low

50
50
40
40
95
95
55
55
55
55
50
50
55
55
35
35
65
65
55
55

50
50
40
40
12
0
55
55
55
55
10
1
21
3
35
25
62
22
54
34

1.00
1.00
1.00
1.00
0.13
0.00
1.00
1.00
1.00
1.00
0.20
0.02
0.38
0.055
1.00
0.71
0.95
0.34
0.98
0.62

50
50
40
40
16
0
55
55
55
54
10
1
21
3
35
25
62
22
42
27

1.00
1.00
1.00
1.00
0.17
0.00
1.00
1.00
1.00
0.98
0.20
0.02
0.38
0.055
1.00
0.71
0.95
0.34
0.76
0.49

Milk chocolate 1
Milk chocolate 2
Milk chocolate 3

Soy flour
Dried egg
Black pepper 1
Black pepper 2
Raw ground turkey

a
b
c

Known positive (McClure, 1990) represents total number of inoculated or naturally-contaminated samples.
Test positive is number of known samples which tested positive and were confirmed.
Sensitivity is test positive to known positive ratio (McClure, 1990).

Table 993.08C Method performance (summary) for

colorimetric, monoclonal, enzyme-linked
immunosorbent assay for Salmonella in foods

Agreement
b
False negative (EIA)
c
False negative (AOAC/BAM)

Food

Serotype

MPN levela

Cells/g

High
Low
High
Low
High
Low
Very high
High
Low
Very low
High
Low
High
Low
High
Low
High
Low

1.100
0.023
0.053
0.016
0.004
<0.003
1.100
0.460
0.093
0.007
0.023
0.003
0.023
0.004
>1.100
0.023
0.093
0.009

High
Low

0.460
0.093

Results

Table 1. Salmonella serotypes, inoculum levels, and

food types used in the collaborative study

97.6
1.6
4.0

Proportion of samples which agree between AOAC/BAM culture

method and EIA method expressed as a percentage.
Proportion of samples confirmed as positive by AOAC/BAM
culture method but negative by EIA method expressed as a
percentage of total number of positive samples.
Proportion of samples confirmed as positive by EIA culture method
but negative by AOAC/BAM method expressed as a percentage
of total number of positive samples.

Nonfat dry milk

S. cubana

Milk chocolate 1

S. senftenberg

Milk chocolate 2

S. senftenberg

Milk chocolate 3

S. senftenberg

Soy flour

S. agona

Dry egg

S. montevideo

Black pepper 1

S. typhimurium

Black pepper 2

S. typhimurium

Raw turkey

a
b

naturally
contaminated

All control samples had MPN/g values of <0.003.

All foods except raw turkey were considered to have low levels of
competitive flora.

Table 2. Laboratory participation in collaborative study

Sample set
Laboratorya
1
2
4
5
6
7
8
9
10
12
13
14
15
16
17
18
19
21
22
23
24
25
26
27
28
29
30
31
32
33
a
b
c
d

NFDMb
c c

x
x
x
x
x
x
x
x
0
x
0
x
x
x
x
x
x
0
0
0
0
0
0
0
0
0
0
0
0
0

Milk
chocolate 1

Milk
chocolate 2

Milk
chocolate 3

x
x
x
x
x
x
x
x
0
x
0
x
x
x
x
x
0
0
0
0
0
0
0
0
0
0
0
0
0
0

x
x
x
x
x
x
0
0
0
x
0
0
x
0
x
x
0
x
x
x
x
x
x
x
x
x
x
0
0
0

d d

Laboratories 3, 11, and 20 withdrew from the study.

Nonfat dry milk.
Sample sent to collaborator.
Sample not sent to collaborator.

0
x
x
x
0
x
0
0
0
x
0
0
0
0
0
x
0
x
0
x
x
x
0
x
x
x
x
x
x
x

Soy flour

Dried egg

Black
pepper 1

Black
pepper 2

Raw
turkey

0
x
x
x
x
x
x
x
0
x
0
x
x
x
x
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

x
x
x
x
x
x
x
x
x
x
0
x
x
x
x
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
x
x
x
x
x
x
x
0
0
0
x
x
x
x
0
x
0
0
0
0
0
0
0
0
0
0
0
0
0

0
x
x
x
0
x
0
x
0
0
0
x
x
0
x
x
0
x
0
x
x
0
x
x
x
x
x
x
0
0

0
x
0
x
x
x
x
x
x
0
x
x
0
x
x
x
x
0
0
0
0
0
0
0
0
0
0
0
0
0

Table 3. Method performance (sensitivity) for Salmonella detection in all foods by colorimetric monoclonal enzyme
immunoassay
ELISA method

Reference method

Food

MPN level

Known positivea

Test positiveb

Sensitivityc

Test positive

Sensitivity

Nonfat dry milk

High
Low
High
Low
High
Low
Very High
High
Low
Very low
High
Low
High
Low
High
Low
High
Low
High
Low

50
50
40
40
95
95
55
55
55
55
50
50
55
55
35
35
65
65
55
55

50
50
40
40
12
0
55
55
55
55
10
1
21
3
35
25
62
22
54
34

1.000
1.000
1.000
1.000
0.126
0.000
1.000
1.000
1.000
1.000
0.200
0.020
0.382
0.055
1.000
0.714
0.954
0.338
0.982
0.618

50
50
40
40
16
0
55
55
55
54
10
1
21
3
35
25
62
22
42
27

1.000
1.000
1.000
1.000
0.168
0.000
1.000
1.000
1.000
0.982
0.200
0.020
0.382
0.055
1.000
0.714
0.954
0.338
0.764
0.491

Milk chocolate 1
Milk chocolate 2
Milk chocolate 3

Soy flour
Dry egg
Black pepper 1
Black pepper 2
Raw turkey

a
b
c

Total number of inoculated or naturally contaminated samples.

Number of known samples which tested positive and were confirmed.
Test positive to known positive ratio (McClure, 1990).

Table 4. Method performance (specificity) for Salmonella detection in all foods by colorimetric monoclonal enzyme
immunoassay
ELISA method
Food
Nonfat dry milk
Milk chocolate 1
Milk chocolate 2
Milk chocolate 3
Soy flour
Dry egg
Black pepper 1
Black pepper 2
a
b
c

Reference method

MPN level

Known
negativea

Test negativeb

Specificityc

Test negative

Specificityc

Control
Control
Control
Control
Control
Control
Control
Control

50
40
95
55
50
55
35
65

46
40
95
52
49
55
35
64

0.920
1.000
1.000
0.945
0.980
1.000
1.000
0.985

50
40
95
55
50
55
35
65

1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000

Total number of expected negative samples.

Number of known negative samples which tested negative.
Test negative to known negative ratio (McClure, 1990).

Table 5. Method performance: agreement and incidence of false negatives among positive samples
Incidence of false negatives
among positive samples,g %

Number of samples

Food

MPN level

ELISA +
Ref +a

ELISA +
Ref b

ELISA
Ref +c

ELISA
Ref d

Method
agreemente

ELISAf

Reference
method

Nonfat dry milk

High
Low
Control
High
Low
Control
High
Low
Control
Very high
High
Low
Very low
Control
High
Low
Control
High
Low
Control
High
Low
Control
High
Low
Control
High
Low
Control

50
50
0
40
40
0
12
0
0
55
55
55
54
0
10
1
0
21
3
0
35
25
0
62
22
0
42
22
5

0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
8
12
6

0
0
0
0
0
0
4
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
4
2

0
0
50
0
0
40
79
95
95
0
0
0
0
55
39
48
50
34
52
55
0
10
35
3
43
65
5
17
42

100.0
100.0
100.0
100.0
100.0
100.0
95.8
100.0
100.0
100.0
100.0
100.0
98.18
100.0
98.0
98.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
85.45
70.91
85.45

0.00
0.00
0.00
0.00
0.00
0.00
25.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
9.09
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
10.53
15.38

0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1.82
0.00
0.00
50.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
16.00
31.58
46.15

Milk chocolate 1

Milk chocolate 2

Milk chocolate 3

Soy flour

Dried egg

Black pepper 1

Black pepper 2

Raw ground turkey

a
b
c
d
e

Samples confirmed positive by both methods.

Samples positive by confirmed ELISA and negative by AOAC cultural reference method.
Samples positive by AOAC cultural reference method and negative by confirmed ELISA.
Samples negative by both methods.
Agreement calculated as sum of samples confirmed positive and negative by both methods divided by the total number of confirmed
samples.
Incidence for ELISA method was samples negative by ELISA and positive by reference method divided by the sum of samples confirmed
positive by any method including controls.
Incidence for ELISA method was samples negative by ELISA and positive by reference method divided by the sum of samples confirmed
positive by any method.

Appendix 1. Analysis of nonfat dry milk samples for Salmonella cubana

High-level samples
Lab.

Low-level samples

Control samples

11

12

10

14

13

15

Salmonella-Tek assay result

a

1
2
4
5
6
7
8
9
12
14
15
c
16
17

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

d b
+

d b
+

d b
+

d b
+

Salmonella-Tek assay confirmation result

1
2
4
5
6
7
8
9
12
14
15
c
16
17

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

d d

d d

AOAC cultural method

1
2
4
5
6
7
8
9
12
14
15
c
16
17
a
b
c
d

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

Improper novobiocin concentration used in M-broth. Data excluded from statistical analysis.
Salmonella-Tek positive assay unable to be confirmed by culture method.
Improper biochemical identification procedures followed. Data excluded from statistical analysis.
Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.

Appendix 2. Analysis of milk chocolate samples for Salmonella senftenberg (trial 1)

High-level samples
Lab.

Low-level samples

Control samples

13

14

11

15

10

12

Salmonella-Tek assay result

1
2
4
5
6
7
8
9
12
14
15
b
16
17

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

d a
+

d a
+

d a
+

Salmonella-Tek assay confirmation result

1
2
4
5
6
7
8
9
12
14
15
b
16
17

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

d a
+

d a
+

d a
+

d a
+

d a

AOAC cultural method

1
2
4
5
6
7
8
9
12
14
15
b
16
17
a
b

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.
Improper biochemical identification procedures followed. Data excluded from statistical analysis.

Appendix 3. Analysis of milk chocolate samples for Salmonella senftenberg (trial 2)

High-level samples
Lab.

Low-level samples

Control samples

11

15

12

13

10

14

d b
+

d a

+
+

d a

d a

d b
+

d a

Salmonella-Tek assay result

1
2
4
5
6
7
12
15
17
18
21
22
23
24
25
26
27
28
29
30

d a

Salmonella-Tek assay confirmation result

1
2
4
5
6
7
12
15
17
18
21
22
23
24
25
26
27
28
29
30

d a
+

+
+

+
+

d a
+

Appendix 3. (continued)
High-level samples
Lab.

Low-level samples

Control samples

11

15

12

13

10

14

+
+

+
+

d c
+

AOAC cultural method

1
2
4
5
6
7
12
15
17
18
21
22
23
24
25
26
27
28
29
30
a
b
c

Salmonella-Tek false negative.

Salmonella-Tek positive assay unable to be confirmed by culture method.
Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.

Appendix 4. Analyses of milk chocolate samples for Salmonella senftenberg (trial 3)

Very high level
Lab. 2

High level

17 23 25

Low level

Very low level

Control

12 19 22

18 21 24

11

14 16 20

10 13 15

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

d b
+
d a
+
d a
+

d a
+

Salmonella-Tek assay result

2
4
5
7
12
18
21
23
24
27
28
29
30
31

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

Salmonella-Tek assay confirmation result

2
4
5
7
12
18
21
23
24
27
28
29
30
31

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

d b
+

d b
+

d b
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
d c

+
+
+
d c

+
+
+
+
d c

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

d b
+

d b
+

d b
+

AOAC cultural method

2
4
5
7
12
18
21
23
24
27
28
29
30
31
a
b
c

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

Salmonella-Tek positive assay result unable to be confirmed by cultural method.

Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.
AOAC reference method false-negative result.

Appendix 5. Analysis of soy flour samples for Salmonella agona

High-level samples
Lab.

Low-level samples

Control samples

10

13

11

14

12

15

+
+

d b

+
+

d a
+

c c
+

Salmonella-Tek assay result

2
4
5
6
7
8
9
12
14
15
16

Salmonella-Tek assay confirmation result

2
4
5
6
7
8
9
12
14
15
16

d a
+

+
+

+
+

d d
+

d d
+

+
+

+
+

e e

AOAC cultural method

2
4
5
6
7
8
9
12
14
15
16
a
b
c
d
e

d a
+

Group E1 Salmonella isolated; Group B Salmonella inoculated into sample.

Salmonella-Tek false negative.
Salmonella-Tek positive assay unable to be confirmed by culture method.
Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.
AOAC reference method false negative.

Appendix 6. Analysis of dried egg samples for Salmonella montevideo

High-level samples
Lab.

Low-level samples

Control samples

10

14

12

13

11

15

+
+

+
+

+
+
+

+
+

a a
+
a a
+

Salmonella-Tek assay result

1
2
4
5
6
7
8
9
10
12
14
15
16

+
+
+

+
+

Salmonella-Tek assay confirmation result

1
2
4
5
6
7
8
9
10
12
14
15
16

+
+
+

+
+

+
+

+
+

+
+
+

+
+

a a
+
a a
+

+
+

+
+

+
+
+

+
+

a a
+
a a
+

AOAC cultural method

1
2
4
5
6
7
8
9
10
12
14
15
16
a

+
+
+

+
+

Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.

Appendix 7. Analysis of ground black pepper samples for Salmonella typhimurium (trial 1)
High-level samples
Lab.

10

11

12

Low-level samples

Control samples

13

14

15

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+

+
+
+

+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+a

+a

+a

+a

Salmonella-Tek assay result

2
4
5
6
7
8
9
14
15
16
18

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

Salmonella-Tek assay confirmation result

2
4
5
6
7
8
9
14
15
16
18

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+

+
+
+

+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+a

+a

+a

+a

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+

+
+
+

+
+
+

+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+a

+a

+a

+a

AOAC cultural method

2
4
5
6
7
8
9
14
15
16
18
a

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.

Appendix 8. Analysis of ground black pepper for Salmonella typhimurium (trial 2)

High-level samples
Lab.

10

Low-level samples

Control samples

11

14

12

13

15

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+

+
+
+

+
+
+

+
+
+

+
+
+
+
+

+
+
+

+
+

a a
+

+
+
+

+
+

a a
+

a a
+

+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+

+
+
+

+
+
+

+
+
+

+
+
+
+
+

+
+
+

+
+

+
+
+

+
+

a b
+

a b
+

a b
+

a b
+

a b

a b
+

a b
+

Salmonella-Tek assay result

2
4
5
7
9
14
15
17
18
21
23
24
26
27
28
29
30
31

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Salmonella-Tek assay Confirmation result

2
4
5
7
9
14
15
17
18
21
23
24
26
27
28
29
30
31

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

a b
+

a b
+

a b
+
a b
+

Appendix 8. (continued)
High-level samples
Lab.

Low-level samples

Control samples

10

11

14

12

13

15

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+

+
+
+

+
+
+

+
+
+
+
+

+
+
+

+
+

+
+
+

a b
+

a b
+

a b
+

a b
+

a b
+

a b
+

a b
+

a b
+

AOAC cultural method

2
4
5
7
9
14
15
17
18
21
23
24
26
27
28
29
30
31
a
b

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

Salmonella-Tek positive assay unable to be confirmed by culture method result.

Salmonella isolated from uninoculated control samples. Data excluded from statistical analysis.

Appendix 9. Analysis of ground raw turkey samples for Salmonella spp.

High-level samples
Lab.

Low-level samples

Control samples

10

15

11

13

12

14

Salmonella-Tek assay result

2
5
7
8
9
10
13
14
b
16
17
18
19

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
a a

+
+

+
+
+
+
+
+

+
+

+
cc a

+
+
+
+
+

c a

+
+
+
+

c a

+
+
+

+
+
+

+
+
+

+
+
+

+
+
+

+
+
+

+
+

c a

+
+
c a

Salmonella-Tek assay confirmation result

2
5
7
8
9
10
13
14
b
16
17
18
19

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+

+
+

+
+

+
+

+
+
+

+
+
+

+
+
+

+
+
+

+
+
+

+
+
+

+
+

+
+

AOAC cultural method

2
5
7
8
9
10
13
14
b
16
17
18
19
a
b
c

+
+
+
+
+
+

c c

+
+
+
+
c c

+
c

c c

+
+
+
+
+
+

c c

+
+
c c

c c

+
+

+
+
+
c c

+
+

+
+
c c

c c

+
+

c c

+
+
+
+
c c

c c

c c

c c

c c

c c

+
+

c c

c c

+
+
+

+
+
+

c c

+
+
+

+
+
+

c c

+
+
c c

+
+

c c

c c

c c

+
+

c c

Salmonella-Tek false negative result.

Data incomplete; improper biochemical procedures followed. Data excluded from statistical analysis.
AOAC reference method false-negative result.

c c

Figure 1. Analysis of raw, fleshy (high-level

competitive microflora products.

Figure 2. Analysis of processed (low-level competitive

microflora) products.