470 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO.

2, 2002

MICROBIOLOGICAL METHODS

Method Extension Study to Validate Applicability of AOAC

â
Official Method 997.03 Visual Immunoprecipitate Assay (VIP )
for Listeria monocytogenes and Related Listeria spp. from
Environmental Surfaces: Collaborative Study
PHILIP T. FELDSINE, ANDREW H. LIENAU, STEPHANIE C. LEUNG, and LINDA A. MUI
BioControl Systems, Inc., 12822 SE 32nd St, Bellevue, WA 98005

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Collaborators: G. Aguilar, J. Aharchi, I. Aldridge, V. Arling, B. Bitner, C. Bullard, P. Carlson, C. Cox, K. Deiss, J. Dillon,
P. Dombroski, J. Ellingson, S. Fitzgerald, R. Forgey, K. Gailbreath, D. Gallagher, V. Geftman, K. Herbst, P. Hillis,
M. Johnson, S. Koch, D. Lewis, J. Luepke, D. Martensen, S. McDonagh, B. McGovern, B. Moon, L. Moreland, L. Murray,
D. Richter, M. Robertson, P. Rogers, C. Rucker, J. Sacca, M.-C. Siu, C. Smith, J. Smith, E. Stoltzfus, C. Summers,
B. Taylor, J. Toth, R. Vess, S. White, J.-L. Witt, S. Young

Test portions from 3 environmental surface types,
representative of typical surfaces found in a food
production facility, were analyzed by the Visual
Immunoprecipitate assay (VIPâ) and the U.S. De-
partment of Agriculture/Food Safety and Inspec-
tion Service (USDA/FSIS) culture method for Liste-
ria monocytogenes and related Listeria species. In
all cases, naturally contaminated environmental
test samples were collected from an actual food
production facility by sponge or swab. Test sam-
ples from concrete surfaces were collected by both
swab and sponge; sponge test samples were col-
lected from rubber surfaces, and swabs were used
to sample steel surfaces. Test portions from each
surface type were simultaneously analyzed by both
methods. A total of 27 laboratories, representing
government agencies as well as private industry in
both the United States and Canada, participated in
the study. During this study, a total of 615 test por-
tions and controls was analyzed and confirmed, of
which 227 were positive and 378 were negative by
both methods. Nine test portions were positive by
culture, but negative by the VIP. Five test portions
were negative by culture, but positive by the VIP.
Four test portions were negative by VIP and by cul-
ture, but confirmed positive when VIP enrichment
broths were subcultured to selective agars. The
data reported here indicate that the VIP method
and the USDA/FSIS culture method are statistically
equivalent for detection of L. monocytogenes and
Submitted for publication December 2001.
The recommendation was approved by the Methods Committee on
Microbiology and Extraneous Materials as First Action. See “Official
Methods Program Actions,” (2002) Inside Laboratory Management,
January/February issue.
Corresponding author’s e-mail: ptf@biocontrolsys.com.
related Listeria species from environmental sur-
faces taken by sponges or swabs.
he Visual Immunoprecipitate assay (VIP) for Listeria
T is currently AOAC Official Method 997.03 for food
products (1). An applicability revision comparative
study (unpublished) was conducted to demonstrate the equiv-
alence of the VIP to the U.S. Department of Agriculture/Food
Safety and Inspection Service (USDA/FSIS) culture
method (2) for the detection of L. monocytogenes and related
Listeria species in environmental surfaces. This study, previ-
ously approved by the AOAC Microbiology Committee,
compared 9 surface types representative of a wide variety of
food production site surfaces. A total of 520 test portions and
controls was analyzed, and no statistically significant differ-
ence was detected between the VIP for Listeria and the refer-
ence culture method. A collaborative study examining natu-
rally contaminated surfaces was conducted to support a
method applicability statement revision to include environ-
mental surfaces.
An assay was developed which uses highly specific anti-
bodies directed against antigens produced by Listeria. This as-
say is configured in a single-use device that produces a visu-
ally detectable reaction on a solid support in the presence of
L. monocytogenes and related Listeria species.
To conduct this assay, the analyst collects a test sample
from the environmental surface by either a sponge or swab
premoistened with an appropriate neutralizing medium. The
sponge is enriched in a sterile plastic bag containing 60 mL
modified Fraser broth with lithium chloride (mFB+LiCl); the
swab is placed in a 10 mL tube of mFB+LiCl. The enrich-
ments for both devices are incubated at 30°C for 28 ± 2 h. A
secondary transfer of 1 mL mFB+LiCl to 9 mL buffered Liste-
ria enrichment broth (BLEB) is made and incubated for 24 ±
2 h at 30°C. After this incubation, a 1.0 mL aliquot is
 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002 471
heat-inactivated at 100°C for 5 min. The analyst transfers
0.1 mL inactivated enriched broth into the sample addition
well of the VIP device. This initiates a lateral flow of broth
along the surface of a solid support. During the initial
hydration of the device, Listeria reacts with an anti-
body-chromogen complex contained in the device. The anti-
gen-antibody-chromogen complex formed flows across lat-
eral flow membrane and is subsequently bound by antibody
immobilized on the device membrane. If Listeria is present in
the test portion, a detection line will form which is positioned
across the solid support in a viewing window of the device.
The formation of a visually detectable line indicates a positive
reaction. Additionally, in a procedural control window, a sec-
ond line is formed, indicating proper test completion. Absence
of a procedural control line indicates an invalid test. After en-
richment, total assay time is about 10 min from addition of in-
cubated broth to sample addition well.
Study Design
The VIP for Listeria was previously validated as AOAC
Official Method 997.03. A comparative study was performed
analyzing 20 diverse food types (unpublished). Additionally,
a 6 food collaborative study was conducted (1).
The VIP method was subjected to 2 separate phases of test-
ing for a method applicability statement revision to include
environmental surfaces. The first completed phase was an
in-house pre-collaborative validation study that has been ap-
proved by the Microbiology and Extraneous Materials
Methods Committee (unpublished). A separate
multi-laboratory collaborative study is reported here. Test
samples from environmental surfaces naturally contaminated
with Listeria were collected from an actual food production
facility. Each sponge or swab test portion was homogenized
and used for both the test and reference methods, allowing for
a true paired sample analysis.
Collaborative Study
Three surface types were analyzed for this study: concrete,
rubber, and steel. All test samples used in this study were ob-
tained from a food production facility with surfaces naturally
contaminated with Listeria. Environmental test samples for
concrete were collected by both the sponge and swab meth-
ods. Test samples from rubber and steel surfaces were col-
lected by only sponge or swab, respectively. Each participat-
ing collaborator analyzed 5 test portions from each
surface/test sample collection device: concrete/sponge, con-
crete/swab, rubber/sponge, and steel/swab. Five uninoculated
Dey-Engley (D/E) broths were included as controls on each
day that a particular surface was analyzed. A minimum of
15 laboratories agreed to analyze each surface/collection de-
vice combination.
Test Portion Preparation
Five separate sponge test samples and 5 separate swab test
samples were collected from surface type 1 (concrete) for the
first week of the study. In the second week of the study, 5 sep-
arate swab test samples were taken from surface type 2 (steel)
and 5 separate sponge test portions were taken from surface
type 3 (rubber). The sponges and swabs were premoistened
with D/E neutralizing broth before collection. A 5 cm2 area
was sampled for each swab and a 100 cm2 area was sampled
for each sponge. After collection, the sponges were trans-
ferred back into sterile plastic bags and the swabs were placed
in tubes containing 10 mL D/E neutralizing broth. All
swabs/sponges were immediately transported on ice to
BioControl Systems. Upon arrival, approximately 60 mL D/E
neutralizing broth was aseptically added to each sponge bag
and homogenized via stomacher. Each swab was homoge-
nized in the tube of D/E broth by vortex mixer. The D/E broth
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from the swab was then aseptically transferred to a sterile con-
tainer and diluted with an additional 60 mL D/E broth. For
each sponge and swab test portion, a 2.25 mL aliquot of D/E
broth was transferred to a sterile screw-cap tube and shipped
to participating laboratories on ice via an overnight delivery
service. Five uninoculated controls consisting of 2.25 mL
tubes of D/E broth were also shipped at the same time.
Test Portion Distribution
Each collaborator received one set of 15 test portions for
each week the study was run. The 10 environmental test por-
tions and 5 uninoculated controls were stored at 2–8°C upon
receipt until tested. Test portions were sent to participating
laboratories to arrive no later than the week preceding the
Monday that analyses were initiated. Collaborators were in-
structed to analyze each test portion by both the USDA/FSIS
culture procedure and the VIP method. Results obtained by
collaborators were submitted on summary forms with the ap-
propriate raw data.
Test Portion Analysis
Test portions from 2 surfaces/collection devices were
tested for each week of the study. For each 2.25 mL test por-
tion, collaborators were instructed to transfer a 1 mL aliquot to
9 mL mFB+LiCl for the VIP method, and another 1 mL to
9 mL University of Vermont Listeria enrichment broth
(UVM) for the USDA/FSIS method. For each week of analy-
sis, 5 test portions of uninoculated D/E broths along with a
positive and a negative control were tested. All test portions
and controls were enriched and analyzed according to the VIP
Official Method 997.03, and the USDA/FSIS culture
method (3).
Test Portion Confirmations
The appropriate enrichment broths from both the test and
reference methods were streaked onto modified Oxford agar
(MOX) plates for confirmation. At least 5 suspect colonies
(isolates with black halos) from the MOX plates were selected
for confirmation according to the Bacteriological Analytical
Manual (4).
472 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002
Statistical Analysis
A paired statistical analysis of the methods was performed
for each surface type/collection device using the method of
McNemar (5). A chi square value >3.84 was indicative of a
significant difference at the 5% probability level. Sensitivity,
specificity, and percent agreement were conducted according
to the method of McClure (6).
AOAC Official Method 997.03
Detection of Listeria monocytogenes
and Related Listeria Species in Selected Foods
and from Environmental Surfaces
Visual Immunoprecipitate Assay (VIP)
First Action 1997
Revised 2001
Final Action 2001
(Applicable to detection of Listeria monocytogenes and re-
lated Listeria species in dairy foods, red meats, pork, poultry
and poultry products, seafood, fruits, vegetables, nutmeats,
pasta, chocolate, eggs, bone meal, and from environmental
surfaces.)
Caution: L. monocytogenes infections can cause fetal
death. It is recommended that pregnant women
avoid handling this organism. Attention should be
given to sterilization of contaminated equipment
and media before disposal or reuse.
See Table 997.03 for the results of the interlaboratory study
supporting acceptance of the method.
A. Principle
In the VIP assay, proprietary antibodies, with high speci-
ficity to antigens of L. monocytogenes and related Listeria
species, are bound to a chromogenic carrier and separately to a
solid support matrix. These reagents are configured in a single
use device that will produce a visually determined reaction in
the presence of Listeria. During the initial hydration of the de-
vice, Listeria will react with an antibody-chromogen complex
contained in the device. Antigen-antibody-chromogen com-
plex is formed, which flows across lateral flow membrane and
is subsequently bound by antibody immobilized on mem-
brane. If Listeria is present in the test portion, a detection line
will form which is positioned across the solid support in a
viewing window of the device. The formation of a visually de-
tectable line indicates a positive reaction. Additionally, a pro-
cedural control window exists wherein a second line is formed
indicating proper test completion. Absence of a procedural
control line indicates an invalid test.
B. Media and Reagents
(a) Visual Immunoprecipitate assay (VIP) unit.—One for
each test portion (available as VIP for Listeria from
BioControl Systems, Inc., 12822 SE 32nd St, Bellevue, WA
98005, USA).
(b) Modified Fraser broth with lithium chloride (mFB +
LiCl).—Suspend 55 g of commercial Fraser broth base into
1 L water. Stir until completely dissolved. If necessary, warm
to dissolve powder. Do not overheat. Only after powder has
completely dissolved, add 4 g LiCl and stir until completely
dissolved. Sterilize by autoclaving at 121°C for 15 min. Do
not overheat. Do not add ferric ammonium citrate additive to
broth. Alternatively, prepare a 45% (w/v) LiCl solution by dis-
solving 45 g LiCl in enough water for final volume of 100 mL.
Filter sterilize the solution through a 0.2 µm filter. Add 2 mL
sterile LiCl stock solution to 225 mL sterilized mFB. If using a
commercially prepared sterile 8M LiCl stock solution (Sigma),
add 2.65 mL per 225 mL sterilized mFB.
(c) Buffered Listeria enrichment broth (BLEB).—Sus-
pend 36.1 g commercial Listeria enrichment broth in 1 L wa-
ter. Add 8.5 g 3-(N-morpholino)propanesulfonic acid
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(MOPS) free acid and 13.7 g MOPS sodium. Mix well and
heat to dissolve if necessary. Dispense in 9 mL aliquots. Ster-
ilize by autoclaving at 121°C for 15 min.
C. Apparatus
(a) Incubators.—Maintaining 30–32°C and 35–37°C.
(b) Micropipets.—Accurately dispensing 0.1 and 1.0 mL.
(c) Vortex mixer.—For mixing test portion tubes.
(d) Waterbath.—Maintaining 100°C. Alternatively, flow-
ing steam autoclave set at 100°C or dry heat block may be
used.
(e) Top loading balance.—For weighing test portions,
measuring up to 1000 g, sensitivity of ±0.1 g.
(f) Blender/stomacher.—For homogenizing test portions.
D. General Instructions
Store VIP units inside foil pouch with desiccant at ambient
temperature (15–25°C). After use, discard units into a decon-
tamination container and sterilize before disposal. Do not re-
use, and do not use VIP units after expiration date.
Run positive and negative control cultures to become fa-
miliar with interpretations of results.
E. Preparation of Test Portions
(a) Primary enrichment.—Food products.—Aseptically
weigh 25 g test portion or pipet a 25 mL aliquot into 225 mL
mFB + LiCl, B(b). Blend or stomach solid food to homoge-
nize test portion.
Environmental monitoring.—For environmental sponges,
ensure that the sponge is in a horizontal position in the bag and
add 60 mL mFB + LiCl, B(b), to sponge bag. If using a swab,
add swab test portion to 10 mL mFB + LiCl, B(b).
Mix well via stomacher or vortex mixer. Incubate 28 ± 2 h at
30°C. If test material produces a viscous liquid (i.e., powdered
cheese and meats), add ≤2.25 mL steamed Triton X-100 per
225 mL mFB + LiCl, B(b), preparation prior to incubation.
(b) Secondary enrichment.—Transfer 1 mL incubated
mFB + LiCl to 9 mL BLEB, B(c). Vortex mix tubes and incu-
bate at 30°C for 24 ± 2 h.
(c) Inactivation.—Vortex mix incubated BLEB tubes and
transfer 1.0 mL to a clean tube. Inactivate microorganisms at
100°C for 5 min. Cool tube 25–37°C before testing. Inacti-
vated tubes can be stored at 2–8°C up to 4 days prior to testing.
Table 997.03. Interlaboratory study results for the detection of Listeria monocytogenes and related Listeria spp. from environmental surfaces (VIP)
Incidence of false Incidence of false
negatives among total Specificity positives among total
Samples positive Sensitivity ratec positive test samples, %d ratee negative test samples, %f

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VIP Agreement between
Surface/ No. of Total No. test USDA/ USDA/ VIP and USDA/FSIS
collection device labs samples Pres.a Conf.a FSIS χ2b VIP FSIS VIP USDA/FSIS VIP VIP methods, %g

Steel/swab 24 120 36 34 36 0.2 90 95 10.0 5.0 98 2 95

Rubber/sponge 24 120 89 88 88 0.2 97 97 3.2 3.2 98 3 95
Uninoculated 24 120 1 0 0 — — — — — — — —
Concrete/sponge 17 85 55 56 57 0.5 96 100 3.5 0.0 100 0 98
Concrete/swab 17 85 51 51 51 NAh 100 100 0.0 0.0 100 0 100
Uninoculated 17 85 0 0 0 — — — — — — — —
a
Pres. = presumptive positive data; Conf. = culturally confirmed data.
b
χ2 as defined by McNemar is (|a – b| – 1)2 / (a + b) where a = test samples positive by VIP and negative by USDA/FSIS, and b = test samples negative by VIP and positive by USDA/FSIS. A χ2
value >3.84 indicates significance at p < 0.05.

FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002 473
c
Sensitivity rate is defined as total number of analyzed positive test portions among “known” positive test portions/laboratory divided by total number of “known” positive test portions/laboratory,
where “known” positive is defined as test samples confirmed positive by the reference method.
d
Incidence of false negatives is 100 – sensitivity rate.
e
Specificity rate is defined as total number of analyzed negative test portions among “known” negative test portions/laboratory divided by total number of “known” negative test portions/laboratory,
where “known” negative is defined as test samples confirmed negative by the reference method and negative controls.
f
Incidence of false positives is 100 – specificity rate.
g
Rate reflects number of confirmed determinations that were equivalent between VIP and USDA/FSIS.
h
Statistical analysis not applicable. Methods gave equivalent results.
474 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002

Store remaining BLEB enrichment tubes at 2–8°C for confir-
mation of presumptive positive tubes.
F. VIP Assay Procedure
(1) Open sealed pouch containing VIP units, B(a), and re-
move required number of tests. One device is necessary for
each test portion. Do not reuse VIP units. Reseal unused VIP
units in pouch containing desiccant. Store at ambient tempera-
ture (15–25°C) in a cool dark location.
(2) Gently mix inactivated enrichment broth. If the broth
has been previously stored in the cold, bring to room tempera-
ture before testing.
(3) Transfer 0.1 mL inactivated broth to sample addition
well.
confirmed positive when VIP secondary enrichment broths
were subcultured to selective agar. These 4 test portions were
counted as false negatives on both the VIP and reference side.
There were 4 presumptive positive test portions by the VIP
method that could not be confirmed culturally.
Tables 2–5 present individual collaborator results. Results
were analyzed by surface type/test sample collection method.
Table 997.03 provides the interlaboratory study results as well
Table 1. Collaborator participation for Listeria VIP by
surface type/test sample collection methoda

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(4) Incubate at ambient temperature for 10 min. Steel/ Rubber/ Concrete/ Concrete/
Lab swab sponge sponge swab
G. Reading and Interpreting Results
1 Y Y N N
Note: Examine device immediately after 10 min incuba-
tion. Otherwise, faint lines may develop because of nonspe- 2 Y Y N N
cific color development that should be disregarded. 3 Y Y Y Y
Examine VIP unit for the presence of distinct detection 4 Y Y Y Y
lines in both test sample and test verification windows. Lines 5 Y Y Y Y
should be dark when contrasted with white background and b
6 Y Y Y Yb
should extend across window. Intensity of test sample and test
7 Y Y Y Y
verification lines may differ. Tests is valid if line is present in
b b b
test verification window. 8 Y Y Y Yb
Test sample is considered positive when lines are present 9 Y Y Y Y
in test sample window and in test verification window. Test 10 Y Y Y Y
sample is considered negative when control is valid and no 11 Y Y Y Y
line is seen in test sample window. If no line is present in test 12 Y Y Y Y
verification window, test is invalid.
13 Y Y N N
Autoclave used VIP units 15 min at 121°C prior to discarding.
14 N N Y Y
H. Confirmation of Positive VIP Test Portions 15 Y Y Y Y
Presumptive positive tests must be confirmed using culture 16 Y Y Y Y
methods as described in the current edition of Bacteriological 17 Y Y Yb Yb
Analytical Manual, AOAC INTERNATIONAL, 18 Y Y Y Y
Gaithersburg, MD 20877, USA, or Microbiology Laboratory
19 Y Y Y Y
Guidebook, U.S. Department of Agriculture/Food Safety In- b b c
20 Y Y Y Yc
spection Service, Athens, GA 30604, USA. Isolate from pre-
viously enriched BLEB tubes. 21 Y Y N N
Refs.: J. AOAC Int. 80, 791(1997); 85, 472–474(2002) 22 Y Y N N
23 Y Y N N
Results 24 Y Y Y Y
25 Y Y Y Y
Twenty-seven collaborators participated in the study.
26 Y Y Y Y
Twenty participants agreed to analyze all 4 surface/test sam-
ple collection methods, and 7 analyzed only 2 of the sur- 27 Y Y Y Y
d
face/test sample collection methods (Table 1). At the end of Total 26 26 21 21
the study, valid data were submitted from 615 test portions: a
Y = collaborator analyzed this surface type; N = collaborator did
410 were naturally contaminated and 205 were controls. Data not analyze this surface type.
collected from the 615 test and control samples indicated that b
Uninoculated control test samples were confirmed as Listeria.
227 were confirmed positive and 378 were negative by both Results were not included in the statistical analysis for the
VIP and the culture methods. Five test portions were con- designated surface types.
c
firmed positive by VIP method but negative by culture, and Laboratory did not follow study instructions. Results were not
included in the statistical analysis for the designated surface types.
9 test portions were negative by VIP and positive by culture. d
Total number of laboratories providing data.
Four test portions were negative by VIP and by culture, but
 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002 475

Table 2. Results of steel/swab test samples analyzed Table 3. Results of rubber/sponge test samples
for the presence of Listeria analyzed for the presence of Listeria
Test sample Test sample
Steel Control Rubber Control
Lab 1 6 9 11 13 2 5 8 12 14 Lab 3 4 7 10 15 2 5 8 12 14
VIP methoda VIP methoda
1 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 1 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
2 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 2 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
3 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 3 +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– –/–
4 +/+ –/– –/– –/– +/+ –/– –/– –/– –/– –/– 4 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
5 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 5 +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– –/–
6 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 6 +/+ –/– +/+ –/– +/+ –/– –/– –/– –/– –/–
7 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 7 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
9 +/+ –/– –/– –/– +/+ –/– –/– –/– –/– –/– 9 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–

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10 +/+ –/– –/– –/– +/+ –/– –/– –/– –/– –/– 10 +/+ +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/–
11 +/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 11 +/+ +/+ +/+ +/+ +/– –/– –/– –/– –/– –/–
12 –/– –/– +/+ –/– +/+ –/– –/– –/– –/– –/– 12 +/+ –/– +/+ +/+ +/+ –/– –/– –/– –/– –/–
13 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 13 +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– –/–
15 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 15 +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– –/–
16 +/+ –/– –/– –/– +/+ –/– –/– –/– –/– –/– 16 +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– –/–
17 –/– –/+ –/– +/– –/+ –/– –/– –/– –/– –/– 17 +/+ –/+ +/+ +/+ –/+ –/– –/– –/– –/– –/–
18 +/+ –/– –/– –/– +/+ –/– –/– –/– –/– –/– 18 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
19 –/– –/– +/+ –/– +/+ –/– –/– –/– –/– –/– 19 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
21 –/– –/– –/+ +/+ +/+ –/– –/– –/– –/– –/– 21 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
22 +/+ –/– –/– –/– +/+ –/– –/– –/– –/– +/– 22 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– +/–
23 +/+ –/– –/– –/– +/+ –/– –/– –/– –/– –/– 23 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
24 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 24 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
25 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 25 –/– –/– +/+ +/+ –/– –/– –/– –/– –/– –/–
26 –/– –/– –/– –/– +/+ –/– –/– –/– –/– –/– 26 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
27 –/– –/– +/+ –/– +/+ –/– –/– –/– –/– –/– 27 +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– –/–
USDA/FSIS method USDA/FSIS method
1 –b –b –b –b + – – – – – 1 + + + + –b – – – – –
2 – – – – + – – – – – 2 + + + + – – – – – –
3 – – – – + – – – – – 3 + – + + – – – – – –
4 + – – – + – – – – – 4 + + + + – – – – – –
5 – – – – + – – – – – 5 + – + + – – – – – –
6 – – – – + – – – – – 6 + – + – + – – – – –
7 – – – – + – – – – – 7 + + + + – – – – – –
9 + – – – + – – – – – 9 + + + + – – – – – –
10 + – – –b + – – – – – 10 + + + + – – – – – –
11 –b – – –b – – – – – 11 + + + + – – – – – –
12 – – + – + – – – – – 12 + – + + + – – – – –
13 – – – – + – – – – – 13 + – + + – – – – – –
15 –b – – – + – – – – – 15 + + + + –b – – – – –
16 + – – – + – – – – – 16 + – + + – – – – – –
17 – – – – – – – – – – 17 + – + + – – – – – –
18 + – – – + – – – – – 18 + + + + – – – – – –
19 – – + – + – – – – – 19 + + + + – – – – – –
21 – – + + + – – – – – 21 + + + + – – – – – –
22 + – – – + – – – – – 22 + + + + – – – – – –
23 + – – – + – – – – – 23 + + + + – – – – – –
24 + – – – + – – – – – 24 + + + + – – – – – –
25 – – – – + – – – – – 25 – – + + – – – – – –
26 – – – – + – – – – – 26 + + + + – – – – – –
27 – – + – + – – – – – 27 + + + + – – – – – –
a a
+, Listeria was detected in test sample; –, Listeria was not +, Listeria was detected in test sample; –, Listeria was not
detected in test sample; ( / ) = first entry is presumptive result / detected in test sample; ( / ) = first entry is presumptive result /
second entry is confirmed result. second entry is confirmed result.
b b
Selective plates had colonies typical of Listeria but were not Selective plates had colonies typical of Listeria but were not
confirmed as Listeria. confirmed as Listeria.
476 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002

Table 4. Results of concrete/sponge test samples Table 5. Results of concrete/swab test samples
analyzed for the presence of Listeria analyzed for the presence of Listeria
Test sample Test sample

Concrete Control Concrete Control

Lab 3 5 9 11 15 1 2 7 8 12 Lab 4 6 10 13 14 1 2 7 8 12

VIP methoda VIP methoda

3 –/– +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– 3 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
4 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 4 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
5 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 5 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
7 –/– +/+ –/+ +/+ +/+ –/– –/– –/– –/– –/– 7 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–

Downloaded from https://academic.oup.com/jaoac/article/85/2/470/5656511 by guest on 17 March 2022

9 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 9 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
10 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 10 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
11 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 11 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
12 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 12 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
14 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 14 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
15 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 15 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
16 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 16 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
18 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 18 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
19 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 19 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
24 –/– +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– 24 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
25 –/– +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/– 25 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
26 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 26 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
27 +/+ +/+ –/– +/+ +/+ –/– –/– –/– –/– –/– 27 –/– +/+ –/– +/+ +/+ –/– –/– –/– –/– –/–
USDA/FSIS method USDA/FSIS method

3 – + + + + – – – – – 3 – + – + + – – – – –
4 – + – + + – – – – – 4 – + – + + – – – – –
b
5 – + – + + – – – – – 5 – + – + + – – – – –
7 – + + + + – – – – – 7 – + – + + – – – – –
9 – + – + + – – – – – 9 – + – + + – – – – –
10 –b + – + + – – – – – 10 – + – + + – – – – –
b
11 – + – + + – – – – – 11 – + – + + – – – – –
12 – + – + + – – – – – 12 – + – + + – – – – –
14 – + – + + – – – – – 14 – + – + + – – – – –
15 –b + – + + – – – – – 15 – + – + + – – – – –
16 – + – + + – – – – – 16 – + – + + – – – – –
18 – + + + + – – – – – 18 – + – + + – – – – –
19 – + – + + – – – – – 19 – + – + + – – – – –
24 – + + + + – – – – – 24 – + – + + – – – – –
25 – + + + + – – – – – 25 – + – + + – – – – –
26 – + – + + – – – – – 26 – + – + + – – – – –
27 + + – + + – – – – – 27 – + – + + – – – – –

a a
+, Listeria was detected in test sample; –, Listeria was not +, Listeria was detected in test sample; –, Listeria was not
detected in test sample; ( / ) = first entry is presumptive result / detected in test sample; ( / ) = first entry is presumptive result /
second entry is confirmed result. second entry is confirmed result.
b
Selective plates had colonies typical of Listeria but were not
confirmed as Listeria.
 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002 477
as sensitivity and specificity data for each surface/test sample
collection method.
Steel Surface Test Portions Collected with Swabs
Twenty-six participants analyzed test portions from steel
surfaces. Laboratories 8 and 20 reported uninoculated controls
as confirmed positive for Listeria. Data from these laborato-
ries were not included in the analysis. Twenty-four collabora-
tors followed study instructions and appeared to have valid
data as indicated by data summary worksheets (Table 2). Frac-
tional recovery was observed for this surface type. Thirty-four
test portions were confirmed positive and 82 were negative by
both methods. Two test portions were negative by culture, but
confirmed positive by VIP, and 4 test portions were confirmed
positive by culture, but negative by VIP. There were 2 uncon-
firmed positive test portions reported by VIP. Two test por-
tions were negative by the culture method and negative by
VIP, but were confirmed positive when subcultured from the
broth. These test portions were reported as false negatives for
both the VIP and culture methods. Chi square analysis for
swab test portions collected from steel surfaces was 0.2, indi-
cating that the methods were equivalent. There was no statisti-
cally significant difference between the VIP method and the
USDA reference method in this test portion set.
Rubber Surface Test Portions Collected with
Sponge
Twenty-six collaborators agreed to participate in the analy-
sis of sponge test portions taken from rubber surfaces. Labora-
tories 8 and 20 reported uninoculated controls as confirmed
positive for Listeria. Data from these laboratories were not in-
cluded in the analysis. The remaining 24 collaborators fol-
lowed study instructions and appeared to have valid data as in-
dicated by data summary worksheets (Table 3). Fractional
recovery was observed for this surface type. Eighty-seven
were confirmed positive and 29 were negative by both meth-
ods. Three test portions were culture negative, but confirmed
VIP positive and 3 test portions were confirmed culture posi-
tive, but VIP negative. There was 1 unconfirmed positive test
portion reported by VIP. Two test portions were negative by
the culture method and negative by VIP, but were confirmed
positive when subcultured from the VIP enrichment broth.
These test portions were reported as false negatives for both
the VIP and culture methods. Chi square analysis for test por-
tions taken from rubber surfaces was 0.2, indicating equiva-
lent results between the VIP and USDA reference methods.
Concrete Surface Test Portions Collected with
Sponge
Twenty-one collaborators agreed to participate in the anal-
ysis of sponge test portions taken from concrete surfaces. Lab-
oratories 6, 8, and 17 reported uninoculated controls as con-
firmed positive for Listeria. Laboratory 20 did not follow
study instructions. Data from these laboratories were not in-
cluded in the analysis. The remaining 17 collaborators fol-
lowed study instructions and appeared to have valid data as in-
dicated by data summary worksheets (Table 4). Fractional
recovery was observed for this surface. Fifty-five test portions
were confirmed positive and 28 were negative by both meth-
ods. No test portions were culture negative and confirmed VIP
positive, and 2 test portions were confirmed culture positive
but VIP negative. There were no unconfirmed positive test
portions reported by VIP. Chi square analysis for concrete sur-
faces sampled by sponge was 0.5, indicating that the VIP and
USDA reference methods were equivalent.
Concrete Surface Test Portions Collected with Swab
Twenty-one collaborators agreed to participate in the anal-
ysis of swab test portions taken from concrete surfaces. Labo-
ratories 6, 8, and 17 reported uninoculated controls as con-
Downloaded from https://academic.oup.com/jaoac/article/85/2/470/5656511 by guest on 17 March 2022
firmed positive for Listeria. Laboratory 20 did not follow
study instructions. Data from these laboratories were not in-
cluded in the analysis. The remaining 17 collaborators fol-
lowed study instructions and appeared to have valid data as in-
dicated by data summary worksheets (Table 5). Fractional
recovery was observed for this surface. Fifty-one test portions
were confirmed positive and 34 were negative by both meth-
ods. No test portions were culture negative and confirmed VIP
positive. There were no unconfirmed presumptive positive
test portions for either the culture method or the VIP. Chi
square analysis for concrete surfaces sampled by swab was
not applicable as the 2 methods produced identical results, in-
dicating that the VIP and USDA reference methods were
equivalent.
Discussion
The analysis of all valid data for each surface type and test
sample collection method shows that of the 615 test portions,
227 were confirmed positive and 378 were negative by both
methods. Five test portions were negative by culture, but con-
firmed positive by VIP. Nine test portions were confirmed
positive by culture, but negative by VIP. All test samples were
from naturally contaminated surfaces taken from an actual
food production facility.
The USDA/FSIS procedure involves streaking all primary
enrichment UVM tubes, and any darkened Fraser broth tubes
from the secondary enrichment. Numerous enrichment broths
were observed where (1) the tubes had darkened yet there was
no growth on the MOX isolation agar plates after 48 h incuba-
tion, or (2) esculin-positive colonies were isolated on the
streaked MOX plates, but did not confirm to be Listeria. The
VIP for Listeria may help to minimize the number of pre-
sumptive positive plates required because it is not based on
esculin hydrolysis.
Conclusions
The data reported here indicate that the VIP method and the
USDA/FSIS culture method are statistically equivalent for de-
tection of L. monocytogenes and related Listeria species from
environmental surfaces sampled by sponges or swabs.
478 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002
Recommendations
It is recommended that the method applicability statement
for VIP for detection of L. monocytogenes and related Listeria
species be revised to include environmental surfaces and be
adopted First Action.
Acknowledgments
The participation of the following collaborators is ac-
knowledged with appreciation:
Gina Aguilar, Breneman Bitner, Dean Martensen, Morgan
Robertson, and Penny Rogers, Otto & Sons, West Jordan, UT
Joseph Aharchi and Cathy Smith, Washington State De-
partment of Agriculture, Olympia, WA
Irene Aldridge, Victoria Geftman, and Delando Lewis,
Strasburger & Siegel, Inc., Hanover, MD
Victoria Arling, Shelagh McDonagh, and Sandy White,
Canadian Food Inspection Agency, Calgary, Canada
Carolyn Bullard and Jerri-Lynn Witt, Tyson Foods,
Springdale, AR
Paul Carlson, Karen Deiss, and Jacqui Dillon, PSI,
Arlington, TX
Chantel Cox, Robin Forgey, and Christine Summers,
Costco Wholesale, Issaquah, WA
Pete Dombroski, Illinois Department of Public Health,
Springfield, IL
Jay Ellingson, Kasey Herbst, and Julie Luepke, Marshfield
Laboratories, Marshfield, WI
Sung Fitzgerald, Peter Pan/Seablends, Seattle, WA
Katherine Gailbreath, Sandy Koch, Brian McGovern, and
Lynn Murray, Analytical Laboratories, Boise, ID
Donna Gallagher, BioControl Systems, Inc., Bellevue,
WA
Paulette Hillis and Corinne Rucker, Valley Fresh, Turlock,
CA
Monique Johnson and John Toth, A&L Analytical Labora-
tories, Memphis, TN
Bonnie Moon, Michigan Department of Agriculture, East
Lansing, MI
Landon Moreland, K&N Meats, Renton, WA
Dawn Richter, Heinz Frozen Food Co., Ontario, OR
Julie Sacca and Robert Vess, Omnitech Laboratories,
Marietta, GA
Mei-Chi Siu, Food and Drug Administration Pacific Re-
gional Laboratory, Los Angeles, CA
Janet Smith, Fieldale Farms Corp., Baldwin, GA
Ellen Stoltzfus, Heinz Frozen Food Co., West Chester, PA
Downloaded from https://academic.oup.com/jaoac/article/85/2/470/5656511 by guest on 17 March 2022
Bob Taylor, Biotec, Inc., Hillsdale, PA
Steve Young, Doskocil Food Service, North Richland
Hills, TX
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