776  Feldsine et al.: Journal of AOAC International Vol. 96, No.
4, 2013
FOOD BIOLOGICAL CONTAMINANTS
Comparative Validation Study To Demonstrate the
Equivalence of a Minor Modification to AOAC Official Method
2005.04 Assurance GDS® E. coli O157:H7 Method to the
Reference Culture Method: 375 Gram Sample Size
Philip T. Feldsine, Megan Montgomery-Fullerton, Nerie Roa, Mandeep Kaur, Andrew H. Lienau,
Markus Jucker, and David E. Kerr
BioControl Systems, Inc., 12822 SE 32nd St, Bellevue, WA 98005
The Assurance GDS® Escherichia coli (E. coli)
SM
O157:H7, AOAC Official Method 2005.04, has been
modified to include a larger sample size of 375 g.
A methods comparison study was conducted to
demonstrate the equivalence of this modification to
the reference culture method. Ninety samples and
controls, representing three foods, were analyzed.
Results show no statistically detectable difference
between the Assurance GDS E. coli O157:H7
assay and the reference culture methods for the
detection of E. coli O157:H7, other than the low
level of inoculation for leaf lettuce, for which the
GDS gave noticeably higher recovery [difference in
probability of detection between candidate methods
(dPODc = +0.45)]. There were also suggestions of
moderate differences (dPODc = +0.15 to +0.20) for
ground beef and the high level of leaf lettuce, but
the study size was too small to detect differences of
this size. Results showed that the Assurance GDS
E. coli O157:H7 method is equivalent to reference
culture methods for the detection of E. coli O157:H7.
T
he Assurance GDS® Escherichia coli O157:H7
(Assurance GDS) is an automated nucleic acid
amplification assay that incorporates multiple levels of
specificity to ensure highly accurate results. The method utilizes
proprietary probes and specific primers directed against a highly
conserved DNA sequence of E. coli O157:H7. Assurance GDS
also utilizes a proprietary device and reagents that concentrate
populations of target microorganisms and eliminate potential
competitive microflora. The method is designed to be highly
selective, and does not detect microorganisms that are potential
cross-reactors in antibody-based assays, including E. coli O157
that are not H7 or nonmotile (NM), and other microorganisms
that express an O157 antigen but are not E. coli O157:H7. The
Assurance GDS method was adopted AOAC Official Method
Final Action in 2007.
The proposed modification to the Assurance GDS is the
addition of a larger sample size for testing 375 g sample sizes.
The 25 g sample size has conventionally been used for testing.
Submitted for publication January 7, 2013.
The recommendation was approved by the Methods Committee on
Microbiology as Revised First Action.
Corresponding author’s e-mail: ptf@biocontrolsys.com
DOI: 10.5740/jaoacint.CS2005_04
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Recently, 375 g samples have become common for testing
E.  coli O157:H7 in select industries. The 375 g sample size
allows testing of either a single larger individual sample or a
composite of smaller combined samples.
There is also an interest in testing both E. coli O157:H7 and
Salmonella from the same enriched sample. The samples for
this study were co-inoculated with both E. coli O157:H7 and
Salmonella. This study will show validation for the Assurance
GDS E. coli O157:H7. A related study (pending) will show
validation for the Assurance GDS Salmonella from these
samples.
Experimental Design
A methods comparison study was conducted to compare the
modified Assurance GDS to the reference culture methods for
select foods. The foods analyzed were raw beef trim, raw ground
beef and leaf lettuce. All three foods were analyzed using an
8–18 h enrichment protocol in modified enterohemorrhagic
E. coli (mEHEC) broth for 375 g sample testing by Assurance
GDS. For foods regulated by the U.S. Department of
Agriculture (USDA), the reference method used for comparison
was the USDA Microbiology Laboratory Guidebook (MLG)
culture method (1). For foods regulated by the U.S. Food and
Drug Administration (FDA), the reference method used for
comparison was the FDA Bacteriological Analytical Manual
(BAM) method (2). All samples for both Assurance GDS and
reference methods were enriched and then plated to selective/
differential agars as described for the appropriate reference
method.
Foods were inoculated with E. coli O157:H7 to achieve
two levels, a low probability of detection (POD) level where
fractional recovery was anticipated (PODC in the range of
0.25–0.75) and a high level where predominantly positive
results were expected (PODC of nearly 1.00). For each food,
20 replicate samples representing the low and five representing
the high contamination levels were analyzed by Assurance GDS
and reference culture methods. Five uninoculated samples were
also analyzed by each method.
Preparation of Inocula and Materials
Foods were initially screened for natural contamination
by target organism. Because no natural contamination was
found, the foods were artificially contaminated with the target
organism.
 Feldsine et al.: Journal of AOAC International Vol. 96, No. 4, 2013  777
The samples for this study were co-inoculated with E.  coli
O157:H7 and Salmonella. Both strains used as sources of
inocula were grown in Brain Heart Infusion broth for 18–24 h
at 35–37°C. For raw beef trim, raw ground beef, and raw
leaf lettuce, foods received inocula of stationary phase cells
estimated to achieve two seed levels: 0.2–2 CFU/25  g (low)
and 5–50  CFU/25 g (high) when measured by most probable
number (MPN) at the time of sample analysis.
After artificial contamination, all foods were held at 2–8°C
for at least 48 h to allow populations to stabilize. For raw beef
trim, raw ground beef, and lettuce, after seeding, the inoculated
foods were aliquoted into samples containing 25 g of food
and additional inoculated food reserved for MPN analysis.
These 25 g samples were randomly distributed among the
test and reference methods. For each Assurance GDS sample,
an additional 350 g of uninoculated food was added to the
25  g of inoculated food. For each USDA reference sample
(raw beef trim and raw ground beef), an additional 300  g of
uninoculated food was added to the 25 g of inoculated food. For
each FDA reference sample (leaf lettuce), an additional 175 g
of uninoculated food was added to the 25 g of inoculated food,
then rinsed with an equal weight of Butterfield’s phosphate
dilution buffer. MPN estimation of contamination levels were
also conducted on the day of initiation of analyses, using a
5-tube 3-level analysis scheme of inoculated food samples.
MPNs were evaluated using the appropriate FDA BAM- or
USDA-specified procedures. MPNs were calculated using the
MPN formula in Appendix A: MPN Analysis of Contaminated
Matrix (3).
Test Portion Analysis and Confirmation
For the Assurance GDS, a 1:5 sample to media ratio was
used for 375 g sample enrichment. Quantities of 375 g of food
were added to 1500 mL mEHEC, prewarmed to 42 ± 1°C.
Each Assurance GDS test portion was tested according to the
Package Insert (Attachment 1). Autoclaved prepared mEHEC
broth was used for this study. All samples, for both Assurance
GDS and reference methods, were confirmed using the USDA
MLG method  (1) for raw beef trim and raw ground beef.
Leaf lettuce samples were confirmed using the FDA BAM
method (2). To confirm E. coli O157:H7 for raw beef trim and
raw ground beef, 1 mL of each primary enrichment sample
was concentrated using immunomagnetic beads and streaked
for isolation on Rainbow agar O157 plates. For leaf lettuce,
1  mL of each primary enrichment sample was concentrated
using immunomagnetic beads and streaked for isolation on
Tellurite-Cefixime-Sorbitol MacConkey and Rainbow agar
plates. Typical colonies were picked from the selective agar
plates, and isolates were biochemically identified using API
20E strips (bioMérieux, Inc., Hazelwood, MO) and serology
using O157 antisera.
Data Analysis
Data analysis was performed for each food. Two methods
were employed to analyze the results: POD and Chi-square
analysis. Four different PODs were calculated: PODR for the
reference method; PODC for the candidate method; PODCP for
the candidate presumptive method; and PODCC for the candidate
confirmation method (4). For each of these cases, the POD was
calculated as the ratio of number positive (x) to the total number
tested (N). The dPOD values, which are the differences between
the two POD values, and the 95% confidence intervals on dPOD
values were also determined. If the confidence interval range
on dPOD does not contain a zero, then the two POD values are
statistically different.
AOAC Official Method 2005.04
Escherichia coli O157:H7 in Selected Foods
Assurance GDS E. coli O157:H7
First Action 2005
Final Action 2007
Revised First Action 2012
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[Applicable to detection of E. coli O157:H7 in raw ground
beef, beef trim, orange juice, apple juice, fresh vegetables and
sprout process water.]
Caution: E. coli O157:H7 are pathogenic bacteria. Symptoms
of infection include bloody diarrhea and cramping, little to no
fever, and hemolytic uremic syndrome. Decontaminate all spent
media and equipment used in test prior to disposal of media or
re-use of equipment.
Note: Use proper environmental and procedural precautions
for the handling of reagents and equipment when performing
genetic-based assays.
A.  Principle
The Assurance GDS E. coli O157:H7 method is a
gene-based assay that uses specific primers and proprietary
probes directed against a highly conserved DNA sequence in
the target organism. The method uses enrichment in mEHEC
broth. After enrichment, populations of target microorganisms
are concentrated by using a proprietary concentration device
and reagents or an automated concentration instrument. The
concentrate is transferred to a conical reaction vessel containing
amplification reagents. The vessel is sealed and placed in an
instrument which allows for simultaneous amplification and
detection. All tests, positive and negative, are indicated at the
end of analysis, as well as the results of a procedural control
which is contained in every reaction vessel.
Assurance GDS E. coli O157:H7 detects strains of E.  coli
O157:H7 and toxigenic E. coli O157:NM. This method
is designed to be highly selective, and does not detect
microorganisms that are potential cross-reactors in antibody-
based assays, including E. coli O157 that are not H7 or NM,
and other microorganisms that express O157 antigen but are not
E. coli O157:H7.
B.  Apparatus
Items (a)–(l) are available from BioControl Systems (12822
SE 32nd St, Bellevue, WA 98005; www.biocontrolsys.com).
Items (m)–(n) are available commercially.
(a)  Amplification and detection instrument.—Assurance
GDS Rotor-Gene®, or equivalent.
(b)  Computer and software.
(c)  Sample concentration device.—PickPen®.
(d)  Vortex mixer.
(e)  Sample wells and sample wells base.
(f)  Resuspension plate.
778  Feldsine et al.: Journal of AOAC International Vol. 96, No. 4, 2013
(g)  Gel cooling block.
(h) PickPen tips in box.
(i)  Sealing film with adhesive backing.
(j)  Dedicated pipets.—Eight-channel pipettor, capable of
dispensing 30 µL; repeater pipettor; and micropipettors capable
of accurately dispensing 10 and 35 µL.
(k)  Repeat pipettor tips.—0.5 and 10 mL.
(l)  Micropipettor tips.—Filter-barrier, volumes 20–200 and
200–1000 µL.
(m)  Incubator.—Maintaining 42 ± 1°C.
(n)  Masticator.—Seward 400 (Seward Ltd, 12 Park Lane,
Worthington, UK; www.seward.co.uk), or equivalent.
C.  Media and Reagents
Item (a) is available from BioControl Systems. Items (b)–(e)
are available in the Assurance GDS E. coli O157:H7 Tq test kit
from BioControl Systems. Item (f) is available commercially.
(a)  BioControl mEHEC broth.—Prewarm 1 L sterile
deionized water at 42 ± 1°C overnight. On day of use, aseptically
transfer 31.6 g mEHEC into the prewarmed sterile water. Gently
mix to dissolve the powder. Use prepared medium within 6 h.
Alternatively, mEHEC can be prepared and autoclaved at 121°C
for 15 min. Autoclaved media must be prewarmed to 42 ± 1°C
overnight prior to sample addition.
(b)  Concentration reagent.
(c)  Resuspension buffer.
(d) Amplification tubes containing lyophilized reagents for
E. coli O157:H7.
(e)  Wash solution.
(f)  Diagnostic media and reagents.—Necessary for cultural
confirmation of positive Assurance GDS samples.
Reagents (b) and (d) must be stored at 2–8°C when not in
use.
D.  Sample Enrichment
(a)  Aseptically weigh 25 g sample into 225 mL prewarmed
(42  ±  1°C) mEHEC, C(a). Homogenize samples well with
masticator, B(n). Incubate samples for 6.5–18 h at 42 ± 1°C. For
sprout irrigation water, incubate for 8–18 h.
(b)  Aseptically weigh 375 g sample into 1500 mL prewarmed
(42 ± 1°C) mEHEC. Homogenize samples well with masticator.
Incubate samples for 8–18 h at 42 ± 1°C.
E.  Sample Preparation Protocol
Note: Wear new gloves prior to handling reagents.
(a)  Mix concentration reagent, C(b), in a vortex mixer.
Transfer 20 µL to each of the required number of Assurance
GDS sample wells (one well/sample), B(e), using a repeater
pipettor, B(j), and 0.5 mL pipet tip, B(k). Cover sample wells
with adhesive film strips, B(i).
(b)  Transfer 1.0 mL of wash solution, C(e), to additional
sample wells (one well/sample), using a repeat pipettor and
10 mL pipet tip. Cover sample wells with adhesive film strips.
(c)  Transfer 45 µL of resuspension buffer, C(c), to the sample
wells in the resuspension plate, B(f), using a repeat pipettor and
0.5 mL pipet tip. Cover resuspension plate with adhesive film
strips.
(d)  From one strip of sample wells, (a), carefully remove
adhesive film. Using a micropipettor, add 1 mL of incubated
sample to each sample well containing concentration reagent.
Avoid transferring food particles. A new pipet tip must be
used for each sample. Cover each strip of sample wells with a
new adhesive film strip prior to adding samples to a new row.
Immediately return samples to incubator.
(e)  Place sealed sample wells held in the sample wells
base atop the vortex mixer, B(d), and vortex at approximately
900 rpm for 5–15 min. If necessary, adjust vortex speed to avoid
liquid contact with adhesive film. After vortexing is completed,
remove sample wells base from vortex mixer.
(f)  Carefully remove and discard an adhesive film strip from
one row of samples. Remove corresponding film strip from strip
of sample wells containing wash solution, (b)(1) or (b)(2).
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(g)  Load PickPen tips, B(h), onto the PickPen tool, B(c),
ensuring that the tips are firmly in place on the PickPen tool.
Extend the PickPen magnets and insert into the first strip of
sample wells containing sample with concentration reagent. Stir
gently for 30 s while continually moving tips up and down from
the surface to the bottom of the well. Gently tap the PickPen
tips against the side of the sample wells to remove excess media
droplets.
(h)  Transfer PickPen tips to corresponding sample wells
containing wash solution and gently swirl for 5 s (do not
release particles into solution). Transfer PickPen tips to the
corresponding row of the prepared resuspension plate wells,
(c). With tips submerged, retract the PickPen magnets and tap
gently to release particles into the resuspension buffer. Cover
resuspension plate with adhesive strips.
(i)  Repeat steps in (f)–(h) for all samples using new tips for
each row of samples.
F.  Amplification and Detection
Note: Wear new gloves prior to handling reagents.
(a)  Prior to initial use, cool the gel cooling block, B(g), in
the freezer (–25 to –15°C) for 6 h. When not in use, the gel
cooling block should be stored at –25 to –15°C. Between each
use, return the gel cooling block to the freezer until it has turned
completely purple, indicating it is ready for use. This may take
up to 2 h.
(b)  Remove amplification tubes, C(d), from foil pouch and
place the amplification tubes in the gel cooling block. Reseal
pouch.
(c)  Open caps of amplification tubes in gel cooling block.
Transfer 30 µL of sample from the resuspension plate wells into
each amplification tube using the eight-channel pipettor and
filter-barrier tip, B(j). Firmly press down on each amplification
tube cap to close. Visually inspect each tube to ensure the cap
is securely sealed.
(d)  Invert the gel cooling block holding amplification
tubes and shake with snapping motion to thoroughly mix tube
contents.
(e)  Place amplification tubes into Assurance Rotor-Gene,
B(a), in sequential order, beginning with position No. 1. Start
Rotor-Gene cycle, B(b). Refer to Assurance GDS user manual
for detailed instructions on operating the Rotor-Gene.
Note: The Assurance GDS Rotor-Gene must be started within
15 min after addition of the samples to the Amplification Tubes.
 Feldsine et al.: Journal of AOAC International Vol. 96, No. 4, 2013  779

Table  1.  Co-inoculating E. coli O157:H7, Salmonella, and BacteriologicalAnalyticalManualBAM/default.htm or USDA/

food type FSIS Microbiology Laboratory Guidebook, Chapter MLG
5.05 “Detection, Isolation and Identification of Escherichia
Sample type Target organism Challenge organism
coli O157:H7 from Meat Products,” http://www.fsis.usda.gov/
Raw beef trim E. coli O157:H7 (WA S. enterica ser. Newport Science/Microbiological_Lab_Guidebook/index.asp.
Department of Health) (ATCC 6962)
Raw ground beef E. coli O157:H7 S. enterica ser. Typhimurium I.  Precautions
(ATCC 35150) (ATCC 14028)
Leaf lettuce E. coli O157:H7 S. enterica ser. Heidelberg (a)  If possible, maintain separate work zones and dedicated
(ATCC 43894) (ATCC 8326)
equipment and supplies for sample preparation, amplification,
and detection.
(b)  Use of both positive and negative control samples is
G.  Assay Results recommended.
(c)  Do not use test kit beyond the expiration date on the

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Upon completion of the run, the Assurance Rotor-Gene
program will provide a results table. Each sample will be
identified as “positive,” indicating that the test sample is positive
for E. coli O157:H7, “negative,” indicating that it is negative for
E. coli O157:H7, or “no amp,” indicating that amplification did
not occur. In the event of a “no amp” result, from the sample
enrichment broth that has been returned to the incubator, E(d),
remove 1 mL of broth and start at E(a). If result continues to
show “no amp,” contact BioControl Systems Technical Service
(800-245-0113).
Caution: When the assay is completed, handle and dispose of
all waste as a biohazard. Never, under any circumstances, open
amplification tubes after the amplification process has started
to prevent discharge of amplified materials into the laboratory,
which could cause contamination of the testing environment.
H.  Confirmation of Positive Results
Assurance GDS positive samples should be confirmed
culturally, C(f), as described in current Bacteriological Analytical
Manual, Chapter 4A “Diarrheagenic Escherichia coli,” http://
www.fda.gov/Food/ScienceResearch/LaboratoryMethods/
product box label.
(d)  Decontaminate and dispose of materials in accordance
with good laboratory practices and in accordance with local,
state, and federal regulations.
(e)  Do not open used amplification tubes. Do not autoclave
used amplification tubes. Prior to disposal, immerse used
amplification tubes in a sealed container with 20% bleach or
1.2% sodium hypochlorite solution.
(f)  Waste may be contaminated with E. coli O157:H7, which
is potentially hazardous to human health. All biohazard waste
should be disposed of appropriately (5).
Results and Discussion
Foods in this study were co-incoculated with E. coli O157:H7
and Salmonella (Table 1). Data for the Assurance GDS E. coli
O157:H7 are presented below; data for the Assurance GDS
Salmonella are presented in a separate paper. Three food types
were analyzed by the Assurance GDS E. coli O157:H7 method.
Fractional recovery was achieved for all food types tested. In
total, there were valid results from 90 samples and controls.
POD statistical analyses of the Assurance GDS samples

Table  2.  Comparison of Assurance GDS E. coli O157:H7 to reference: Assurance GDS presumptive versus confirmed—POD

Assurance GDS Presumptive Assurance GDS Confirmed

a b c
Matrix MPN/25 g N x PODCPd 95% CI x PODCCe 95% CI dPODCPf 95% CIg
h
Raw beef trim 0.0 5 0 0.00 0.00, 0.43 0 0.00 0.00, 0.43 0.00 –0.43, +0.43
0.37 20 10 0.50 0.30, 0.70 10 0.50 0.30, 0.70 0.00 –0.28, +0.28
12.3 5 5 1.00 0.57, 1.00 5 1.00 0.57, 1.00 0.00 –0.43, +0.43
Raw ground beef 0.0 5 0 0.00 0.00, 0.43 0 0.00 0.00, 0.43 0.00 –0.43, +0.43
0.65 20 13 0.65 0.43, 0.82 14 0.70 0.48, 0.85 –0.05 –0.32, +0.23
5.1 5 5 1.00 0.57, 1.00 5 1.00 0.57, 1.00 0.00 –0.43, +0.43
Leaf lettuce 0.0 5 0 0.00 0.00, 0.43 0 0.00 0.00, 0.43 0.00 –0.43, +0.43

0.21 20 11 0.55 0.34, 0.74 11 0.55 0.34, 0.74 0.00 –0.28, +0.28
1.6 5 4 0.80 0.38, 0.96 4 0.80 0.38, 0.96 0.00 –0.45, +0.45
a
 MPN = Most probable number is based on the POD of reference method test portions across laboratories using the AOAC MPN calculator, with 95%
confidence interval (CI).
b
  N = Number of test portions.
c
  x = Number of positive test portions.
d
  PODCP = Candidate method presumptive positive outcomes divided by the total number of samples.
e
  PODCC = Candidate method confirmed positive outcomes divided by the total number of samples.
f
  dPODCP = Difference between the candidate method presumptive result and candidate method confirmed result POD values.
g
  95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.
h
  Indicates the range of lowest to highest values, with a dash (–), between the two values.
780  Feldsine et al.: Journal of AOAC International Vol. 96, No. 4, 2013

Table  3.  Comparison of Assurance GDS E. coli O157:H7 to reference: method comparison results—POD

Assurance GDS Presumptive FDA/USDA Reference

a b c d
Matrix MPN/25 g N x PODC 95% CI x PODRe 95% CI dPODCf 95% CIg
h
Raw beef trim 0.0 5 0 0.00 0.00, 0.43 0 0.00 0.00, 0.43 0.00 –0.43, +0.43
0.37 20 10 0.50 0.30, 0.70 9 0.45 0.26, 0.66 0.05 –0.25, +0.33
12.3 5 5 1.00 0.57, 1.00 5 1.00 0.57, 1.00 0.00 –0.43, +0.43
Raw ground beef 0.0 5 0 0.00 0.00, 0.43 0 0.00 0.00, 0.43 0.00 –0.43, +0.43
0.65 20 13 0.65 0.43, 0.82 10 0.50 0.30, 0.70 0.15 –0.15, +0.41
5.1 5 5 1.00 0.57, 1.00 5 1.00 0.57, 1.00 0.20 –0.43, +0.43
Leaf lettuce 0.0 5 0 0.00 0.00, 0.43 0 0.00 0.00, 0.43 0.00 –0.43, +0.43

0.21 20 11 0.55 0.34, 0.74 2 0.10 0.03, 0.30 0.45 +0.16, +0.65

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1.6 5 4 0.80 0.38, 0.96 3 0.60 0.20, 0.88 0.20 –0.31, +0.60
a
 MPN = Most probable number is based on the POD of reference method test portions across laboratories using the AOAC MPN calculator, with 95%
confidence interval (CI).
b
 N = Number of test portions.
c
 x = Number of positive test portions.
d
 PODC = Confirmed candidate method positive outcomes divided by the total number of samples.
e
 PODR = Confirmed reference method positive outcomes divided by the total number of samples.
f
 dPODC = Difference between the candidate method and reference method POD values.
g
 95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.
h
 Indicates the range of lowest to highest values, with a dash (–), between the two values.

indicated that, for all foods and surfaces analyzed, there was no
statistical difference between the Assurance GDS presumptive
and the Assurance GDS confirmed (Table 2). For the low level
of ground beef, one sample was GDS negative, but confirmed
positive.
The recovery of the Assurance GDS and reference methods
were compared. There were 48 samples that were positive by
Assurance GDS (assay positive and subsequently confirmed)
and 34 that were positive by reference culture. There were 42
samples that were negative by Assurance GDS and 56 that were
negative by reference culture (Table 3). POD statistical analyses
of the unmatched samples indicated that for all foods analyzed,
there was no statistical difference between the Assurance GDS
presumptive and the Assurance GDS confirmed. Furthermore,
Assurance GDS results were not statistically different from the
reference culture method for the detection of E. coli O157:H7,
except for the low level of leaf lettuce. For the low inoculation
level of leaf lettuce, Assurance GDS had statistically significant
higher recovery than the reference method. The reference
method for leaf lettuce uses a rinse-off step of the lettuce leaves,
followed by incubation of the rinse material. By comparison, the
Assurance GDS preparation methodology specifies mastication
of the lettuce leaves and incubation of the entire sample. The
incubation of a rinsate compared to the entire sample may
account for the lower recovery for the reference method.
Conclusions
It is recommended that the minor modification to AOAC
Official Method 2005.04 be adopted revised First Action. The
modification is the increased sample size testing of composite-
sized (375 g) raw beef trim, raw ground beef, and leaf lettuce.
References
  (1) U.S. Department of Agriculture/Food Safety Inspection
Services, Microbiological Laboratory Guidelines. http://www.
fsis.usda.gov/PDF/MLG_5_05.pdf (access date 1/5/12)
  (2) U.S. Food and Drug Administration, Bacteriological
Analytical Manual. http://www.fda.gov/Food/ScienceResearch/
LaboratoryMethods/BacteriologicalAnalyticalManualBAM/
ucm070080.htm (access date 1/5/12)
  (3) Association of Analytical Communities (2012) Methods
Committee Guidelines for Validation of Microbiological
Methods for Food and Environmental Surface, Appendix X-A,
Analysis of Contaminated Matrix, AOAC INTERNATIONAL,
Gaithersburg, MD
  (4) Association of Analytical Communities (2012) Methods
Committee Guidelines for Validation of Microbiological
Methods for Food and Environmental Surface, Appendix
X-C, Calculation of POD and dPOD Values from Qualitative
Method Single Laboratory Data, AOAC INTERNATIONAL,
Gaithersburg, MD
  (5) Feldsine, P.T., Green, S.T., Lienau, A.H., Stephens, J., Jucker,
M.T., & Kerr, D.E. (2005) J. AOAC Int. 88, 1334–1348