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B
ird
et al
.:
J
ournal of
AOAC I
nternational
V
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100, N
o
.
2, 2017
455
melon (whole), raw chicken leg pieces (25 g); raw chicken
breast fillet (25 g), sealed concrete (sponge, 225 and 100 mL),
stainless steel (sponge, 225 mL), and plastic (high-density
polyethylene; 3M EnviroSwab, 10 mL).
Additional PTM parameters (inclusivity, exclusivity,
ruggedness, stability, and lot-to-lot variability) tested in the
PTM studies satisfied the performance requirements for
PTM approval. The method was awarded PTM Certification
No. 081501 on August 18, 2015.
The purpose of this collaborative study was to compare the
reproducibility of the 3M MDA 2 –
Listeria monocytogenes
method to the U.S. Department of Agriculture (USDA) Food
Safety Inspection Service (FSIS)
Microbiology Laboratory
Guidebook
(MLG) Chapter 8.09 “Isolation and Identification
of
Listeria monocytogenes
from Red Meat, Poultry, and Egg
Products, and Environmental Samples” (USDA/FSIS MLG)
reference method (5) for deli turkey (125 g) and raw chicken
breast fillet (25 g).
Collaborative Study
Study Design
In this collaborative study, two matrixes, deli turkey and
raw chicken breast fillet, were evaluated. The matrixes were
obtained from a local retailer and screened for the presence of
L. monocytogenes
by the USDA/FSIS MLG reference method.
The raw chicken breast fillet was artificially contaminated with
fresh unstressed cells of
L. monocytogenes
,AmericanType Culture
Collection (ATCC) 7644, and the deli turkey was artificially
contaminated with heat-stressed cells of
L. monocytogenes
,
ATCC 19115 (Table 1), at two inoculation levels: a high
inoculation level of approximately 2–5 CFU/test portion and a
low inoculation level of approximately 0.2–2 CFU/test portion. A
set of uninoculated control test portions (0 CFU/test portion) was
also included.
Twelve replicate samples from each of the three inoculation
levels were analyzed by each method. Two sets of samples
(72 total) were sent to each laboratory for analysis by the 3M
MDA 2 –
Listeria monocytogenes
and the USDA/FSIS MLG
reference method due to the different sample enrichment
procedures required for each method. In addition, collaborators
were sent a 60 g test portion and instructed to conduct a total
aerobic plate count (APC) using the 3M Petrifilm™ Rapid
Aerobic Count Plate (AOAC
Official Method
SM
2015.13
; 6) on
the day samples were received for the purpose of determining
the total APC.
A detailed collaborative study packet outlining all necessary
information related to the study, including media preparation,
test portion preparation, and documentation of results, was sent
to each collaborating laboratory before the initiation of the study.
A conference call was then conducted to discuss the details of
the collaborative study packet and answer any questions from
the participating laboratories.
Preparation of Inocula and Test Portions
The
L. monocytogenes
cultures (ATCC 7644 and ATCC
19115) used in this evaluation were propagated onto TSA with
5% sheep blood from a Q Laboratories frozen stock culture
stored at −70°C. Each organism was incubated for 24 ± 2 h at
35 ± 1°C. Isolated colonies were picked to 10 mL brain heart
infusion broth and incubated for 18 ± 0.5 h at 35 ± 1°C. Raw
chicken breast fillet was inoculated in bulk using the fresh
broth culture. Before inoculation of the deli turkey, the culture
suspension was heat-stressed at 55 ± 1°C in a water bath for
15 ± 0.5 min to obtain 50–80% injury, as determined by plating in
duplicate onto selective modified Oxford agar and nonselective
TSA with yeast extract. The degree of injury was estimated as
1
100
−
×
n
n
select
nonselect
where
n
select
= the number of colonies on selective agar; and
n
nonselect
=thenumberofcoloniesonnonselectiveagar.Appropriate
dilutions of each culture were prepared in Butterfield’s phosphate
diluent based on previously established growth curves for both
the low and high inoculation levels. Bulk portions of each matrix
were inoculated with the diluted liquid inoculum and mixed
thoroughly to ensure an even distribution of microorganisms. The
inoculated raw chicken breast fillet was packaged into separate
30 g samples in sterile Whirl-Pak
®
bags and shipped to the
collaborators. For the analysis of the deli turkey, 25 g inoculated
test product was mixed with 100 g uninoculated test product to
prepare 125 g test portions, which were packaged in sterileWhirl-
Pak bags and shipped to collaborators.
To determine the level of
L. monocytogenes
in the matrixes, a
five-tube most probable number (MPN) test was conducted by
the coordinating laboratory on the day of the initiation of analysis
using the USDA/FSIS MLG reference method. For deli turkey, the
MPN was determined by analyzing five 250 g test portions, the
reference method test portions from the collaborating laboratories,
and five 65 g test portions. For the raw chicken breast fillet, the
MPN of the high and low inoculated levels was determined by
analyzing five 50 g test portions, the reference method test portions
from the collaborating laboratories, and five 10 g test portions.
The MPN and 95% confidence intervals were calculated using
the Least Cost Formulations, Ltd, MPN Calculator–Version 1.6,
provided by AOAC Research Institute (7).
Test Portion Distribution
All samples were labeled with a randomized, blind-coded
three-digit number affixed to the sample container. Test portions
Table 1. Heat-stress injury results
Matrix
Test organism
CFU/MOX
(selective
agar)
a
CFU/TSA
(nonselective
agar)
b
Degree of
injury, %
Deli
turkey
L. monocytogenes
9.1 × 10
8
2.5 × 10
9
60.8
ATCC 19115
Raw
chicken
breast
fillet
c
L. monocytogenes
Not
applicable
Not
applicable
—
d
ATCC 7644
a
MOX = Modified Oxford agar.
b
TSA = Tryptic soy agar.
c
Cultures were heat-stressed for 10 min at 55°C in a recirculating
water bath.
d
— = Raw chicken breast fillet sample is not heat treated, so it is not
necessary to injure the cells.