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ird
et al
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ournal of
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nternational
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ol
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99, n
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4, 2016
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instant nonfat dry milk (25 g), black pepper (25 g), cocoa powder
(25 g), pasteurized liquid whole egg (100 g), raw head on shrimp
(25 g), raw bagged spinach (25 g), spent sprout irrigation water
(375 mL), cooked breaded chicken (325 g), creamy peanut
butter (25 and 375 g), dry dog food (25 and 375 g), pasteurized
American cheese (25 g), sealed concrete environmental surface
[4 × 4 in. area with 3M hydrated sponge stick with Dey/Engeley
(D/E)], stainless steel environmental surface (1 × 1 in. area with
3M Enviroswab), and sealed ceramic tile environmental surface
(4 × 4 in. area with 3M hydrated sponge stick with D/E).
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 number
091501 on September 21, 2015.
The purpose of this collaborative study was to compare the
reproducibility of the 3M MDA 2 –
Salmonella
method to
the U.S. Department of Agriculture (USDA), Food Safety and
Inspection Service (FSIS)
Microbiology Laboratory Guidebook
(MLG) Chapter 4.08 “Isolation and Identification of
Salmonella
from Meat, Poultry, Pasteurized Egg, and Catfish Products
and Carcass and Environmental Sponges” reference method
(USDA/FSIS MLG; 7) for raw ground beef (73% lean) and to
the U.S. Food and Drug Administration (FDA)
Bacteriological
Analytical Manual
(BAM) Chapter 5 “
Salmonella
” reference
method (FDA BAM; 8) for creamy peanut butter.
Collaborative Study
Study Design
In this collaborative study, two matrixes, raw ground beef
(73% lean) and creamy peanut butter (50% fat, 22% sugar
content), were evaluated. The matrixes were obtained from a
local retailer and screened for the presence of
Salmonella
by the
appropriate reference method before analysis. The raw ground
beef was artificially contaminated with nonheat-stressed cells of
Salmonella
Agona, American Type Culture Collection (ATCC)
51957, and the creamy peanut butter was artificially contaminated
with heat-stressed cells of
Salmonella
Muenchen, ATCC BAA-
1594, 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 3M MDA
2 –
Salmonella
and by either the USDA/FSIS MLG (raw ground
beef) or FDA BAM (creamy peanut butter) reference method
due to the different sample enrichment procedures for each
method. In addition, collaborators were sent a 60 g test portion
and instructed to conduct a total aerobic plate count (APC) using
3M Petrifilm™Aerobic Count Plate (AOAC
Official Method
SM
990.12
; 9) on the day samples were received for the purpose of
determining the total aerobic microbial load.
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
Salmonella
cultures used in this evaluation were
propagated onto tryptic soy agar (TSA) with 5% sheep blood
agar from a Q Laboratories, Inc. (Cincinnati, OH) 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. Before inoculation, the culture suspension for the
creamy peanut butter was heat-stressed at 55 ± 1°C in a water bath
for 15 ± 0.5 min to obtain an injury of 50–80% [as determined
by plating onto selective xylose lysine deoxycholate (XLD) agar
and nonselective TSA]. The degree of injury was estimated as
−
×
n
n
1
100
select
nonselect
where
n
select
is the number of colonies on selective agar, and
n
nonselect
is the number of colonies onnonselective agar.Appropriate
dilutions of each culture were prepared in Butterfield’s phosphate
diluent based on previously established growth curves for both
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
creamy peanut butter was packaged into separate 30 g test portions
in sterile Whirl-Pak
®
bags and shipped to the collaborators. For
the analysis of the raw ground beef, 25 g inoculated test product
was mixed with 300 g uninoculated test product to prepare 325 g
test portions, which were packaged in sterile Whirl-Pak bags and
shipped to the collaborators.
To determine the level of
Salmonella
in the matrixes,
a most probable number (MPN) assay was conducted by
the coordinating laboratory on the day of the initiation of
analysis using the USDA/FSIS MLG reference method for
raw ground beef and the FDA BAM reference method for
the creamy peanut butter. For raw ground beef, the MPN was
determined by analyzing 5 × 650 g test portions, the reference
method test portions from the collaborating laboratories, and
5 × 160 g test portions by the USDA/FSIS MLG reference
method. For the creamy peanut butter, the MPN of the high
and low inoculated levels was determined by analyzing 5 ×
50 g test portions, the reference method test portions from the
collaborating laboratories, and 5 × 10 g test portions by the
FDA BAM reference method. The MPN and 95% confidence
intervals were calculated using the Least Cost Formulations,
Ltd MPN Calculator, Version 1.6
(www.lcftld.com/customer/LCFMPNCalculator.exe), provided by the AOAC Research
Institute (10). Confirmation of the samples was conducted
according to the USDA/FSIS MLG or the FDA BAM reference
method, depending on the matrix.
Test Portion Distribution
All samples were labeled with a randomized, blind-coded
three-digit number affixed to the sample container. Test portions
were shipped on a Thursday via overnight delivery according
to the Category B Dangerous Goods shipment regulations set
forth by the International Air Transportations Association. Upon
receipt, raw ground beef samples were held by the collaborating
laboratory at refrigeration temperature (2–8°C) until the following
Monday when analysis was initiated after a total equilibration