![Show Menu](styles/mobile-menu.png)
![Page Background](./../common/page-substrates/page0055.jpg)
B
ird
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
3, 2016
665
Additional PTM parameters (ruggedness, stability, and
lot-to-lot variability) tested in the PTM studies satisfied the
requirements for PTM approval. The method was awarded
PTM certification No. 121403 on December 29, 2014.
The purpose of this collaborative study was to compare the 3M
Petrifilm RAC Plate to the U.S. Food and Drug Administration
(FDA)
Bacteriological Analytical Manual
(BAM) Chapter 3
(Aerobic Plate Count; 4) and the Standard Methods for the
Examination of Dairy Products (SMEDP) Chapter 6 (Standard
Plate Count; 5), using BPD as the diluent for raw easy-peel
shrimp, pasteurized skim milk, and instant NFDM.
Collaborative Study
Study Design
In this collaborative study, three matrixes—raw easy-peel
shrimp, pasteurized skim milk, and instant NFDM—were
evaluated. The matrixes were obtained from local retailers and
screened for the presence of naturally occurring aerobic organisms
by BAM or the SMEDP reference methods. Three separate levels
of contamination were targeted for the evaluation of each matrix
using naturally occurring aerobic microflora. The target levels for
the naturally contaminated matrixes were low (10–100 CFU/g),
medium (100–1000 CFU/g), and high (1000–10 000 CFU/g). To
obtain the required contamination levels, bulk lots of the target
matrixes were temperature-abused by heat-stressing to elevate
the naturally occurring aerobic bacteria present in the matrixes,
or diluted using lots containing low numbers of aerobic bacteria.
Two replicate samples from each of the three contamination
levels were analyzed by both candidate and reference methods
in a paired study design. One set of paired samples (six total)
per matrix was sent to each laboratory for analysis by the 3M
Petrifilm RAC Plate and BAM or SMEDP reference methods.
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 prior to the initiation of the
study. A conference call was conducted prior to the initiation of
the study to discuss the collaborative study packet and answer
any questions from the participating laboratories.
Preparation of the Test Portions
For raw easy-peel shrimp and pasteurized skim milk test
portions, a single bulk lot of the matrix was evaluated for total
aerobic plate count following BAM or SMEDP, respectively, to
determine baseline aerobic bacterial counts. For both raw easy-
peel shrimp and pasteurized skim milk, two 1000 g portions were
removed from the bulk lot and temperature-abused to increase
the aerobic bacterial counts. For raw easy-peel shrimp, one set
of 1000 g was placed in a 35 ± 1°C incubator for 1 h, and the
second set of 1000 g was placed at room temperature (20–25°C)
for 4 h. For pasteurized skim milk, one set of 1000 g was placed
at room temperature (24 ± 2°C) for 4 h, and the second set of
1000 g was placed at room temperature (24 ± 2°C) for 12 h.
Following temperature abuse, five replicate test portions from
each lot were evaluated for total aerobic count using the specified
reference method. Results from both test matrixes indicated that
the temperature abuse had increased aerobic bacterial counts to
produce two additional levels of contamination. The raw easy-
peel shrimp samples were subsampled into 60 g test portions and
the pasteurized skim milk samples were subsampled into 15 mL
test portions to be sent to collaborators for use in the evaluation.
For instant NFDM, several lots of product were evaluated for
the presence of aerobic bacteria. Initial testing identified two lots
(one that produced aerobic plate counts in the high contamination
level and one that produced counts in the low contamination level)
to be used in the evaluation. The medium contamination level
was prepared by mixing a portion of the high contamination lot
with the low contamination lot. The instant NFDM samples were
subsampled into 15 g test portions to be used in the evaluation.
Test Portion Distribution
All samples were labeledwith a randomized, blind-coded three-
digit number affixed to the sample container. Test portions were
shipped in leak-proof insulated containers via overnight delivery
according to the Category B Dangerous Goods Regulations
(DGR) as set forth by the International Air Transport Association
(56th edition). Raw easy-peel shrimp and pasteurized skim milk
samples were packed with cold packs to ensure refrigeration
temperature (2–8°C) during shipment. Upon receipt, these
test portions were held at refrigeration (2–8°C) until analyses
were initiated the following day. Instant NFDM samples were
packed and shipped at ambient temperature (24 ± 2°C). Upon
receipt, samples were held at room temperature (24 ± 2°C) until
analysis was initiated. In addition to each of the test portions,
collaborators also received a test portion for each matrix labeled
as “temperature control” for all three matrixes. Participants
were instructed to record the temperature of this portion upon
receipt of the shipment, document results on the Sample Receipt
Confirmation form provided, and fax to the study director.
Test Portion Analysis
Collaborators followed the appropriate preparation and
analysis protocol according to the method specified for each
matrix. For all three matrixes, each collaborator received six
test portions (two high, two medium, and two low). For the
analysis of the raw easy-peel shrimp by the 3M Petrifilm
RAC Plate, a 50 g test portion was diluted with 450 mL BPD
and homogenized by blending for 2 min. For pasteurized
skim milk, 11 g test portion was diluted with 99 mL BPD and
homogenized by shaking 25 times in a 30 cm arc within 7 s. For
instant NFDM, 11 g sample was added to 99 mL tempered BPD
(40–45°C) ensuring that the entire sample was visibly dissolved
throughout the diluent prior to homogenizing by shaking 25 times
in a 30 cm arc within 7 s. Ten-fold serial dilutions of each
sample were prepared for each matrix and a 1.0 mL aliquot of
each dilution was plated onto 3M Petrifilm RAC Plates. For raw
easy-peel shrimp, four replicate 3M Petrifilm RAC Plates were
prepared for each dilution, with two plates being incubated at
32 ± 1°C for 24 ± 2 h and two plates being incubated at 35 ± 1°C
for 24 ± 2 h. For the evaluation of the pasteurized skim milk, two
replicate 3M Petrifilm RAC Plates for each dilution were prepared
and incubated at 32 ± 1°C for 24 ± 2 h. For the evaluation of the
instant NFDM, two replicate 3M Petrifilm RAC Plates for each
dilution were prepared and incubated at 32 ± 1°C for 48 ± 3 h.
Seafood test portions were evaluated at two temperatures (32 and
35°C) to allow end users the option to choose either temperature
for incubation. After incubation, 3M Petrifilm RAC Plates were
removed from the incubator and typical colonies (all colonies
regardless of size, color, or intensity) were enumerated using a