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Aerobic Count Plate (AOAC Official Method 990.12) [6] on the day samples were received as a
1
quality measure to verify appropriate shipping conditions.
2
A detailed collaborative study packet outlining all necessary information related to the study
3
including media preparation, test portion preparation and documentation of results was sent to
4
each collaborating laboratory prior to the initiation of the study. A conference call prior to the
5
initiation of the study was conducted to discuss the collaborative study packet and answer any
6
questions from the participating laboratories.
7
8
Preparation of Inocula and Test Portions
9
10
The
Trichosporon mucoides
used in this evaluation was propagated in 10 mL of Brain Heart
11
Infusion (BHI) broth from a frozen stock culture stored at -70°C at Q Laboratories, Inc. The
12
broth was incubated for 48 ± 2 hours at 30 ± 1°C. Appropriate dilutions of the cultures were
13
prepared in Butterfields Phosphate Buffer (PBW) based on previously established growth curves
14
to obtain low, medium and high contamination levels. For the raw frozen ground beef patties,
15
a bulk lot of the matrix was thawed, inoculated with the diluted liquid inoculum and mixed
16
thoroughly by hand kneading to ensure an even distribution of microorganisms. The inoculated
17
raw frozen ground beef patties samples were separated into 30 g test portions and held at
18
-20 ± 2°C for 2 weeks when testing was initiated.
19
The
Aspergillus aculeatus
used in this evaluation was propagated on Potato Dextrose Agar
20
(PDA) in a culture tissue flask from a frozen stock spore suspension stored at -70°C at
21
Q Laboratories, Inc. The culture tissue flask was incubated for 7 days at 25 ± 1°C. The mold
22
spore suspension was prepared by rinsing the culture tissue flask twice with 20 mL of 0.9%
23
saline containing 0.05% Tween 20. Each suspension was centrifuged at 4,000 x
g
for 10 ± 0.5
24
minutes to pellet the spores and the supernatant was decanted. The two separate spore pellets
25
were resuspended with 2.5 mL of PBW and combined. The spore suspension was combined with
26
a cryoprotectant, reconstituted Non-Fat Dry Milk (NFDM), homogenized by vortex to mix and
27
placed onto a freeze dry system for 72 ± 4 hours to lyophilize the culture. After the lyophilization
28
process, appropriate dilutions of the culture were prepared in sterile NFDM powder to produce
29
the low, medium and high contamination levels. A bulk lot of whole raw almonds were
30
inoculated with the lyophilized inoculum and mixed thoroughly by hand mixing to ensure an
31
even distribution of microorganisms. The inoculated almonds were separated into 30 g test
32
portions and held at ambient temperature (24 ± 2°C) so that the organism had equilibrated for 2
33
weeks when testing was initiated.
34
The shipment conditions and hold times of the inoculated test materials were verified as a
35
quality control measure prior to study initiation
36
37
Test Portion Distribution
38
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All samples were labeled with a randomized, blind-coded 3 digit number affixed to the
40
sample container. Test portions were shipped on a Thursday via overnight delivery according to
41
the Category B Dangerous Goods shipment regulations set forth by International Air Transport
42
Association (IATA). Frozen raw ground beef patties samples were packed with cold packs to
43
ensure the samples remained frozen (-20 ± 2°C) during shipment. Upon receipt, raw frozen
44
ground beef patties samples were held at -20 ± 2°C until the following Monday when analysis
45
was initiated. Raw almond samples were packed and shipped at ambient temperature. Upon
46
receipt, samples were held at room temperature (24 ± 2
o
C) until the following Monday when
47
analysis was initiated. In addition to each of the test portions and the total plate count replicate,
48
collaborators also received a test portion for each matrix labeled as “temperature control”.
49
Candidates for 2016 Method of the Year
77