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3
conducted to discuss the details of the collaborative study packet and answer any questions from
1
the participating laboratories.
2
3
Preparation of Inocula and Test Portions
4
5
The
Listeria monocytogenes
cultures used in this evaluation were propagated onto Tryptic Soy
6
Agar with 5% Sheep Blood (SBA) from a Q Laboratories frozen stock culture stored at -70°C.
7
Each organism was incubated for 24 ± 2 hours at 35 ±1°C. Isolated colonies were picked to 10
8
mL of Brain Heart Infusion (BHI) broth and incubated for 18 ± 0.5 hours at 35 ±1°C. Raw
9
chicken breast fillet was inoculated in bulk using the fresh, broth culture. Prior to inoculation of
10
the deli turkey, the culture suspension was heat stressed at 55 ± 1
o
C in a water bath for 15
11
±0.5minutes to obtain a percent injury of 50-80% (as determined by plating onto selective
12
Modified Oxford agar (MOX) and non-selective tryptic soy agar with yeast (TSA/ye). The
13
degree of injury was estimated as:
14
15
where n
select
= number of colonies on selective agar and n
nonselect
= number of colonies on non-
16
selective agar. Appropriate dilutions of each culture were prepared in Butterfield’s Phosphate
17
Diluent (BPD) based on previously established growth curves for both low and high inoculation
18
levels. Bulk portions of each matrix were inoculated with the diluted liquid inoculum and mixed
19
thoroughly to ensure an even distribution of microorganisms. The inoculated raw chicken
20
breastfillet was packaged into separate 30 g test portions in sterile Whirl-Pak
®
bags and shipped
21
to the collaborators. For the analysis of the deli turkey, 25 g of inoculated test product was
22
mixed with 100 g of un-inoculated test product to prepare 125 g test portions which were
23
packaged in sterile Whirl-Pak
®
bags and shipped to collaborators.
24
To determine the level of
Listeria monocytogenes
in the matrices, a 5-tube most probable
25
number (MPN) was conducted by the coordinating laboratory on the day of the initiation of
26
analysis using the USDA/FSIS-MLG 8.09 reference method. For deli turkey, the MPN was
27
determined by analyzing 5 x 250 g test portions,thereference method test portions from the
28
collaborating laboratories and 5 x 65 g test portions. For the raw chicken breast fillet, the MPN
29
of the high and low inoculated levels was determined by analyzing 5 x 50 g test portions, the
30
reference method test portions from the collaborating laboratories and 5 x 10 g test portions. The
31
MPN and 95% confidence intervals were calculated using the LCF MPN Calculator, Version 1.6,
32
( www.lcftld.com/customer/LCFMPNCaclucator.exe ), provided by AOAC Research Institute
33
(RI) [7].
34
35
Test Portion Distribution
36
37
All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample
38
container. Test portions were shipped on a Thursday via overnight delivery according to the
39
Category B Dangerous Goods shipment regulations set forth by the International Air
40
Transportations Association(IATA).The two matrices were shipped consecutively, with
41
collaborating laboratories performing analysis on one matrix at a time. Upon receipt, samples
42
were held by the collaborating laboratory at refrigeration temperature (2-8 °C) until the
43
following Monday when analysis was initiatedafter a total equilibration time of 96 hours.All
44
samples were packed with cold packs to target a temperature of < 7°C during shipment.
45
In addition to each of the test portions and a separate APCsample, collaborators received a test
46
portion for each matrix labeled as ‘temperature control’. Participants were instructed to obtain
47
the temperature of this portion upon receipt of the package, document the results on the Sample
48
100 )
1(
x
n
n
nonselect
select
−
AOACResearch Institute
Expert Review Panel Use Only
OMAMAN-30 A/ Collaborative Study Manuscript
OMA ERP June 2016
ERP Use Only