99 / 596
3
Preparation of Inocula and Test Portions
1
2
The
Listeria
cultures used in this evaluation were propagated onto Tryptic Soy Agar with 5%
3
Sheep Blood (SBA) from a Q Laboratories frozen stock culture stored at -70°C. Each organism
4
was incubated for 24 ± 2 hours at 35 ±1°C. Isolated colonies were picked to 10 mL of Brain
5
Heart Infusion (BHI) broth and incubated for 18 ± 0.5 hours at 35 ±1°C. Raw chicken breast
6
fillet was inoculated in bulk using the fresh, broth culture. Prior to inoculation of the deli turkey,
7
the culture suspension was heat stressed at 55 ± 1
o
C in a water bath for 15 ±0.5minutes to obtain
8
a percent injury of 50-80% (as determined by plating onto selective Modified Oxford agar
9
(MOX) and non-selective tryptic soy agar with yeast (TSA/ye). The degree of injury was
10
estimated as:
11
12
where n
select
= number of colonies on selective agar and n
nonselect
= number of colonies on non-
13
selective agar. Appropriate dilutions of each culture were prepared in Butterfield’s Phosphate
14
Diluent (BPD) based on previously established growth curves for both low and high inoculation
15
levels. Bulk portions of each matrix were inoculated with the diluted liquid inoculum and mixed
16
thoroughly to ensure an even distribution of microorganisms. The inoculated raw chicken
17
breastfillet was packaged into separate 30 g test portions in sterile Whirl-Pak
®
bags and shipped
18
to the collaborators. For the analysis of the deli turkey, 25 g of inoculated test product was
19
mixed with 100 g of un-inoculated test product to prepare 125 g test portions which were
20
packaged in sterile Whirl-Pak
®
bags and shipped to collaborators.
21
To determine the level of
Listeria
in the matrices, a 5-tube most probable number (MPN) was
22
conducted by the coordinating laboratory on the day of the initiation of analysis using the
23
USDA/FSIS-MLG 8.09 reference method. For deli turkey, the MPN was determined by
24
analyzing 5 x 250 g test portions,thereference method test portions from the collaborating
25
laboratories and 5 x 65 g test portions. For the raw chicken breast fillet, the MPN of the high and
26
low inoculated levels was determined by analyzing 5 x 50 g test portions, the reference method
27
test portions from the collaborating laboratories and 5 x 10 g test portions. The MPN and 95%
28
confidence intervals were calculated using the LCF MPN Calculator, Version 1.6,
29
( www.lcftld.com/customer/LCFMPNCaclucator.exe ), provided by AOAC Research Institute
30
(RI) [7].
31
32
Test Portion Distribution
33
34
All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample
35
container. Test portions were shipped on a Thursday via overnight delivery according to the
36
Category B Dangerous Goods shipment regulations set forth by the International Air
37
Transportations Association(IATA).The two matrices were shipped consecutively, with
38
collaborating laboratories performing analysis on one matrix at a time. Upon receipt, samples
39
were held by the collaborating laboratory at refrigeration temperature (2-8 °C) until the
40
following Monday when analysis was initiatedafter a total equilibration time of 96 hours.All
41
samples were packed with cold packs to target a temperature of < 7°C during shipment.
42
In addition to each of the test portions and a separate APCsample, collaborators received a test
43
portion for each matrix labeled as ‘temperature control’. Participants were instructed to obtain
44
the temperature of this portion upon receipt of the package, document the results on the Sample
45
Receipt Confirmation form provided and fax or email it back to the study director.The shipment
46
and hold timesof the inoculated test material had been verified as a quality control measure prior
47
to study initiation.
48
100 )
1(
x
n
n
nonselect
select
−
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29 A/ Collaboartive Study Manuscript
OMA ERP June 2016
ERP Use Only