475 / 596
3
Petrifilm™Rapid Aerobic Count Plate (AOAC Official Method 2015.13) [6] on the day samples
1
were received for the purpose of determining the total aerobic
microbimicrobe\-pp
al load.
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 was then
5
conducted to discuss the details of the collaborative study packet and answer any questions from
6
the participating laboratories.
7
8
Preparation of Inocula and Test Portions
9
10
The
Listeria monocytogenes
cultures
(ATCC 7644 and ATCC 19115)
used in this evaluation
11
were propagated onto Tryptic Soy Agar with 5% Sheep Blood (SBA) from a Q Laboratories
12
frozen stock culture stored at -70°C. Each organism was incubated for 24 ± 2 hours at 35 ±1°C.
13
Isolated colonies were picked to 10 mL of Brain Heart Infusion (BHI) broth and incubated for 18
14
± 0.5 hours at 35 ±1°C. Raw chicken breast fillet was inoculated in bulk using the fresh, broth
15
culture. Prior to inoculation of the deli turkey, the culture suspension was heat stressed at 55 ±
16
1
o
C in a water bath for 15 ±0.5minutes to obtain a percent injury of 50-80% (as determined by
17
plating onto selective Modified Oxford agar (MOX) and non-selective tryptic soy agar with yeast
18
(TSA/ye). The degree of injury was estimated as:
19
20
where n
select
= number of colonies on selective agar and n
nonselect
= number of colonies on non-
21
selective agar. Appropriate dilutions of each culture were prepared in Butterfield’s Phosphate
22
Diluent (BPD) based on previously established growth curves for both low and high inoculation
23
levels. Bulk portions of each matrix were inoculated with the diluted liquid inoculum and mixed
24
thoroughly to ensure an even distribution of microorganisms. The inoculated raw chicken
25
breastfillet was packaged into separate 30 g test portions in sterile Whirl-Pak
®
bags and shipped
26
to the collaborators. For the analysis of the deli turkey, 25 g of inoculated test product was
27
mixed with 100 g of un-inoculated test product to prepare 125 g test portions which were
28
packaged in sterile Whirl-Pak
®
bags and shipped to collaborators.
29
To determine the level of
Listeria monocytogenes
in the matrices, a 5-tube most probable
30
number (MPN) was conducted by the coordinating laboratory on the day of the initiation of
31
analysis using the USDA/FSIS-MLG 8.09 reference method. For deli turkey, the MPN was
32
determined by analyzing 5 x 250 g test portions,thereference method test portions from the
33
collaborating laboratories and 5 x 65 g test portions. For the raw chicken breast fillet, the MPN
34
of the high and low inoculated levels was determined by analyzing 5 x 50 g test portions, the
35
reference method test portions from the collaborating laboratories and 5 x 10 g test portions.The
36
MPN and 95% confidence intervals were calculated using the LCF MPN Calculator, Version 1.6,
37
( www.lcftld.com/customer/LCFMPNCaclucator.exe ), provided by AOAC Research Institute
38
(RI) [7].
39
40
Test Portion Distribution
41
42
All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample
43
container. Test portions were shipped on a Thursday via overnight delivery according to the
44
Category B Dangerous Goods shipment regulations set forth by the International Air
45
Transportations Association(IATA).The two matrices were shipped consecutively, with
46
collaborating laboratories performing analysis on one matrix at a time. Upon receipt, samples
47
were held by the collaborating laboratory at refrigeration temperature (2-8 °C) until the
48
100 )
1(
x
n
n
nonselect
select
−