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Culture Collection (ATCC) 19114at two inoculation levels: a high inoculation level of
1
approximately 2-5 colony-forming units (CFU)/test portion and a low inoculation level of
2
approximately 0.2-2 CFU/test portion. A set of un-inoculated control test portions were also
3
included at 0 CFU/test portion. Twelve replicate samples from each of the three inoculation
4
levels were analyzed. Two sets of samples (72 total) were sent to each laboratory for analysis by
5
3M MDA
Listeria
and the AOAC OMA 993.12 reference method due to the different sample
6
enrichment procedures for each method. Additionally, collaborators were sent a 30 g test portion
7
and instructed to conduct atotal aerobic plate count (APC) using 3M
™
Petrifilm Aerobic Count
8
Plate (AOAC Official Method 990.12) [7] on the day samples were received for the purpose of
9
determining the total aerobic microbial load.
10
A detailed collaborative study packet outlining all necessary information related to the study
11
including media preparation, test portion preparation and documentation of results was sent to
12
each collaborating laboratory prior to the initiation of the study. A conference call was conducted
13
to discuss the collaborative study packet and answer any questions from the participating
14
laboratories.
15
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Preparation of Inocula and Test Portions
17
18
The
Listeriamonocytogenes
culture used in this evaluation was propagated in 10 mL of Brain
19
Heart Infusion (BHI) broth from a frozen stock culture stored at -70°C at Q Laboratories, Inc.
20
The broth was incubated for 18 ± 0.5 hours at 35 ±1°C. Appropriate dilutions of the culture were
21
prepared based on previously established growth curves for both the low and high inoculation
22
levels. The full fat cottage cheese was inoculated at a low and high inoculation level with the
23
diluted inoculum and mixed thoroughly by hand mixing to ensure an even distribution of
24
microorganisms. The inoculated test product was divided into separate 30 g portions which were
25
packaged into sterile whirl-pak
®
bags.
26
To determine the level of
Listeria monocytogenes
in the full fat cottage cheese, a 5-tube most
27
probable number (MPN) was conducted on the day of initiation of analysis. From both the high
28
and low inoculated batches, 5 x50 g test portions, the reference method test portions from the
29
collaborating labs, and 5 x10 g test portions were analyzed. Each test portion was enriched at a
30
1:10 dilution and evaluated following the AOAC 993.12 reference method. The MPN and 95%
31
confidence intervals were calculated from the high, medium, and low levels using the LCF MPN
32
Calculator, Version 1.6, provided by AOAC RI
33
( www.lcfltd.com/customer/LCFMPNCalculator.exe )[8]. Confirmation of the samples was
34
conducted according to the AOAC OMA 993.12 reference method.
35
36
Test Portion Distribution
37
38
All samples were labeled with a randomized, blind-coded 3 digit number affixed to the sample
39
container. Test portions were shipped on a Thursday via overnight delivery according to the
40
Category B Dangerous Goods shipment regulations set forth by the International Air
41
Transportations Association(IATA). Upon receipt, samples were held by the collaborating
42
laboratory at refrigeration temperature (3-5 °C) until the following Monday when analysis was
43
initiated a total of 96 hours after inoculation. All samples were packed with cold packs to target
44
a temperature of < 7°C during shipment. In addition to each of the test portions and the total
45
plate count replicate, collaborators also received a test portion for each matrix labeled as
46
‘temperature control’. Participants were instructed to obtain the temperature of this portion upon
47
receipt of the package, document results on the Sample Receipt Confirmation form provided and
48
Candidates for 2016 Method of the Year
113