185 / 471
candidate method and the reference methods. Additionally, collaborators were sent a 30 g test
1
portion and instructed to conduct a total aerobic plate count (APC) using 3M™ Petrifilm Aerobic
2
Count Plate (AOAC Official Method 990.12) [6] on the day samples were received for the
3
purpose of determining the total aerobic microbial load.
4
A detailed collaborative study packet outlining all necessary information related to the study
5
including media preparation, test portion preparation and documentation of results was sent to
6
each collaborating laboratory prior to the initiation of the study. A conference call was conducted
7
to discuss the collaborative study packet and answer any questions from the participating
8
laboratories.
9
10
Preparation of Inocula and Test Portions
11
12
The
Listeria monocytogenes
cultures used in this evaluation were propagated in 10 mL of
13
Brain Heart Infusion (BHI) broth from a Q Laboratories frozen stock culture held at -70°C. Each
14
organism was incubated for 18 ± 0.5 hours at 35 ±1°C. Prior to inoculation, the culture
15
suspension for the deli turkey was heat stressed at 50 ± 1
o
C in a water bath for 10 ±0.5minutes to
16
obtain a percent injury of 50-80% (as determined by plating onto selective modified Oxford agar
17
(MOX) and non-selective Tryptic Soy agar with yeast extract (TSA/ye). The degree of injury
18
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 Butterfields’ 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 by hand mixing to ensure an even distribution of microorganisms. The inoculated full
25
fat cottage cheese was divided into separate 30 g portions packaged in sterile whirl-pak
®
bags
26
and shipped to the collaborators. For the analysis of the deli turkey, 25 g of inoculated test
27
product was mixed with 100g of un-inoculated test product to prepare 125 g test portions which
28
were packaged in sterile whirl-pak
®
bags..
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 initiation of analysis
31
using the USDA/FSIS-MLG 8.09 reference method for deli turkey or the AOAC OMA 993.12
32
for the full fat cottage cheese. For deli turkey, the MPN of the high and low inoculated levels
33
was determined by analyzing 5 x 250 g test portions, the reference method test portions from the
34
collaborating laboratories and 5 x 60 g test portions by the USDA/FSIS-MLG 8.09 reference
35
method. For the full fat cottage cheese, the MPN of the high and low inoculated levels was
36
determined by analyzing5 x50g test portions, the reference method test portions from the
37
collaborating laboratories and 5 x 10g test portions by the AOAC OMA 993.12 reference
38
method. Each test portion was enriched at a 1:10 dilution and evaluated following the
39
appropriate reference method. The MPN and 95% confidence intervals were calculated using the
40
LCF MPN Calculator, Version 1.6, provided by AOAC
41
RI
( www.lcftld.com/customer/LCFMPNCaclucator.exe )[7].Confirmation of the samples was
42
conducted according to the USDA/FSIS-MLG 8.09 or the AOAC OMA 993.12 reference
43
method, dependent on the matrix.
44
45
Test Portion Distribution
46
47
100 )
1(
x
n
n
nonselect
select
−
Candidates for 2016 Method of the Year
149