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A detailed collaborative study packet outlining all necessary information related to the study including media
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preparation, test portion preparation and documentation of results was sent to each collaborating laboratory prior to
2
the initiation of the study. A conference call was then conducted to discuss the details of the collaborative study
3
packet and answer any questions from the participating laboratories. 3M Food Safety Technical Service provided
4
on-site hands on training to 12 of the 15 participating laboratories prior to the start of the collaborative study.
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6
Preparation of Inocula and Test Portions
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The
E. coli
O157:H7cultureATCC43895 used in this evaluation were propagated onto Tryptic Soy Agar (TSA)
9
from a Vanguard Sciences(collaborating laboratory) frozen stock culture stored at -70°C. The organism was
0
incubated for 24 ± 2 hours at 35 ±1°C. Isolated colonies were picked to 10 mL of Brain Heart Infusion (BHI)
1
broth and incubated for 18 ± 0.5 hours at 35 ±1°C. The cultures were stored at 2-8°C for 24 h while the inoculum
2
level was determined. Appropriate dilutions of each culture were prepared in Butterfield’s Phosphate Diluent
3
(BPD) for both low and high inoculation levels. Bulk portions of each matrix were inoculated with the diluted
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liquid inoculum and mixed thoroughly to ensure an even distribution of microorganisms. For the analysis of the
5
raw ground beef, 25 g of inoculated test product was mixed with 300 g of un-inoculated test product to prepare 325
6
g test portions which were packaged in sterile Smasher
®
XLbags and shipped to collaborators.
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A most probable number (MPN) was conducted by the coordinating laboratory on the day of the initiation of
8
analysis using the USDA/FSIS-MLG 5.09 reference method for raw ground beef.The MPN was determined by
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analyzing 5 x 650 g test portions,thereference method test portions from the collaborating laboratories and 5 x 160
0
g test portions by the USDA/FSIS-MLG 5.09 reference method for the low level inoculated samples and the
1
reference method test portions from the collaborating laboratories, 5 x 160 g, and 5 x 80 g test portions for the high
2
samples. The MPN and 95% confidence intervals were calculated using the LCF MPN Calculator, Version 1.6,
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( www.lcftld.com/customer/LCFMPNCalculator.exe ), provided by AOAC RI [6]. Confirmation of the samples was
4
conducted according to the USDA/FSIS-MLG 5.09 reference method.
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Test Portion Distribution
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All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample container. Test
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portions were shipped on a Thursday via overnight delivery according to the Category B Dangerous Goods
0
shipment regulations set forth by the International Air Transportations Association(IATA). All raw ground beef
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samples were packed with cold packs to target a temperature of < 7°C during shipment.Upon receipt, raw ground
2
beef samples were held by the collaborating laboratory at refrigeration temperature (2-8 °C) until the following
3
Monday when analysis was initiatedafter a total equilibration time of 96 hours.
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In addition to each of the test portions and a separate APCsample, collaborators received a test portion for each
5
matrix labeled as ‘temperature control’.Participants were instructed to measurethe temperature of this portion upon
6
receipt of the package, record the results on the Sample Receipt Confirmation form provided and send the filled
7
form back to the study directorby fax or email.
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Test Portion Analysis
0
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Collaborators were instructed to follow the appropriate preparation and analysis protocol for both the 3M MDA 2 –
2
E. coli
O157 (including H7) method and reference methods. Each collaborator received 72 test portions (12 high,
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12 low and 12 un-inoculated controls) for each method to be performed. For the analysis of the raw ground beef
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test portions by the 3M MDA 2 –
E. coli
O157 (including H7) method, a 325 g portion was enriched with 975 mL
5
of pre-warmed (41.5 ±1
o
C) BPW ISO, manually homogenized by hand for 2 minutes and incubated for 10 hours at
6
41.5 ±1
o
C.
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Following enrichment, samples were assayed by the 3M MDA 2 –
E. coli
O157 (including H7) method and,
8
regardless of presumptive result, confirmed following the appropriate reference method. The matrix evaluated by
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OMAMAN-35 A : Collaborative Study Manuscript
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only