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For this collaborative study, one matrix, queso fresco (soft Mexican Cheese), was analyzed
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using two test portion sizes: 25 gand 125 g.The queso fresco was obtained from local retailers
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and screened for the absence of
Listeria
by the AOAC OMA 993.12 reference method prior to
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analysis. The 25 g and 125 g test portions of queso fresco were inoculated with a different strain
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of
Listeria
at two inoculation levels: a high inoculation level of approximately
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2-5 colony-forming units (CFU)/test portionand a low inoculation level of approximately 0.2-2
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CFU/test portion. A set of un-inoculated control test portions were also included for each matrix
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at 0 CFU/test portion. The 25 g test portions were artificially contaminated with
Listeria innocua
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ATCC 33090 and the 125 g test portions with
Listeria monocytogenes
ATCC 19115.
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Twelvereplicate samples from each of the three inoculation levels of product were analyzed.
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Two sets of samples (72 total) were sent to each laboratory for analysis by VIDAS LPTand
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theAOAC OMA 993.12 reference method due to different sample enrichments for each method.
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A detailed collaborative study packet outlining all necessary information related to the study
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including media preparation, method specific test portion preparation and documentation of
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results was sent to each collaborating laboratory prior to the initiation of the study.
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Preparation of Inocula and Test Portions
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The
Listeria
cultures used in this evaluation were propagated in 10 mL of Brain Heart Infusion
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(BHI) broth from a frozen stock culture stored at -70°C atQ Laboratories, Inc. The broth was
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incubated for 18-24 hours at 35 ±1°C. The inoculum was heat stressed in a 50 ± 1
o
C water bath
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for 10 minutes to obtain a percent injury of 50-80% (as determined by plating onto selective
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Oxford agar and non-selective Tryptic Soy agar). The degree of injury was estimated as
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, where n
select
= number of colonies on selective agar and n
nonselect
= number of
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colonies on non-selective agar.Appropriate dilutions of the heat stressed cultures were prepared
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based on previously established growth curves for both low and high inoculation levels, resulting
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in fractional positive outcomes for at least one level. For both test portion sizes, a bulk lot of the
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queso frescowas inoculated with a liquid inoculum and mixed thoroughly by hand kneading to
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ensure an even distribution of microorganisms.The queso fresco was inoculated on the day of
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shipment so that all test portions would have been held for 96 hours by the day testing was
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initiated. The shipment and hold times of the inoculated test material had been verified through
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120 hours as a quality control measure prior to study initiation. For the analysis of the 25 g test
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portions, the bulk lot of test material was divided into 30 g portions for shipmentto the
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collaborators. For the analysis of the 125 g test portions, 25 g of inoculated test product was
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mixed with 100 g of un-inoculated test product for shipment to the collaborators for the analysis
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by the VIDAS LPT method. Collaborators received 30 g portions for analysis by the AOAC
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OMA 993.12 method.Validation criterion are satisfied when inoculated test portions produce
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fractional recovery of the spiked organism, defined as either the reference or candidate method
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yielding 25-75% positive results. To determine the level of
Listeria
spp. in thequeso fresco, a 5-
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tube MPN was conducted on the day of initiation of analysis. From both the high and low
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inoculated batches of queso fresco, five100g test portions, the reference method test portions
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from the collaborating laboratories, and five 10 g test portions were analyzed following the
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AOAC OMA 993.12 reference method.The most probable number (MPN) and 95% confidence
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intervals were calculated from the high, low and uninoculated levels using the LCF MPN
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Calculator provided by AOAC
( w ww.lcfltd.com/customer/LCFMPNCalculator.exe )45
[7].Confirmation of the samples was conducted according to the AOAC OMA 993.12reference
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method.
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48
100 )
1(
x
n
n
nonselect
select
−
3