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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
Listeriamonocytogenes
culture used in this evaluation was propagated in 10 mL of Brain
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Heart Infusion (BHI) broth from a frozen stock culture stored at -70°C atQ Laboratories, Inc.
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The broth was incubated for 18-24 hours at 35 ±1°C. The inoculum was heat stressed in a 50
o
C
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water bath for 10 minutes to obtain a percent injury of 50-80% (as determined by plating onto
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selective 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
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n
nonselect
= number of colonies on non-selective agar.Appropriate dilutions of the heat stressed
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cultures were prepared based on previously established growth curves for both low and high
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inoculation levels, resulting in fractional positive outcomes for at least one level. For both test
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portion sizes, a bulk lot of the queso frescowas inoculated with a liquid inoculum and mixed
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thoroughly by hand kneading to ensure an even distribution of microorganisms. The queso fresco
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was inoculated on the day of shipment so that all test portions would have been held for 96 hours
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by the day testing was initiated. The shipment and hold times of the inoculated test material had
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been verified through 120 hours as a quality control measure prior to study initiation. For the
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analysis of the 25 gram test portionsby the VIDAS LMX and the AOAC 993.12 methods, the
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bulk lot of test material was divided into separate 30 gram portions for shipmentto the
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collaborators. For the analysis of the 125 gram test portions by the VIDAS LMX method, 25
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gram of inoculated test product was mixed with 100 gram of un-inoculated test product for
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shipment to the collaborators. Validation criterion are satisfied when inoculated test portions
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produce fractional recovery of the spiked organism, defined as either the reference or candidate
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method yielding 25-75% positive results. To determine the level of
Listeriamonocytogenes
in
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thequeso fresco, a 5-tube MPN was conducted on the day of initiation of analysis. From both the
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high and low inoculated batches of queso fresco, five100gram test portions, the reference method
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test portions from the collaborating labs, and five 10 gram test portions were analyzed following
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the AOAC 993.12 reference method.The most probable number (MPN) and 95% confidence
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intervals were calculated from the high, medium, and low levels using the LCF MPN Calculator
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provided by AOAC
( w ww.lcfltd.com/customer/LCFMPNCalculator.exe )[7].Confirmation of the
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samples was conducted according to the AOAC OMA 993.12reference 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
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container. Test portions were shipped on a Thursday via overnight delivery according to the
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Category B Dangerous Goods shipment regulations set forth by IATA. Upon receipt, samples
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were held by the collaborating laboratory at refrigeration temperature (3-5 °C) until the
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following Monday when analysis was initiated. All samples were packed with cold packs to
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target a temperature of < 7°C during shipment. In addition to each of the test portions and the
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total plate count replicate, collaborators also received a test portion for each matrix labeled as
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‘temperature control’. Participants were instructed to obtain the temperature of this portion upon
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receipt of the package, document results on the Sample Receipt Confirmation form provided and
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fax to the study director.
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100 )
1(
x
n
n
nonselect
select
−
3