AOAC-RI ERP Book Micro Jan 19 2017

analysis was setup after the 48 hr stabilization period in order to mimic all handling and treatment of matrices. For spinach, five 100 g samples, five 10 g samples and five 1 g samples were weighed into sterile Whirl-Pak bags and hydrated with 900 mL, 90 mL and 9 mL of mBPWp, respectively. For sprouts, five 50 g samples and five 10 g samples were weighed into sterile Whirl-Pak bags and hydrated with 450 mL and 90 mL of mBPWp, respectively. The samples were then processed according to the reference method procedures described above in Section 2.4. Note: for sprouts MPN test samples, the second set of five samples for the reference testing will be used (25 g, respectively). Samples that confirmed positive for this procedure were noted and the LCF MPN Calculator Version 1.6 (Least Cost Formulations, Ltd., Virginia Beach, VA) was used to calculate the MPN/g and 95% confidence intervals for each portion using the mass of each sample and the number of reference method confirmed positives at each level of the MPN setup. 2.7 Probability of Detection Statistical Analysis After results from the “screening” and “confirmation” stages were obtained for the candidate method and “confirmed” results were obtained for the reference method, the data was analyzed according to the POD methods of the 2012 version of the AOAC International Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces document (1). The POD for the candidate method’s “screening stage” (presumptive) results (POD CP ) was calculated for each level of inoculation, and compared with the POD for the candidate method’s “confirmation stage” (confirmed) results (POD CC ) at each level of inoculation in order to attain a difference in POD for the two stages (dPOD CP ) at each level of inoculation. A 95% confidence interval was also calculated for each dPOD CP value calculated, and was used to determine whether the “screening stage” results wa s significantly different from the “confirmation stage” results for each level of inoculation. According to the methodology, if the 95% confidence interval of a dPOD CP value does not contain zero, then the difference between the POD CP and the POD CC of the candidate method at that level of inoculation is statistically significant at the 5% level. In addition to these analyses, the rate of true positives (samples with a “screening stage” positive result and “confirmation stage” positive result), the rate of true negatives (samples with a “screening stage” negative result and “confirmation stage” neg ative result), the rate of false positives (samples with a “screening stage” positive result and “confirmation stage” negative result), and the rate of false negatives (samples with a “screening stage” negative result and “confirmation stage” positive resu lt) were calculated. Additionally, PODs were calculated for the reference method’s “confirmed” results (POD R ) at each level of inoculation and were compared to the PODs that were calculated for each level of inoculation for the candidate method where the “screening stage” and

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