AOAC-RI ERP Book MICRO.pdf

CONFIDENTIAL INFORMATION

Page 5 of 17

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

)

was calculated for each level of inoculation, and compared with the POD for the

candidate method’s “confirmation stage” (confirmed) results (POD

) at each level

of inoculation in order to attain a difference in POD for the two stages (dPOD

) at

each level of inoculation. A 95% confidence interval was also calculated for each

dPOD

value calculated, and was used to determine whether

the “screening

stage” results wa

significantly different from the “confirmation stage” results for

each level of inoculation. According to the methodology, if the 95% confidence

interval of a dPOD

value does not contain zero, then the difference between the

POD

and the POD

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

) 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