AOAC-RI ERP Book MICRO.pdf

CONFIDENTIAL INFORMATION

Page 5 of 13

For ground beef, five 650 g samples and five 162 g samples were weighed into

sterile Whirl-Pak bags and hydrated with 1,955 mL and 486 mL, respectively, of

mTSB. The samples were then processed according to the reference method

procedures described above in Section 2.4. Note: for the MPN test samples, the

second set of five samples for the reference testing were used. 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” negative 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 result)

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

“confirmation stage” results

were both positive for a particular sample (POD

). The

difference in POD for the two values at each level of inoculation were calculated

(dPOD

) along with its corresponding 95% confidence interval. As noted above, if

the 95% confidence interval of a dPOD

value does not contain zero, then the

difference between the POD

of the candidate method and the POD

of the