Methods to be Reviewed by OMB in 2016

ird

et al

ournal of

AOAC I

nternational

99, N

3, 2016

Additional PTM parameters (ruggedness, stability, and

lot-to-lot variability) tested in the PTM studies satisfied the

requirements for PTM approval. The method was awarded

PTM certification No. 121403 on December 29, 2014.

The purpose of this collaborative study was to compare

the 3M Petrifilm RAC Plate to the U.S. Food and Drug

Administration (FDA)

Bacterial Analytical Manual

(BAM)

Chapter 3 (Aerobic Plate Count; 4) and the Standard Methods

for the Examination of Dairy Products (SMEDP) Chapter 6

(Standard Plate Count; 5), using BPD as the diluent for raw

easy-peel shrimp, pasteurized skim milk, and instant NFDM.

Collaborative Study

Study Design

In this collaborative study, three matrixes—raw easy-peel

shrimp, pasteurized skim milk, and instant NFDM—were

evaluated. The matrixes were obtained from local retailers and

screened for the presence of naturally occurring aerobic organisms

by BAM or the SMEDP reference methods. Three separate levels

of contamination were targeted for the evaluation of each matrix

using naturally occurring aerobic microflora. The target levels for

the naturally contaminated matrixes were low (10–100 CFU/g),

medium (100–1000 CFU/g), and high (1000–10 000 CFU/g). To

obtain the required contamination levels, bulk lots of the target

matrixes were temperature-abused by heat-stressing to elevate

the naturally occurring aerobic bacteria present in the matrixes,

or diluted using lots containing low numbers of aerobic bacteria.

Two replicate samples from each of the three contamination

levels were analyzed by both candidate and reference methods

in a paired study design. One set of paired samples (six total)

per matrix was sent to each laboratory for analysis by the 3M

Petrifilm RAC Plate and BAM or SMEDP reference methods.

A detailed collaborative study packet outlining all necessary

information related to the study including media preparation,

test portion preparation, and documentation of results was sent

to each collaborating laboratory prior to the initiation of the

study. A conference call was conducted prior to the initiation of

the study to discuss the collaborative study packet and answer

any questions from the participating laboratories.

Preparation of the Test Portions

For raw easy-peel shrimp and pasteurized skim milk test

portions, a single bulk lot of the matrix was evaluated for total

aerobic plate count following BAM or SMEDP, respectively, to

determine baseline aerobic bacterial counts. For both raw easy-

peel shrimp and pasteurized skim milk, two 1000 g portions were

removed from the bulk lot and temperature-abused to increase

the aerobic bacterial counts. For raw easy-peel shrimp, one set

of 1000 g was placed in a 35 ± 1°C incubator for 1 h, and the

second set of 1000 g was placed at room temperature (20–25°C)

for 4 h. For pasteurized skim milk, one set of 1000 g was placed

at room temperature (24 ± 2°C) for 4 h, and the second set of

1000 g was placed at room temperature (24 ± 2°C) for 12 h.

Following temperature abuse, five replicate test portions from

each lot were evaluated for total aerobic count using the specified

reference method. Results from both test matrixes indicated that

the temperature abuse had increased aerobic bacterial counts to

produce two additional levels of contamination. The raw easy-

peel shrimp samples were subsampled into 60 g test portions and

the pasteurized skim milk samples were subsampled into 15 mL

test portions to be sent to collaborators for use in the evaluation.

For instant NFDM, several lots of product were evaluated for

the presence of aerobic bacteria. Initial testing identified two lots

(one that produced aerobic plate counts in the high contamination

level and one that produced counts in the low contamination level)

to be used in the evaluation. The medium contamination level

was prepared by mixing a portion of the high contamination lot

with the low contamination lot. The instant NFDM samples were

subsampled into 15 g test portions to be used in the evaluation.

Test Portion Distribution

All samples were labeledwith a randomized, blind-coded three-

digit number affixed to the sample container. Test portions were

shipped in leak-proof insulated containers via overnight delivery

according to the Category B Dangerous Goods Regulations

(DGR) as set forth by the International Air Transport Association

(56th edition). Raw easy-peel shrimp and pasteurized skim milk

samples were packed with cold packs to ensure refrigeration

temperature (2–8°C) during shipment. Upon receipt, these

test portions were held at refrigeration (2–8°C) until analyses

were initiated the following day. Instant NFDM samples were

packed and shipped at ambient temperature (24 ± 2°C). Upon

receipt, samples were held at room temperature (24 ± 2°C) until

analysis was initiated. In addition to each of the test portions,

collaborators also received a test portion for each matrix labeled

as “temperature control” for all three matrixes. Participants

were instructed to record the temperature of this portion upon

receipt of the shipment, document results on the Sample Receipt

Confirmation form provided, and fax to the study director.

Test Portion Analysis

Collaborators followed the appropriate preparation and

analysis protocol according to the method specified for each

matrix. For all three matrixes, each collaborator received six

test portions (two high, two medium, and two low). For the

analysis of the raw easy-peel shrimp by the 3M Petrifilm

RAC Plate, a 50 g test portion was diluted with 450 mL BPD

and homogenized by blending for 2 min. For pasteurized

skim milk, 11 g test portion was diluted with 99 mL BPD and

homogenized by shaking 25 times in a 30 cm arc within 7 s. For

instant NFDM, 11 g sample was added to 99 mL tempered BPD

(40–45°C) ensuring that the entire sample was visibly dissolved

throughout the diluent prior to homogenizing by shaking 25 times

in a 30 cm arc within 7 s. Ten-fold serial dilutions of each

sample were prepared for each matrix and a 1.0 mL aliquot of

each dilution was plated onto 3M Petrifilm RAC Plates. For raw

easy-peel shrimp, four replicate 3M Petrifilm RAC Plates were

prepared for each dilution, with two plates being incubated at

32 ± 1°C for 24 ± 2 h and two plates being incubated at 35 ± 1°C

for 24 ± 2 h. For the evaluation of the pasteurized skim milk, two

replicate 3M Petrifilm RAC Plates for each dilution were prepared

and incubated at 32 ± 1°C for 24 ± 2 h. For the evaluation of the

instant NFDM, two replicate 3M Petrifilm RAC Plates for each

dilution were prepared and incubated at 32 ± 1°C for 48 ± 3 h.

Seafood test portions were evaluated at two temperatures (32 and

35°C) to allow end users the option to choose either temperature

for incubation. After incubation, 3M Petrifilm RAC Plates were

removed from the incubator and typical colonies (all colonies

regardless of size, color, or intensity) were enumerated using a

Candidates for 2016 Method of the Year

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