AOAC-RI ERP Book MICRO.pdf

768

ird

et al

ournal of

AOAC I

nternational

. 98, N

. 3, 2015

repeatability was observed between the 3M Petrifilm RYMCount

Plate method and the reference methods. For the 3M Petrifilm

RYM Count Plate method PTM evaluation, 10 matrixes were

evaluated: yogurt (1.5% fat), sour cream, raw almonds, sliced

apples, frozen bread dough, ready-made cherry pie, ready-to-

eat deli sandwiches, dehydrated chicken noodle soup, fermented

salami, and raw frozen ground beef patties (77% lean).

All other PTM parameters (inclusivity, exclusivity,

ruggedness, stability, and lot-to-lot variability) tested in the

PTM studies satisfied the performance requirements for PTM

approval. The method was awarded PTM certification No.

121301 on December 18, 2013.

The purpose of this collaborative study was to compare

the 3M Petrifilm RYM Count Plate method to a harmonized

version of the U.S. Food and Drug Administration (FDA)

Bacterial Analytical Manual

(BAM) Chapter 18,

Yeasts, Molds

and Mycotoxins

(1) and the ISO 21527:2008

Microbiology

of Food and Animal Feeding Stuffs – Horizontal Method for

the Enumeration for Yeast and Molds

– Part 1:

Colony Count

Technique in Products with Water Activity Greater Than 0.95

(4) and Part 2:

Colony Count Technique in Products with Water

Activity Less Than or Equal to 0.95

(5) methods using 0.1%

peptone water as the diluent for raw almonds and raw frozen

ground beef patties (77% lean).

Collaborative Study

Study Design

In this collaborative study, twomatrixes, rawfrozen ground beef

patties (77% lean) and raw almonds, were evaluated. The matrixes

were obtained from local retailers and screened for the presence

of yeast and molds by the BAM and ISO reference methods. The

raw frozen ground beef patties were artificially contaminated

with a yeast,

Trichosporon mucoides

American Type Culture

Collection (ATCC, Manassas, VA) 201382, and the almonds with

a mold,

Aspergillus aculeatus

ATCC 56925. Four separate levels

of contamination, including an uninoculated control level and

three levels of artificial contamination, were evaluated for each

matrix. The target for the three levels of artificial contamination

was as follows: low, 10–100 CFU/g; medium, 100–1000 CFU/g;

high 1000–10000 CFU/g. Two replicate samples from each of

the four levels were analyzed by both the candidate and reference

methods. One set of paired samples (eight total) per matrix was

sent to each laboratory for analysis by the 3M Petrifilm RYM

Count Plate method and the BAM and ISO reference methods.

For both matrixes, collaborators were sent an additional 60 g

test portion and instructed to conduct a total aerobic plate count

(APC) using 3M

Petrifilm

Aerobic Count Plate (AOAC

Official Method

990.12

; 6) on the day samples were received as a

quality measure to verify appropriate shipping conditions.

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 prior to the initiation of the study was

conducted to discuss the collaborative study packet and answer

any questions from the participating laboratories.

Preparation of Inocula and Test Portions

The

Trichosporon mucoides

used in this evaluation was

propagated in 10 mL of Brain Heart Infusion (BHI) broth

from a frozen stock culture stored at –70°C at Q Laboratories,

Inc. (Cincinnati, OH) The broth was incubated for 48±2 h at

30±1°C. Appropriate dilutions of the cultures were prepared

in Butterfields Phosphate Buffer (PBW) based on previously

established growth curves to obtain low, medium and high

contamination levels. For the raw frozen ground beef patties, a

bulk lot of the matrix was thawed, inoculated with the diluted

liquid inoculum and mixed thoroughly by hand kneading to

ensure an even distribution of microorganisms. The inoculated

raw frozen ground beef patties samples were separated into 30 g

test portions and held at –20±2°C for 2 weeks when testing was

initiated.

The

Aspergillus aculeatus

used in this evaluation was

propagated on Potato Dextrose Agar (PDA) in a culture tissue

flask from a frozen stock spore suspension stored at –70°C at

Q Laboratories, Inc. The culture tissue flask was incubated for

7 days at 25±1°C. The mold spore suspension was prepared by

rinsing the culture tissue flask twice with 20 mL of 0.9% saline

containing 0.05%Tween 20. Each suspension was centrifuged at

4000 ×

for 10±0.5 min to pellet the spores and the supernatant

was decanted. The two separate spore pellets were resuspended

with 2.5 mL of PBW and combined. The spore suspension was

combined with a cryoprotectant, reconstituted non-fat dry milk

(NFDM), homogenized by vortex to mix and placed onto a

freeze dry system for 72±4 h to lyophilize the culture. After the

lyophilization process, appropriate dilutions of the culture were

prepared in sterile NFDM powder to produce the low, medium

and high contamination levels. A bulk lot of whole raw almonds

were inoculated with the lyophilized inoculum and mixed

thoroughly by hand mixing to ensure an even distribution of

microorganisms. The inoculated almonds were separated into

30 g test portions and held at ambient temperature (24±2°C)

so that the organism had equilibrated for 2 weeks when testing

was initiated.

The shipment conditions and hold times of the inoculated

test materials were verified as a quality control measure prior

to study initiation.

Test Portion Distribution

All samples were labeled with a randomized, blind-coded

3 digit number affixed to the sample container. Test portions

were shipped on a Thursday via overnight delivery according to

the Category B Dangerous Goods shipment regulations set forth

by International Air Transport Association. Frozen raw ground

beef patties samples were packed with cold packs to ensure

the samples remained frozen (–20±2°C) during shipment.

Upon receipt, raw frozen ground beef patties samples were

held at –20±2°C until the following Monday when analysis

was initiated. Raw almond samples were packed and shipped

at ambient temperature. Upon receipt, samples were held at

room temperature (24±2°C) until the following Monday when

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

and the total plate count replicate, collaborators also received