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B

ird

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

98, N

o

. 3, 2015 

769

a test portion for each matrix labeled as “temperature control”.

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 both matrixes, each collaborator received

eight test portions (two high, two medium, two low, and two

uninoculated replicates). For the analysis of the each matrix

by the 3M Petrifilm RYM Count Plate method, a 25 g test

portion was diluted with 225 mL of 0.1% peptone water and

homogenized for 2 min. Ten-fold serial dilutions of each sample

were prepared and a 1.0 mL aliquot of each dilution was plated

onto four separate 3M Petrifilm RYM Count Plates. For each

dilution, two of the plates were incubated at 25±1°C and two of

the plates were incubated at 28±1°C. Plates were removed from

incubation after 48±2 h and typical yeast and mold colonies in

the countable range (plates that contain up to 150 colonies) were

enumerated using a standard colony counter. Plates containing

greater than 150 colonies were either estimated or recorded as

too numerous to count (TNTC). All plates were re-incubated at

the appropriate temperatures for an additional 10–14 h (to reach

a total incubation time of 60 h) and colonies were enumerated a

second time in the same manner as the 48 h time point.

Both matrixes analyzed by the 3M Petrifilm RYM Count

Plate method were also analyzed using the BAM and ISO

reference methods in a paired study design. Serial dilutions for

each sample were spread plated in triplicate onto Dichloran-rose

Bengal Chloramphenicol (DRBC) agar (raw frozen ground beef

patties) or Dichloran 18% Glycerol (DG18) agar (raw almonds).

Agar plates were incubated for 5 days at 25±1°C and typical

colonies in the countable range (10–150) were enumerated

using a standard colony counter. Plates containing no colonies

were re-incubated for 2 days and observed for typical growth.

Statistical Analysis

Each collaborating laboratory recorded the CFU/g results

for the reference methods and the 3M Petrifilm RYM Count

Plate method on the electronic spreadsheet provided in the

collaborator study outline. The data sheets were submitted

to the study director at the end of each week of testing for

analysis. The data from each duplicate plates (3M Petrifilm

RYM Count Plate) or triplicate plates (BAM and ISO) were

averaged. The averaged counts were converted into logarithms

for data analysis. Outliers were identified using the Cochran

and Grubbs’ tests. A paired

t

-test was conducted to determine

if the mean of replicate samples at each contamination level for

each matrix was different between the 3M Petrifilm RYM Count

Plate method and the reference methods. (3). A

P

-value greater

than the standard alpha value of 0.05 indicated no statistical

difference between the two methods. The repeatability (s

r

),

reproducibility (s

R

), RSD

r

, and RSD

R

of the 3M Petrifilm RYM

Count Plate method and reference methods were determined

by the mean of the logarithm transformations of the counts for

each contamination level of each matrix (7). A lower standard

deviation value indicated a greater propensity for repeatability

of a method. In addition, the difference of means and reverse

transformed difference of means for each contamination level

was determined (3).

AOAC Official Method 2014.05

Enumeration of Yeast and Mold

in Food

3M™ Petrifilm™ Rapid Yeast and Mold Count Plate

First Action 2014

[Applicable to the enumeration of yeast and mold in the

following high-water activity matrices: yogurt, frozen bread

dough, fermented salami, sour cream, ready-made pie, raw

frozen ground beef patties (77% lean), ready-to-eat deli

sandwiches, sliced apples, and the following low-water activity

matrixes: raw almonds and dehydrated soup.]

See

Tables

2014.05A

and

2014.05B

for a summary of results

of the collaborative study. The result for each collaborating

laboratory’s aerobic plate count analysis for each matrix is

shown in Table

2014.05C

.

See

Tables 2–9 for detailed results of the collaborative study.

A. Principle

The 3M Petrifilm Rapid Yeast and Mold Count (RYM) Plate

is a sample-ready culture medium system, which contains

nutrients supplemented with antibiotics, a cold-water-soluble

gelling agent, and an indicator system that facilitates yeast and

mold enumeration. 3M Petrifilm RYM Count Plates are used

for the enumeration of yeast and mold in as little as 48 h in the

food and beverage industries. 3M Food Safety is certified to

ISO (International Organization for Standardization) 9001 for

design and manufacturing.

B. Apparatus and Reagents

(

a

)

 3M Petrifilm RYM Count Plate.

—25 plates/pouch; two

pouches/box (3M Food Safety, St. Paul, MN).

(

b

)

 Sterile diluents.—

0.1% peptone water.

(

c

)

 Pipets.—

Capable of 1000 µL or a serological pipet.

(

d

)

 Sterile pipet tips.—

Capable of 1000 µL.

(

e

)

 Stomacher.—

Seward or equivalent.

(

f

)

 Filter stomacher bags.

—Seward or equivalent.

(

g

)

 3M Petrifilm Flat Spreader.

(

h

)

 Incubators.—

Capable of maintaining 25 ± 1°C and 28 ±

1°C and having a solid front to maintain a dark interior.

(

i

)

 Refrigerator.—

Capable of maintaining 2–8°C, for storing

the 3M Petrifilm RYM Plates.

(

j

)

 L-shaped spreaders.

(

k

)

 Standard colony counter or illuminated magnifier.

C. General Instructions

(

a

)

Store unopened 3M Petrifilm RYM Plate pouches

refrigerated or frozen (–20 to 8°C/–4 to 46°F). Just prior to use,

allow unopened pouches to come to room temperature before

opening (20–25°C/<60% RH). Return unused 3M Petrifilm

RYM Plates to the pouch. Seal by folding the end of the

pouch over and applying adhesive tape. To prevent exposure

to moisture, do not refrigerate opened pouches. Store resealed

pouches in a cool dry place (20–25°C/<60% RH) for no longer

than 4 weeks. It is recommended that resealed pouches of 3M