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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