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upon prolonged incubation.
See
Table
2014.05D
for yeast and
mold appearance.
(
11
) The circular growth area is approximately 30 cm
2
. Plates
containing greater than 150 colonies can be either estimated
or recorded as TNTC (too numerous to count). Estimation
can only be done by counting the number of colonies in one
or more representative squares and determining the average
number per square. The average number can be multiplied by
30 to determine the estimated count per plate. If a more accurate
count is required, the sample will need to be retested at higher
dilutions. When the sample contains substantial amounts of
mold, depending on the type of mold, the upper countable limit
may be at user discretion.
(
12
) Food samples may occasionally show interference on
the 3M Petrifilm RYM Count Plates, for example:
(
a
) Uniform blue background color (often seen from the
organisms used in cultured products). These should not be
counted as TNTC.
(
b
) Intense pinpoint blue specks (often seen with spices or
granulated products).
(
c
) Report final results as colony-forming units/gram
(CFU/g).
(
13
) If required, colonies may be isolated for further
identification by direct microscopy or biochemical analysis. Lift
the top film and pick the colony from the gel.
Results of the Collaborative Study
In this collaborative study, the 3M Petrifilm RYM Count
Plate method was compared to two reference methods for
enumerating total yeast and mold: FDA BAM Chapter 18
and ISO 21527 Part 1 or Part 2. A total of 15 laboratories
throughout the United States participated, with 11 laboratories
submitting valid data for the frozen raw ground beef patties and
14 laboratories submitting valid data for the raw almonds as
presented in Table 1. For the frozen raw ground beef patties,
laboratories 4 (failed to enumerate 3M Petrifilm RYM Count
Plates at 60 h), 6 and 8 (plated the reference method samples in
duplicate and not the required triplicate plating) and 12 [plated
the 3M Petrifilm RYM Count Plates and reference method
plates on two different days (enrichments were held at 2–8°C
overnight and the 3M Petrifilm RYM Plates were plated 24 h
after the reference method plates)] reported deviations from the
protocol and were therefore excluded from statistical analysis.
For the raw almonds, laboratory 4 reported a deviation from the
protocol (failure to enumerate 3M Petrifilm RYM Count Plates
at 60 h) and was therefore excluded from the statistical analysis.
The 3M Petrifilm RYM Count Plate results along with FDA
BAMand ISO results reported by each laboratorywere converted
to logarithmic values for statistical analysis and were plotted
using a Youden’s plot (
see
Figures 1–4). The log
10
individual lab
results are presented in Tables 2–9. Using the Youden’s plots, an
initial review of the data to determine outliers was conducted
by observing the mean replicate results for each laboratory at
each contamination level for each matrix. The transformed
data was than statistically analyzed for outliers by the Cochran
and Grubb’s tests. No evidence of physical cause or suspicion
of cause was noted, so all outliers identified were included in
the statistical analysis. A paired
t
-test, the difference of means,
and the reverse transformed mean difference were calculated
on each contamination level for each matrix to determine if a
statistically significant difference existed between the methods.
Repeatability (s
r
), s
R
, RSD
r
, and RSD
R
were determined for
each contamination level for both the 3M Petrifilm RYM Count
Plate and FDA BAM/ISO 21527 methods. The results of the
interlaboratory data analyses are presented in Tables
2014.05A
and
2014.05B
. The result for each collaborating laboratory’s
aerobic plate count analysis for each matrix is presented in
Table
2014.05C
.
Frozen Raw Ground Beef Patties (77% Lean)
Frozen raw ground beef patties test portions were inoculated
at a low, medium and high contamination level and were
analyzed (Tables 2–5) for the enumeration of yeast and mold.
Uninoculated controls were included in each analysis. Fifteen
laboratories participated in the analysis of this matrix.
3M RYM Count Plate incubated at 25
°
C and enumerated
at 48 h.—
The mean log
10
counts of the 3M Petrifilm RYM
Count Plate and FDA BAM/ISO 21527 results were compared
Table 2014.05D. Appearance of yeast and mold on 3M
Petrifilm RYM Plates
Yeast
Mold
Small colonies
Large colonies
Colonies have defined edges
Colonies have diffused edges
Pink/tan to blue/green in color
Blue/green to variable upon pro-
longed incubation
Colonies appear raised
(3-dimensional)
Colonies appear flat
Colonies have a uniform color
Colonies have a dark center with
diffused edges
Table 1. Participation of each collaborating laboratory
a
Laboratory
Raw ground beef
Almonds
1
Y
Y
2
Y
Y
3
Y
Y
4
Y
b
Y
b
5
Y
Y
6
Y
b
Y
7
Y
Y
8
Y
b
Y
9
Y
Y
10
Y
Y
11
Y
Y
12
Y
b
Y
13
Y
Y
14
Y
Y
15
Y
Y
a
Y= Collaborator analyzed the food type; N= collaborator did not ana-
lyze the food type.
b
Results were not used in statistical analysis due to deviation of testing
protocol or laboratory error.