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ird
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.
3, 2016
standard colony counter. Plates containing >300 colonies were
either estimated or recorded as too numerous to count (TNTC).
Estimations were conducted by counting the number of colonies
in two or more representative squares and determining the average
number per square. The average was multiplied by 30 to determine
the estimated count per plate.
All matrixes analyzed by the 3M Petrifilm RAC Plate were
also analyzed using BAM (shrimp) or SMEDP (milk and
NFDM) reference methods in a paired study design. Serial
dilutions for each sample were plated in duplicate onto plate
count agar (BAM) or standard methods agar (SMEDP). For raw
easy-peel shrimp and pasteurized skim milk, agar plates were
incubated for 48 ± 4 h at 35 ± 1°C or 32 ± 1°C, respectively.
Instant NFDM agar plates were incubated for 72 ± 3 h at
32 ± 1°C. Typical colonies in the countable range (25–250)
were enumerated using a standard colony counter.
Statistical Analysis
Each collaborating laboratory recorded the CFU/g results
for the reference methods and the 3M Petrifilm RAC Plate 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 set of plates were averaged. A logarithmic
transformation of the averaged counts was conducted for data
analysis. Outliers were identified using Cochran and Grubbs’
tests. The differences of means, including 95% upper and lower
confidence limits, were determined for each contamination
level for each matrix (6). If the confidence interval (CI) for
the difference of means passed through the point 0, there was
no statistical difference between the two methods (7). The
reversed transformed difference of means, with a 95% CI, and
the repeatability (s
r
) and reproducibility (s
R
) of the 3M Petrifilm
RAC Plate and reference methods were also determined (7).
AOAC Official Method 2015.13
Enumeration of Aerobic Bacteria in Food
3M Petrifilm Rapid Aerobic Count Plate
First Action 2015
(Applicable to the enumeration of aerobic bacteria from raw
groundbeef, rawgroundpork, rawground turkey, chickencarcass
rinsate, fresh swai, fresh tuna, fresh tiger shrimp, raw easy-peel
shrimp, cherry tomato wash, frozen blueberries, Mediterranean
apricots, creamy salad dressing, fresh pasta, vanilla ice cream,
instant NFDM, and pasteurized skim milk.)
See
Tables
2015.13A
and
2015.13B
for a summary of results
of the collaborative study.
See
Tables
2015.13C–G
for detailed results of the
collaborative study.
A. Principle
The 3M Petrifilm RAC Plate is a sample-ready culture
medium system that contains nutrients, a cold-water-soluble
gelling agent, and an indicator system that facilitates aerobic
bacterial enumeration. 3M Petrifilm RAC Plates are used for the
enumeration of aerobic bacteria in as little as 24 h for most food
matrixes. 3M Food Safety is certified to ISO 9001 for design
and manufacturing.
B. Apparatus and Reagents
(a)
3M Petrifilm RAC Plate
.—25 plates per pouch, two
pouches per box. Available from 3M Food Safety (St. Paul, MN;
Cat. No. 6478).
(b)
Sterile diluent
.—Butterfield’s phosphate-buffered diluent.
(c)
Pipets
.—Capable of pipetting 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 (Cat. No. 6425).
(h)
Incubators
.—Capable of maintaining 32 ± 1°C and
35 ± 1°C and having a solid front to maintain a dark interior.
(i)
Refrigerator or freezer
.—Capable of maintaining
temperature between –20 to 8°C for storing unopened 3M
Petrifilm RAC Plates.
(j)
Freezer
.—Capable of maintaining temperature at
less than −15°C for storing 3M Petrifilm RAC pouches after
incubation.
(k)
Standard colony counter or illuminated magnifier.
Table 2015.13A. Interlaboratory study results of 3M Petrifilm RAC Plate vs FDA BAM Chapter 3 method for raw easy-peel
shrimp
Matrix
raw
easy-peel
shrimp
3M Petrifilm RAC Plate
FDA BAM Chapter 3
Difference
of means
Difference of
means
d
Reverse-
transformed
difference of
the mean,
CFU/g
Reverse-
transformed
difference of
means LCL,
UCL
Lot
N
a
s
r
b
s
R
c
Lot
N
Mean
log
10
CFU/g s
r
s
R
95% LCL,
UCL
32°C Low 16 2.96 0.132 0.280 Low 16 3.02 0.218 0.356 0.06
–0.11, 0.24 139.47
0.77, 1.72
Medium 16 4.29 0.202 0.215 Medium 16 4.23 0.095 0.298 –0.06 –0.18, 0.06 –2424.10 0.67, 1.15
High 16 5.56 0.110 0.248 High 16 5.76 0.097 0.214 0.20
–0.01, 0.42 214352.79 0.97, 2.61
35°C Low 16 2.80 0.121 0.335 Low 16 3.02 0.218 0.356 0.22
–0.03, 0.48 422.68
0.92, 3.03
Medium 16 4.22 0.172 0.273 Medium 16 4.23 0.095 0.298 0.01
–0.08, 0.11 539.37
0.83, 1.28
High 16 5.67 0.141 0.174 High 16 5.76 0.097 0.214 0.09
–0.09, 0.26 105217.30 0.82, 1.83
a
Number of laboratories that reported complete results.
b
s
r
= Repeatability.
c
s
R
= Reproducibility.
d
95% lower and upper confidence limits. A 95% CI that contains the point 0, indicates no statistical significant difference between methods.