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17.5.08 (f) Sodium hydroxide solution.—Sterile 1M. Dissolve 20 g
AOAC Official Method 2003.07 NaOH in 500 mL water in 500 mL autoclavable Nalgene container.
Enumeration of Staphylococcus aureus Autoclave 15 min at 121°C.
in Selected Types of Processed and Prepared Foods ( g ) P h o s p h a t e - b u f f e re d d i l u t i o n w a t e r. — ( 1 ) St o c k
TM TM
3M Petrifilm Staph Express Count Plate Method solution.—Dissolve 34 g KH2PO4 in 500 mL H2O, adjust to pH 7.2
First Action 2003 with ca 175 mL 1M NaOH, and dilute to 1 L. Store in refrigerator. (2)
Final Action 2006
Diluent.—Dilute 1.25 mL stock solution to 1 L with H2O. Prepare
(Applicable to the enumeration of S. aureus in frozen lasagna, dilution blanks with this solution, dispensing enough to allow for
custard, frozen mixed vegetables, frozen hashbrowns, and frozen losses during autoclaving. Autoclave 15 min at 121°C.
batter-coated mushrooms.) (h) Blender.—High-speed blender (16 000–18 000 rpm) with
sterile jar.
Caution: Autoclave materials after use.
(i) Incubator.—Maintaining 35° ± 1°C or 37° ± 1°C.
See Table 2003.07 for the results of the interlaboratory study (j) Balance.—2000 ± 0.1 g capacity.
supporting acceptance of the method. (k) pH indicator strips.—To measure a range of 6.0–8.0.

A. Principle C. Preparation of Test Suspensions

The Petrifilm Staph Express Count plate is a sample-ready culture Use balance, B(j), to aseptically weigh 50 g test portion into
medium system which contains a cold-water-soluble gelling agent. blender jar, B(h). Add 450 mL diluent, B(g)(2), and blend at
The chromogenic, modified Baird-Parker medium in the plate is 16 000–18 000 rpm for 2 min to homogenize. If entire test sample is
selective and differential for S. aureus. Diluted test portions are added <50 g, weigh portion of test sample and add diluent water to make a
at a volume of 1.0 mL per plate. The gelling agent is allowed to solidify 1:10 dilution. As required, adjust pH of diluted test portion to
after inoculation, and the plate is then incubated for 24 ± 2 h at 35° ± 6.0–8.0 with 1M NaOH, B(f), (ca 0.1 mL/g test portion). Do not use
diluents containing citrate, bisulfite, or thiosulfate as they can
1°C or 37° ± 1°C. Red-violet colonies on the plate are S. aureus. When
inhibit growth. Prepare all decimal dilutions with 90 mL diluent plus
only red-violet colonies are present, count the colonies; the test is
10 mL from the previous dilution. Pipets, B(d), must accurately
complete.
deliver the required volume. Mix all dilutions by shaking 25 times
If background flora are encountered in testing, the Petrifilm Staph through 30 cm arc in 7 s.
Express disk is used to identify S. aureus from all suspect colonies.
Use the Petrifilm Staph Express disk whenever colonies other than D. Analysis
red-violet are present on the plate, for example, black colonies or Place Petrifilm Staph Express Count plate, B(a), on flat surface.
blue-green colonies. The Petrifilm Staph Express disk contains a dye Lift top film and inoculate 1 mL test suspension onto center of bottom
and deoxyribonucleic acid. S. aureus produces deoxyribonuclease film. Carefully roll top film down onto inoculum. Distribute test
(DNase), which reacts with the dye to form pink zones. The disk is suspension over 30 cm2 growth area with downward pressure on
inserted into the plate, and the plate and disk are then incubated for a handle of plastic spreader, B(c). Leave plate undisturbed to permit
minimum of 1 h and a maximum of 3 h at 35° ± 1°C or 37° ± 1°C. gelling agent to solidify. Incubate plates at 35° ± 1°C or 37° ± 1°C for
S. aureus (and occasionally, S. hyicus and S. intermedius, which can 24 ± 2 h. In incubator, B(i), place plate in horizontal position in stacks
produce enterotoxins) produce a pink zone. Count the pink zones as not exceeding 20 units. Count plates with colony counter, B(e).
S. aureus, regardless of the size of the zone. Observe colony colors. If no colonies or only red-violet colonies are
B. Apparatus and Reagents present after 24 ± 2 h, count red-violet colonies on the plate as
S. aureus; the test is complete. If any colony colors other than
(a) 3M Petrifilm Staph Express Count plate.—Plates, available
red-violet are present, use a Petrifilm Staph Express disk, B(b).
from 3M Microbiology (St. Paul, MN 55144, USA), contain
chromogenic modified Baird-Parker medium and a Insert Petrifilm Staph Express disk into plate. Apply pressure by
cold-water-soluble gelling agent. sliding a gloved finger firmly across entire disk area (including
edges) to ensure uniform contact of disk with gel and to eliminate
(b) 3M Petrifilm Staph Express disk.—Disks, available from 3M
any air bubbles. Incubate plates and disks, in stacks of no more than
Microbiology, contain toluidine blue-O and DNA.
20 units, for at least 60 min and no longer than 3 h at 35° ± 1°C or 37°
(c) Plastic spreader.—Spreader with handle, available from 3M
± 1°C. Enumerate pink zones as S. aureus whether or not colonies
Microbiology, has a smooth flat side and is designed to spread the
are present. Pink zones are usually associated with S. aureus but may
test suspension evenly over plate growth area.
indicate S. hyicus or S. intermedius. Colonies not associated with a
(d) Pipets.—Calibrated 1.0 and 10.0 mL serological pipets with pink zone are not S. aureus and should not be counted.
0.1 mL graduations. Electronic pipettor and tips, or equivalent, may Safety note: The test kit itself does not contain any pathogenic
be used to deliver 1.0 mL. Do not use pipets to deliver <10% of their components but the enriched test suspension may contain S. aureus.
total volume. For example, to deliver 1 mL, do not use pipet >10 mL; Therefore, discard all test samples according to standard laboratory
to deliver 0.1 mL, do not use pipet >1 mL. hazardous waste procedures.
(e) Col ony coun ter.—Stan dard ap pa ra tus, Que bec Model,
avail able from many suppliers, or one providing equivalent Reference: J. AOAC Int. 86, 954(2003).
magnification and visibility. Posted: April 2006

ã 2006 AOAC INTERNATIONAL

 Table 2003.07. Interlaboratory study results of 3M™ Petrifilm™ Staph Express Count plate method and the Baird-Parker agar method for detection of S. aureus in foods
Petrifilm Staph Express Count plate Baird-Parker agar
a b c b c
Food Lev el N M ean sr RSDr, % r sR RSDR, % R N M ean sr RSDr, % r sR RSDR, % R
L as ag na Low 12 1 .8 4 0 .2 4 1 3 .2 9 0 .6 8 0 .3 4 1 8 .6 2 0 .9 6 13 1 .8 3 0 .3 2 1 7 .7 7 0 .9 1 0 .4 1 2 2 .5 3 1 .1 5
M ed 13 3 .2 1 0 .2 7 8 .3 9 0 .7 5 0 .2 7 8 .3 9 0 .7 5 11 3 .2 8 0 .2 0 5 .9 5 0 .5 5 0 .2 0 5 .9 5 0 .5 5
d
M ed+ 12 3 .1 6 0 .0 8 2 .5 9 0 .2 3 0 .2 0 6 .4 0 0 .5 7 13 3 .1 3 0 .1 7 5 .2 9 0 .4 6 0 .2 7 8 .7 5 0 .7 7
Custard Low 12 1 .7 2 0 .2 3 1 3 .5 1 0 .6 5 0 .2 3 1 3 .5 1 0 .6 5 12 1 .8 0 0 .2 2 1 2 .1 4 0 .6 1 0 .2 5 1 3 .6 4 0 .6 9
d
M ed 12 2 .8 1 0 .0 6 2 .1 7 0 .1 7 0 .1 3 4 .6 4 0 .3 7 12 2 .8 0 0 .1 2 4 .4 2 0 .3 5 0 .2 0 7 .1 7 0 .5 6

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M ed+ 11 2 .8 0 0 .0 9 3 .1 4 0 .2 5 0 .0 9 3 .1 4 0 .2 5 11 2 .8 2 0 .0 9 3 .2 4 0 .2 6 0 .1 5 5 .3 5 0 .4 2
d
Mixed vegetables Low 11 2 .7 3 0 .0 6 2 .0 7 0 .1 6 0 .0 8 3 .0 8 0 .2 4 12 2 .7 4 0 .1 2 4 .4 2 0 .3 4 0 .1 5 5 .5 5 0 .4 3
M ed 11 3 .7 2 0 .0 6 1 .5 9 0 .1 7 0 .0 8 2 .0 6 0 .2 2 10 3 .7 6 0 .0 7 1 .8 3 0 .1 9 0 .1 1 2 .8 7 0 .3 0
M ed+ 12 3 .7 3 0 .0 8 2 .1 4 0 .2 2 0 .0 8 2 .3 4 0 .2 5 11 3 .7 8 0 .0 9 2 .5 0 0 .2 7 0 .1 1 2 .8 6 0 .3 0
Hashbrowns Low 11 2 .3 5 0 .1 2 5 .1 1 0 .3 4 0 .1 3 5 .3 4 0 .3 5 11 2 .3 9 0 .1 2 5 .1 9 0 .3 5 0 .1 7 7 .0 8 0 .4 7
M ed 12 3 .3 4 0 .1 0 2 .9 9 0 .2 8 0 .1 5 4 .4 6 0 .4 2 12 3 .3 4 0 .1 2 3 .6 2 0 .3 4 0 .2 1 6 .2 9 0 .5 9
M ed+ 13 3 .3 2 0 .1 2 3 .5 8 0 .3 3 0 .1 5 4 .5 2 0 .4 2 13 3 .3 6 0 .1 4 4 .1 2 0 .3 9 0 .1 8 5 .4 1 0 .5 1
Batter-coated mushrooms Low 11 2 .0 9 0 .1 5 7 .1 9 0 .4 2 0 .1 8 8 .6 1 0 .5 0 9 2 .2 3 0 .1 5 6 .6 1 0 .4 1 0 .1 5 6 .6 1 0 .4 1
M ed 10 3 .1 6 0 .1 5 4 .6 1 0 .4 1 0 .1 5 4 .6 1 0 .4 1 11 3 .1 1 0 .1 6 5 .0 0 0 .4 3 0 .2 3 7 .4 3 0 .6 5
d
M ed+ 9 3 .1 7 0 .1 0 3 .0 5 0 .2 7 0 .1 0 3 .1 4 0 .2 8 11 3 .0 7 0 .3 0 9 .6 8 0 .8 3 0 .3 0 9 .6 8 0 .8 3
a
Inoculation levels include a low level, medium level, and medium with a background organism.
b
Number of laboratories used in the analysis after the outlier tests.
c
Log10 S. aureus count/g.
d
Significantly better repeatability (p < 0.05).