3M™ Petrifilm™ Staph Express Count Plate for the Rapid Enumeration of

Staphylococcus aureus in Foods – Collaborative Study

Barbara Horter and Kathryn Lindberg, 3M Microbiology, St. Paul, MN, 55144, USA

ABSTRACT If background flora are encountered in testing, the Petrifilm Staph MATERIALS AND METHODS RESULTS
Express disk is used to identify S. aureus from all suspect colonies. Microbiological Analyses
This interlaboratory collaborative study evaluated and compared the The Petrifilm Staph Express disk is used whenever colonies other Test Organisms Thirteen laboratories analyzed the samples for S. aureus using both Twelve or thirteen laboratories participated in the study,
AOAC INTERNATIONAL method for detection of Staphylococcus than red-violet are present on the plate; for example, black colonies The S. aureus organisms were obtained from the American Type the Petrifilm plate method and the AOAC method (Figure 5). depending on the food studied.
aureus in foods with a commercial product, the 3M™ Petrifilm™ or blue-green colonies (see Figure 2). Culture Collection (ATCC, Manassas, VA 20110) and are listed in
Staph Express Count Plate and Disk, for recovery of S. aureus in Table 1. Enterococcus faecalis ATCC 14506 was inoculated to Figure 5. Microbiological analysis The mean log counts of the Petrifilm Staph Express Count plate
foods. In the study, 14 food types — smoked salmon, pepperoni, achieve background competitive flora. method were not statistically different from the mean log counts
Sample
cured ham, cooked and diced chicken, frozen lasagna, custard, of the BPA method.
frozen mixed vegetables, frozen hash browns, frozen batter coated Table 1. Staphylococcus aureus used to inoculate test samples
ATCC Product
mushrooms, raw milk, yogurt, cheese, whey powder, and ice cream 8095 Frozen lasagna, pepperoni, smoked salmon, whey powder Prepare dilutions: 1:10, 1:100, 1:1000 The repeatability variances of the Petrifilm Staph Express Count
— were analyzed for S. aureus by several collaborating laboratories. 13565 Frozen hash browns, cured ham, mozzarella cheese
Baird-Parker agar plates plate method were not significantly different from those for the
25923 Frozen mixed vegetables, vanilla yogurt Petrifilm Staph Express Count Plate
For each food tested, the collaborators received eight blind samples 27664 Raw milk BPA method in 26 of the 41 groups tested. In 13 groups, the
49476 Cooked & diced chicken
consisting of a control sample and three levels of inoculated sample, 51740 Frozen batter coated mushrooms, custard, strawberry ice cream
1mL
0.3 mL 0.3 mL 0.4 mL
repeatability variance of the Petrifilm Staph Express Count plate
each in duplicate. Each sample was tested for S. aureus using the method was smaller in value than that of the BPA method
Petrifilm Staph Express Count plate method, as well as AOAC® (Tables 2-4).
Official MethodSM 975.55, the 3-plate Baird-Parker agar plus Inoculation of Foods and Preparation of Test Samples
The test portions were prepared according to Figure 4. Table 2. Interlaboratory study results from processed and prepared foods
coagulase test (BPA). The precision estimates (repeatability or Incubate 35 + 1C for 24 + 2 h Incubate 35 -37 C for 45 - 48 h
Food Level a Resultb Food Level Result
within-laboratory variation, and reproducibility or between- Figure 2. Petrifilm Staph Express Count plate with red-violet and blue-green colonies Lasagna Low Hash Low
Figure 4. Inoculation of foods and preparation of test samples Med browns Med
laboratory variation) were calculated using standard statistical Med+ Med+
Food Read plate Read plates
techniques. The mean log counts for the two methods were not 6000g
Custard Low Batter- Low
Med coated Med
statistically different for all foods and levels tested. The Med+ mushrooms Med+
repeatability variances of the 24-h Petrifilm Staph Express Count The Petrifilm Staph Express disk contains a dye and Only red-violet colonies? Mixed Low
No
plate method were significantly better than those of the 72-h BPA deoxyribonucleic acid. S. aureus produces deoxyribonuclease Grind
vegetables Med
Med+
method 32% of the time and not significantly different 63% of the (DNase), and the DNase reacts with the dye to form pink zones. Yes
a
Low=low level of inoculation of S. aureus; Med=medium level of inoculation of S. aureus;
Insert disk & Med+=medium level of inoculation of S. aureus & high level of inoculation of E. faecalis
time. When the disk is inserted into the plate and after the plate and disk incubate @
b
Yellow indicates no significant difference in repeatability from the 2 methods; green indicates better
Divide into repeatability of Petrifilm Staph Express Count plate method.
are incubated for up to 3 hours at 35 + 1oC or 37 + 1oC, 4 equal
35 + 1C for 1-3 h
S. aureus (and occasionally, Staphylococcus hyicus and portions Table 3. Interlaboratory study results from dairy foods
Food Level a Resultb Food Level Result
Select one colony Select one colony
Staphylococcus intermedius) produce a pink zone (see Figure 3). Low Low
INTRODUCTION Count the pink zones as S. aureus, regardless of the size of the
Read plate suspected to be
S. aureus
suspected to be
S. aureus
Ice cream
Med
Whey
powder Med
55 g Med+ Med+
portion
Staphylococcus aureus has been recognized as a cause of foodborne zone. Divide into
Raw milk Low Yogurt Low
Uninoculated control Med coated Med
subsamples
illness since the late nineteenth century. Identification of this Streak to BPA & Med+ mushrooms Med+
55 g Cheese Low
potential pathogen is important for food safety because portion incubate overnight
Med
@ 35 + 1C
approximately 35% of S. aureus strains produce at least one type of a
Med+
Low=low level of inoculation of S. aureus; Med=medium level of inoculation of S. aureus;
heat-stable enterotoxin that can cause intoxication and food 55 g
Med+=medium level of inoculation of S. aureus & high level of inoculation of E. faecalis
b
Plant in 0.2 mL of BHI & Yellow indicates no significant difference in repeatability from the 2 methods; green indicates better
poisoning. The 3MTM PetrifilmTM Staph Express Count plate and Low level: portion
incubate 18-24 h @ 35 + 1C repeatability of Petrifilm Staph Express Count plate method; blue indicates better repeatability of BPA
Inoculate with 100- Divide into method.
the 3MTM PetrifilmTM Staph Express disk have been developed to 1000 cells/g of subsamples
enumerate S. aureus in foods. S. aureus 55 g Table 4. Interlaboratory study results from meat, poultry, & seafood
Add rabbit plasma with EDTA & Food Level a Resultb Food Level Result
portion
reincubate @ 35 + 1C for additional 6 h Chicken Low Salmon Low
Send to
collaborators Med Med
The Petrifilm Staph Express Count plate is a sample-ready culture for testing Med+ Med+
medium system which contains a cold-water-soluble gel. The 55 g Read clot formation Ham Low Pepperoni Low NAc
Med level: portion Med Med
chromogenic, modified Baird-Parker medium in the plate is Inoculate with 1001- Divide into Med+ Med+
10,000 cells/g of subsamples
selective and differential for S. aureus. Diluted samples are added S. aureus
a
Low=low level of inoculation of S. aureus; Med=medium level of inoculation of S. aureus;
Med+=medium level of inoculation of S. aureus & high level of inoculation of E. faecalis
55 g
at a volume of 1.0 ml per plate. The gel is allowed to solidify after portion
b
Yellow indicates no significant difference in repeatability from the 2 methods; green indicates better
repeatability of Petrifilm Staph Express Count plate method.
inoculation, and the plate is then incubated for 24 + 2 hours at 35 + c
No S. aureus colonies were detected as the inoculation level was missed.

1oC or 37 + 1oC. Red-violet colonies on the plate are S. aureus (see Data Analysis e
Figure 3. Petrifilm Staph Express Count plate and disk with pink zones Med+ level: 55 g
Figure 1). When only red-violet colonies are seen, count the Inoculate with 1001- portion Raw counts were converted to log10 counts to more nearly match
colonies; the test is complete. 10,000 cells/g of Divide into the underlying assumption of normality. Repeatability and
S. aureus & 10,001-
100,000 cells/g of
subsamples 55 g
reproducibility precision estimates were calculated according to CONCLUSION
portion
E. faecalis
AOAC INTERNATIONAL procedures once outliers were
The objective of this interlaboratory study was to evaluate and The mean log counts of the 24-h Petrifilm Staph Express Count
determined by the Cochran and Grubbs tests. Repeatability
compare AOAC® Official MethodSM 975.55 with the Petrifilm Staph plate method and the repeatability and reproducibility variances
variances were compared using an F-ratio test. Mean log counts
Express Count plate method for the recovery of S. aureus in foods. of that method were similar to those of the 72-h, 3-plate BPA
were compared by analysis of variance. In all statistical tests, a
resulting value of p<0.05 was taken to indicate a significant method for analysis of selected pre-packaged and processed
difference. foods, dairy foods, and meat, poultry, and seafood. The Petrifilm
Staph Express Count plate method has been adopted official First
Action by AOAC for the enumeration of S. aureus.

ACKNOWLEDGEMENTS
The authors wish to thank the participating laboratories for their efforts leading to the successful
completion of this collaborative study.

Figure 1. Petrifilm Staph Express Count plate with red-violet colonies

3 Microbiology
© 3M 2003 70-2009-5907-3