Test OrganismsThe S. aureus organisms were obtained from the American Type Culture Collection (ATCC, Manassas, VA 20110) and are listed in Table 1. Enterococcus faecalis ATCC 14506 was inoculated to achieve background competitive flora.

ABSTRACT

This interlaboratory collaborative study evaluated and compared the AOAC INTERNATIONAL method for detection of Staphylococcus aureus in foods with a commercial product, the 3M™ Petrifilm™Staph Express Count Plate and Disk, for recovery of S. aureus in foods. In the study, 14 food types — smoked salmon, pepperoni, cured ham, cooked and diced chicken, frozen lasagna, custard, frozen mixed vegetables, frozen hash browns, frozen batter coated mushrooms, raw milk, yogurt, cheese, whey powder, and ice cream — were analyzed for S. aureus by several collaborating laboratories. For each food tested, the collaborators received eight blind samples consisting of a control sample and three levels of inoculated sample, each in duplicate. Each sample was tested for S. aureus using the Petrifilm Staph Express Count plate method, as well as AOAC®Official MethodSM 975.55, the 3-plate Baird-Parker agar plus coagulase test (BPA). The precision estimates (repeatability orwithin-laboratory variation, and reproducibility or between-laboratory variation) were calculated using standard statisticaltechniques. The mean log counts for the two methods were not statistically different for all foods and levels tested. The repeatability variances of the 24-h Petrifilm Staph Express Count plate method were significantly better than those of the 72-h BPA method 32% of the time and not significantly different 63% of the time.

INTRODUCTION

Staphylococcus aureus has been recognized as a cause of foodborne illness since the late nineteenth century. Identification of this potential pathogen is important for food safety because approximately 35% of S. aureus strains produce at least one type of heat-stable enterotoxin that can cause intoxication and food poisoning. The 3MTM PetrifilmTM Staph Express Count plate and the 3MTM PetrifilmTM Staph Express disk have been developed to enumerate S. aureus in foods.

The Petrifilm Staph Express Count plate is a sample-ready culture medium system which contains a cold-water-soluble gel. The chromogenic, modified Baird-Parker medium in the plate is selective and differential for S. aureus. Diluted samples are added at a volume of 1.0 ml per plate. The gel is allowed to solidify after inoculation, and the plate is then incubated for 24 + 2 hours at 35 +1oC or 37 + 1oC. Red-violet colonies on the plate are S. aureus (see Figure 1). When only red-violet colonies are seen, count the colonies; the test is complete.

MATERIALS AND METHODS

CONCLUSION

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

If background flora are encountered in testing, the Petrifilm Staph Express disk is used to identify S. aureus from all suspect colonies. The Petrifilm Staph Express disk is used whenever colonies otherthan red-violet are present on the plate; for example, black colonies or blue-green colonies (see Figure 2).

Table 1. Staphylococcus aureus used to inoculate test samples

The Petrifilm Staph Express disk contains a dye and deoxyribonucleic acid. S. aureus produces deoxyribonuclease (DNase), and the DNase reacts with the dye to form pink zones. When the disk is inserted into the plate and after the plate and disk are incubated for up to 3 hours at 35 + 1oC or 37 + 1oC, S. aureus (and occasionally, Staphylococcus hyicus and Staphylococcus intermedius) produce a pink zone (see Figure 3). Count the pink zones as S. aureus, regardless of the size of the zone.

Figure 3. Petrifilm Staph Express Count plate and disk with pink zones

The objective of this interlaboratory study was to evaluate and compare AOAC® Official MethodSM 975.55 with the Petrifilm Staph Express Count plate method for the recovery of S. aureus in foods.

Microbiological AnalysesThirteen laboratories analyzed the samples for S. aureus using both the Petrifilm plate method and the AOAC method (Figure 5).

Data AnalysisRaw counts were converted to log10 counts to more nearly match the underlying assumption of normality. Repeatability and reproducibility precision estimates were calculated according toAOAC INTERNATIONAL procedures once outliers were determined by the Cochran and Grubbs tests. Repeatability variances were compared using an F-ratio test. Mean log counts were compared by analysis of variance. In all statistical tests, a resulting value of p<0.05 was taken to indicate a significant difference.

RESULTS

Twelve or thirteen laboratories participated in the study, depending on the food studied.

The mean log counts of the Petrifilm Staph Express Count plate method were not statistically different from the mean log countsof the BPA method.

The repeatability variances of the Petrifilm Staph Express Countplate method were not significantly different from those for theBPA method in 26 of the 41 groups tested. In 13 groups, the repeatability variance of the Petrifilm Staph Express Count plate method was smaller in value than that of the BPA method (Tables 2-4).

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

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

Inoculation of Foods and Preparation of Test SamplesThe test portions were prepared according to Figure 4.

e

The mean log counts of the 24-h Petrifilm Staph Express Count plate method and the repeatability and reproducibility variancesof that method were similar to those of the 72-h, 3-plate BPA method for analysis of selected pre-packaged and processed foods, dairy foods, and meat, poultry, and seafood. The Petrifilm Staph Express Count plate method has been adopted official FirstAction by AOAC for the enumeration of S. aureus.

ATCC Product 8095 Frozen lasagna, pepperoni, smoked salmon, whey powder

13565 Frozen hash browns, cured ham, mozzarella cheese 25923 Frozen mixed vegetables, vanilla yogurt 27664 Raw49476 Cooked & diced chicken 51740 Frozen batter coated mushrooms, custard, strawberry ice cream

Food6000g

Grind

Divide into4 equalportions

Uninoculated control

Low level:Inoculate with 100-

1000 cells/g ofS. aureus

Med level:Inoculate with 1001-

10,000 cells/g ofS. aureus

Med+ level:Inoculate with 1001-

10,000 cells/g ofS. aureus & 10,001-100,000 cells/g of

E. faecalis

Divide intosubsamples

Divide intosubsamples

Divide intosubsamples

Divide intosubsamples

55 gportion

55 gportion

55 gportion

55 gportion

55 gportion

55 gportion

55 gportion

55 gportion

Send tocollaborators

for testing

Figure 4. Inoculation of foods and preparation of test samples

Sample

Prepare dilutions: 1:10, 1:100, 1:1000

1mL0.3 mL 0.3 mL 0.4 mL

Incubate 35 -37 C for 45 - 48 h

Read plate Read plates

Only red-violet colonies?

Insert disk &incubate @

35 + 1C for 1-3 h

Read plateSelect one colonysuspected to be

S. aureus

Streak to BPA &incubate overnight

@ 35 + 1C

Plant in 0.2 mL of BHI &incubate 18-24 h @ 35 + 1C

Incubate 35 + 1C for 24 + 2 h

Add rabbit plasma with EDTA &reincubate @ 35 + 1C for additional 6 h

Read clot formation

Select one colonysuspected to be

S. aureus

No

Yes

Petrifilm Staph Express Count PlateBaird-Parker agar plates

Figure 5. Microbiological analysis

3Microbiology© 3M 2003 70-2009-5907-3

Figure 2. Petrifilm Staph Express Count plate with red-violet and blue-green colonies

Table 2. Interlaboratory study results from processed and prepared foods Food Levela Resultb Food Level Result Lasagna Low Hash Low

Med browns Med Med+ Med+

Custard Low Batter- Lo Med

w Med coated Med+ mushrooms Med+

Mixed Lowvegetables Med

Med+ aLow=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 bYellow indicates no significant difference in repeatability from the 2 methods; green indicates better repeatability of Petrifilm Staph Express Count plate method. Table 3. Interlaboratory study results from dairy foods Food FoodIce cream Whey

powder

Raw milk Yogurt

Cheese

aLow=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 bYellow indicates no significant difference in repeatability from the 2 methods; green indicates better repeatability of Petrifilm Staph Express Count plate method; blue indicates better repeatability of BPA method. Table 4. Interlaboratory study results from meat, poultry, & seafood

Chicken Salmon

Ham Pepperoni NAc

aLow=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 bYellow indicates no significant difference in repeatability from the 2 methods; green indicates better repeatability of Petrifilm Staph Express Count plate method. cNo S. aureus colonies were detected as the inoculation level was missed.

milk

Levela Resultb Level Result Low Low Med Med Med+ Med+ Low Lo

Medw

Med coated Med+ mushrooms Med+

LowMed

Med+

Food Food

Levela Resultb Level Result Low Low Med Med Med+ Med+ Low Lo

Medw

Med Med+ Med+