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Aerobic count bacteria are ubiquitously present in a variety of food matrices and serve as indicators for food spoilage. These microorganisms can grow in a wide range of temperatures and pH values thus having the potential for substantial economic losses to the food industry. These organisms have become important indicators for monitoring food quality but the 48 hour incubation required by traditional enumeration methods can prevent food producers from getting results in an actionable time frame. A novel dehydrated film medium was developed to address the need for rapid (24 hour) detection of aerobic bacteria. The detection technology was optimized to overcome the inherent limitations associated with over expression of extracellular enzymes by certain spore formers that results in liquefaction of the dehydrated film media.

Performance of 3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plates at 30°C, 32°C and 35°C was compared to a reference medium commonly used for aerobic bacteria [Standard Methods Agar (SMA)]. The study involved a total of thirty-six bacterial strains which included Gram positive and Gram negative bacteria. Each organism was serially diluted in Butterfield’s buffer. 1mL each of the appropriate sample dilution was inoculated in duplicate onto 3M Petrifilm RAC and the reference method (SMA; pour plate method; reference method for lactic acid bacteria (LAB): MRS agar). Counts obtained at 24 hours on the 3M Petrifilm RAC Plates were compared to results obtained on SMA at 48 hours (at 32°C or 35°C) or 72 hours (ISO 4833 at 30°C). All test organisms were incubated in an aerobic environment, except for the LAB, which were incubated under facultative conditions in an anaerobic jar.

Studies were conducted to assess the performance of the 3M Petrifilm RAC Plate method, in the presence of potential interferences from sample matrix and other organisms. The method comparison was conducted using a variety of naturally contaminated food matrices (n=78) with the new dehydrated film test method and a reference method (U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) and ISO 4833). Each food sample was serially diluted in Butterfield’s buffer. 1mL each of the appropriate sample dilution was inoculated in duplicate onto 3M Petrifilm RAC Plate and the reference method (SMA; pour plate method). Counts obtained at 24 hours on the 3M Petrifilm RAC Plate were compared to results obtained on SMA at 48 hours (at 32°C or 35°C) or 72 hours (ISO 4833 at 30°C).

Environmental samples were evaluated using air, swab and direct surface contact surface methodologies with the 3M Petrifilm RAC Plate and the reference method where applicable. The plates used for air and direct-contact surface samples were hydrated with 1mL of Butterfield’s buffer and Bi-sulfite free Letheen broth respectively. Hydrated plates were stored for three days in the freezer (<-20°C) prior to sampling.

Environmental swab samples were obtained using 3M™ Quick Swab with Letheen broth and 3M™ Hydrated-Sponge with neutralizing buffer.

• Quantitative results obtained on the 3M Petrifilm RAC Plates at 24 hours were comparable to those obtained on the Standard Methods Agar in 48–72 hours.

• The detection technology performed comparably at incubation temperatures of 30°C, 32°C and 35°C.

• The new method was found to provide actionable results in a shorter period of time, with significantly improved interpretation without sacrificing performance which is critical for food processors.

We would like to thank Darcy Herman, Haley Decker and Ashley Smith for their contributions to laboratory experimentation.

ABSTRACT

CONCLUSION

ACKNOWLEDGMENTS

RESULTS

EXPERIMENTAL APPROACH

FOOD STUDIES

ENVIRONMENTAL STUDIES

P2-135: Rapid Quantitative Enumeration of Aerobic Count Bacteria in Foods3M Company, St. Paul, MN, United States: Sailaja Chandrapati, Tera Nordby

Adjunct Associate Professor, Department of Food Science and Nutrition, UMN, St. Paul, MN, United States: Sailaja Chandrapati

Table 1: Comparative Recoveries (Log CFU/mL) from a Wide Variety of Naturally Contaminated Matrices on 3M™ Petrifilm™ Rapid Aerobic Count Plates (24 Hours at 30°C, 32°C and 35°C) and Standard Methods Agar (72 Hours at 30°C; 48 Hours at 32°C and 35°C).

Log CFU/mL3M Petrifilm RAC (30°C)

SMA (30°C)

3M Petrifilm RAC (32°C)

SMA (32°C)

3M Petrifilm RAC (35°C)

SMA (35°C)

DRES

SING

S Thousand Island 4.9 5.5 5.2 5.5 5.3 5.4Creamy French 5.0 5.4 5.3 5.5 5.4 5.4Blue Cheese 2.3 2.7 2.5 2.8 2.4 2.6Honey Mustard 2.7 3.0 3.4 2.9 2.8Buttermilk Ranch 5.2 5.5 5.4 5.5 5.4 5.6

Mean Log Difference (Alt-Ref) -0.4 N/A -0.8 N/A -0.1 N/A

MIS

CELL

ANEO

US/P

OWDE

RS/B

AKER

Y

Snicker Pie 3.2 3.2 3.1 3.3 3.0 3.2Caramel Flavor 2.1 2.2 2.0 2.1 2.2 2.2Brownie Pie 3.4 3.4 3.4 3.3 3.5 3.5Lava Cake 4.1 3.8 3.9 3.9 4.1 3.9German Chocolate Pie 2.6 2.6 2.5 2.5 2.6 2.9Pasta and Cheese Sauce 7.9 7.9 7.9 8.0 7.9 7.9Buffalo Powder 4.5 4.7 4.4 4.7 6.2 4.7Garlic Powder 3.8 4.3 3.9 4.2 4.1 4.1Pizza Powder 4.2 3.3 4.1 4.0 4.0 4.0Southwest Queso Powder 3.5 5.2 3.6 5.3 3.5 5.1Mustard Powder 3.9 3.9 3.9 4.0 3.9 4.1Chicken Dumpling Powder 4.1 4.2 3.9 4.1 3.8 4.0Dehydrated Onion 4.2 4.5 3.4 4.5 3.3 4.5Oolong Tea Leaves 6.5 7.1 6.5 7.1 6.5 7.1BBQ Powder 3.6 3.5 3.6 3.6 3.8 3.6Peanut Butter 2.2 2.4 2.0 4.9 2.3 5.3Non Fat Dry Milk 2.2 2.6 2.4 2.4 2.4 2.4Spicy Hummus 4.0 4.5 4.0 4.6 3.2 4.4Soy Protein 9.2 9.2 9.1 9.2 8.4 9.4Refried Bean Mix 8.6 8.5 8.6 8.5 8.6 8.2

Mean Log Difference (Alt-Ref) -0.2 N/A -0.4 N/A -0.4 N/ABay Scallop 4.2 4.4 4.0 3.9 3.7 3.9

SEAF

OOD

Atlantic Salmon 5.3 5.5 5.0 5.1 4.7 4.8Tiger Shrimp 6.8 6.8 6.6 6.6 6.0 6.6Sea Scallop 4.4 4.8 4.2 4.2 3.5 3.5Easy-Peel Shrimp 7.2 7.2 7.1 7.1 5.9 6.1Sockeye Salmon 3.5 4.1 3.4 3.8 3.7 3.8Yellowfin Tuna 5.6 5.4 5.5 5.3 5.1 4.7Swai 5.1 5.5 5.1 5.3 5.0 4.9Monkfish 5.5 5.5 5.1 5.1 5.0 4.7Cooked Shrimp, Frozen 1.5 3.0 1.9 2.6 1.7 2.7Uncooked Shrimp 3.1 3.8 3.1 3.6 3.1 3.7Key West Shrimp 3.0 3.7 3.0 3.7 2.8 3.5Swai Frozen 3.6 3.5 3.6 3.5 3.8 3.6Cod 1.3 1.9 1.0 2.0 1.7 0.7Pollock 3.6 4.1 2.9 4.0 2.9 4.0Tilapia 3.2 3.5 3.1 4.3 3.0 3.5

Mean Log Difference (Alt-Ref) -0.4 N/A -0.4 N/A -0.2 N/A

Log CFU/mL3M Petrifilm RAC (30°C)

SMA (30°C)

3M Petrifilm RAC (32°C)

SMA (32°C)

3M Petrifilm RAC (35°C)

SMA (35°C)

Breakfast Links 4.6 5.4 5.5 4.8 4.4 4.8

MEA

T/PO

ULTR

Y

Ground Pork 5.6 5.8 5.7 5.6 5.2 5.6Ground Chicken 3.1 5.1 3.1 4.4 3.1 4.5Chicken Breast 3.7 4.1 3.6 3.8 3.3 3.7Ground Turkey, 99% Lean 4.7 5.0 4.6 4.5 4.4 4.3Ground Turkey, 93% Lean 7.3 7.5 7.3 7.3 7.2 7.3Turkey Sausage 8.7 8.5 8.8 8.9 8.8 8.9Chicken Salad 4.4 4.5 4.5 4.2 4.4 4.5Ground Beef, 75% Lean 5.6 5.9 5.5 5.9 5.4 5.9Ground Beef, 80% Lean 4.0 3.3 3.1 3.2 3.1 3.4Ground Beef, 85% Lean 4.7 4.2 4.4 4.2 4.3 3.9Ground Beef, 93% Lean 4.3 2.7 4.7 2.7 4.4 2.7Ham Salad 1.8 2.0 2.1 2.0

Mean Log Difference (Alt-Ref) -0.1 N/A 0.1 N/A -0.1 N/A

DAIR

Y

Vanilla Chocolate Chip Ice Cream 7.9 8.1 7.9 8.0 7.8 8.0

Black Raspberry Chocolate Chip Ice Cream 5.4 5.4 5.4 5.5 5.4 5.5

Chocolate Peanut Butter Ice Cream 3.2 2.8 3.4 3.2 3.5 3.1

Strawberry Ice Cream 7.9 7.8 7.6 7.7 6.9 6.9Vanilla Ice Cream 8.1 8.7 6.9 8.4 6.6 7.8Cookies and Cream Ice Cream 8.3 8.6 8.7 8.3 8.3 8.3

Chocolate Chip Vanilla Ice Cream 9.1 9.1 9.1 9.2 9.1 9.2

Chocolate Chip Cookie Dough Ice Cream 5.8 5.5 5.8 5.2 5.8 5.4

Orange Sherbet 3.1 3.2 3.1 3.2 3.2 3.2Raspberry Chocolate Chip Ice Cream 8.2 8.8 8.2 8.2 8.1 8.2

Colby Jack Cheese 8.1 8.2 8.2 8.2 8.1 8.1Raw Milk 1 3.1 4.0 3.1 3.6 3.2 3.8Raw Milk 2 3.4 4.1 3.4 3.8 3.4 3.9Raw Milk 3 3.0 3.3 3.0 3.2 3.1 3.1Raw Milk 4 3.2 3.8 3.2 3.5 3.2 3.6Raw Milk 5 3.2 3.4 3.2 3.3 3.1 3.4Raw Milk 6 3.0 2.9 3.0 2.9 3.0 3.0Raw Milk 7 3.6 2.7 3.7 3.7 3.7 3.7Raw Milk 8 3.3 3.8 3.5 3.7 3.6 3.7Raw Milk 9 3.6 4.0 3.6 4.0 3.6 4.0Raw Milk 10 2.8 2.9 2.8 2.9 2.9 2.9Raw Milk 11 2.8 3.0 2.6 3.0 2.6 3.0Raw Milk 12 4.0 3.9 4.0 3.9 4.0Raw Milk 13 3.0 3.1 2.9 3.0 3.1 3.1

Mean Log Difference (Alt-Ref) -0.3 N/A -0.2 N/A -0.2 N/A

Mean Log Difference — Average of the difference in Log CFU between the 3M™ Petrifilm™ Rapid Aerobic Count Plates and the Reference Method.

Table 2: Descriptive Summary Statistics for Quantitative Recovery from Naturally Contaminated Foods (n=78) on 3M™ Petrifilm™ Rapid Aerobic Count Plates (24 Hours at 30°C, 32°C and 35°C) and Standard Methods Agar (72 Hours at 30°C; 48 Hours at 32°C and 35°C).

Summary Statistics: All Foods (n=78)N Average STD Dev SE Mean Mean Log Difference R2 Slope Intercept

30°C 3M Petrifilm RAC, 24 Hours 77 4.55 1.98 0.23 -0.30 0.93 0.99 -0.15

SMA, 72 Hours 78 4.76 1.93 0.22 N/A N/A N/A N/A

32°C 3M Petrifilm RAC, 24 Hours 76 4.56 1.96 0.22 -0.30 0.91 1.00 -0.21

SMA, 48 Hours 77 4.76 1.86 0.21 N/A N/A N/A N/A

35°C 3M Petrifilm RAC, 24 Hours 78 4.42 1.87 0.21 -0.20 0.89 0.95 0.03

SMA, 48 Hours 78 4.63 1.86 0.21 N/A N/A N/A N/AMEAN LOG Difference — Average of the difference in LOG CFU between the 3M™ Petrifilm™ Rapid Aerobic Count Plates and the Reference Method.

Figure 1: Appearance of Aerobic Bacteria on the 3M™ Petrifilm™ Rapid Aerobic Count Plates at 24 Hours. Figure 2: Improved Interpretation of a Raw Milk Isolate on the 3M™ Petrifilm™ Rapid Aerobic Count Plates compared to Standard Methods Agar.

Figure 1A: Pasta Cheese Sauce Figure 2A: 24 HoursFigure 1B: Pizza Powder Figure 2B: 48 HoursFigure 1C: Turkey Sausage Figure 1D: Ground Beef, 75% Figure 1E: Mustard Powder Figure 1F: Tiger Shrimp Figure 1G: Enviro. Sponge Sample Figure 1H: Enviro. Swab Sample

Figure 3: Regression Graph for Inclusivity Panel (n=36) Including a Wide Variety of Gram Positive and Gram Negative Bacteria at 24 Hours at 30°C, 32°C and 35°C) and Standard Methods Agar (72 Hours at 30°C; 48 Hours at 32°C and 35°C).

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Log CFU/mL (Reference Method: SMA)

0

100

200

300

400

500

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0 100 200 300 400 500 600

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3M Petrifilm RAC (35°C)

3M Petrifilm RAC (32°C)

3M Petrifilm RAC (30°C)

Figure 4: Regression Graph for Naturally Contaminated Foods (n=78) Across a Wide Variety of Matrices on 3M™ Petrifilm™ Rapid Aerobic Count Plates (24 Hours at 30°C, 32°C and 35°C) and Standard Methods Agar (72 Hours at 30°C; 48 Hours at 32°C and 35°C).

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Log

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rifilm

RAC

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toty

pes)

Log CFU/mL (Reference Method: SMA)

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y = 0.9474x + 0.0344R² = 0.89278

y = 0.9984x – 0.21R² = 0.90994

y = 0.986x – 0.1507R² = 0.93355

3M Petrifilm RAC (35°C)

3M Petrifilm RAC (32°C)

3M Petrifilm RAC (30°C)