June 24, Q Laboratories, Inc Harrison Avenue Cincinnati, OH 45214

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1 June 24, 2011 A Comparative Evaluation of the Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates with Two Reference Methods for the Enumeration of Lactic Acid Bacteria in Food and Environmental Surfaces Kiel Fisher, Erin Crowley, Patrick Bird, Megan Boyle, Katherine Goetz, M. Joseph Benzinger Jr., Mark Juenger, Travis Huffman, James Agin, David Goins Q Laboratories, Inc Harrison Avenue Cincinnati, OH A validation study of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M Petrifilm Aerobic Count (AC) Plates for the enumeration and detection of lactic acid bacteria was conducted at Q Laboratories, Inc., Cincinnati, Ohio. The PLAB counts were compared to the counts obtained by the agars used in two reference methods: The Compendium of Methods for the Microbiological Examination of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of mesophilic lactic acid bacteria Colony-count technique. The Compendium reference agar, All Purpose Tween agar plus 2% sucrose and bromocresol purple (APTS) and the ISO reference agar, deman, Rogasa, Sharpe plus bromocresol purple (MRS) were used in the in the analysis of both food and environmental swab samples. Four food matrices and one environmental surface were used in the comparative evaluation: salad dressings, ready to eat (RTE) salads, RTE meats, raw meats and stainless steel surfaces. For those matrices that were inoculated, various brands of each matrix were spiked with Lactobacillus fermentum ATCC or Weissella viridescens ATCC at two target recovery levels on 3M Petrifilm AC Plates: a low level of 1 15 CFU/ plate and a medium level of CFU/ plate. Natural lactic acid bacteria contamination was recovered in five brands of RTE meat and in all brands of raw meat. A paired t-test, comparing the PLAB procedure and the reference methods at varying time points, was conducted at the 95% confidence level. Statistically comparable mean log values were determined for salad dressings, RTE salads, RTE meats, and stainless steel surfaces. The PLAB procedure demonstrated reliability as a rapid and selective quantitative method for the enumeration of lactic acid producing bacteria. This report presents the analytical results for comparison of the Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M Petrifilm Aerobic Count (AC) Plates to agars used in The Compendium of Methods for the Microbiological Examination of Foods, Chapter 19 and ISO 15214: Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of mesophilic lactic acid bacteria Colony-count technique. All analyses were conducted at Q Laboratories, Inc. (Cincinnati, OH). 3M Petrifilm AC Plates and Aerobic Lactic Bacteria Broth were provided by 3M (Minneapolis, MN). Q Laboratories, Inc. is an independent company and is not affiliated with, nor a subsidiary of 3M. Q Laboratories, Inc. was contracted to perform an independent study by and for 3M and the data generated by Q Laboratories, Inc. during the execution of the study does not represent an endorsement of any product(s) by Q Laboratories, Inc. Page 1

2 Materials and Methods The methodology for this study was followed as outlined in the protocol: Comparison of a 3M Petrifilm Aerobic Count Plate Method with Three Methods for the Enumeration of Lactic Acid Bacteria in Food and Environmental Samples, provided by 3M. The study objective was to evaluate the performance of the Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count (AC) Plates to the ISO and Compendium Reference Methods for detecting and enumerating lactic acid bacteria. The following matrices were analyzed: salad dressings, ready to eat (RTE) salads, RTE meats, raw meats, and stainless steel surfaces. A detailed list of the brands used in the food analysis is presented in Table A. Table A. Matrix Details Identification Brand Salad Dressings RTE Salads Caesar 1 Cardini s Caesar Dressing Mustard Potato 1 Meijer Markets Mustard Potato Salad Caesar 2 Newman s Own Caesar Dressing Mustard Potato 2 Grandma s Mustard Potato Salad LoFat Caesar 1 Cardini s Light Caesar Dressing Mustard Potato 3 Kroger Mustard Potato Salad LoFat Caesar 2 Ken s Steak House Light Caesar Dressing American Potato 1 Grandma s Potato Salad with Egg Ranch 1 Kroger Creamy Ranch Dressing American Potato 2 Meijer Markets American Potato Salad Ranch 2 Ken s Steak House Ranch Dressing American Potato 3 Kroger American Potato Salad LoFat Ranch 1 Kroger Lite Creamy Ranch Dressing Macaroni 1 Kroger Macaroni Salad LoFat Ranch 2 Kraft Light Ranch Reduced Fat Dressing Macaroni 2 Meijer Macaroni Salad Catalina 1 Kraft Classic Catalina Dressing Plus Dip Macaroni 3 Grandmas Macaroni Salad Catalina 2 Kroger California French Dressing Bean 1 Kroger Baked Beans Blue Cheese 1 Kraft Roka Brand Blue Cheese Dressing Bean 2 Gordon Food Services Baked Beans Blue Cheese 2 Kroger Chunky Blue Cheese Dressing Bean 3 Meijer Baked Beans Blue Cheese 3 Ken s Steak House Chunky Blue Cheese Dressing Coleslaw 1 Meijer Cole Slaw Zesty Italian 1 Kroger Zesty Italian Dressing Coleslaw 2 Kroger Cole Slaw Zesty Italian 2 Kraft Zesty Italian Dressing Coleslaw 3 Grandma s Cole Slaw RTE Meats Raw Meats O/R Turkey 1 Sara Lee Fresh Ideas Oven Roasted Turkey Breast Ground Turkey 1 Honeysuckle White Ground Turkey 93/7 O/R Turkey 2 Kroger Deli Thin Sliced Oven Roasted Turkey Breast Ground Turkey 2 Kroger Ground Turkey 85/15 Roast Beef 1 Kroger Deli Thin Sliced Roast Beef Chicken 1 Tyson All Natural Chicken Breast Tenderloins 85/15 Roast Beef 2 Private Selection Homestyle Slow Roasted Roast Beef Chicken 2 Perdue Fresh Ground Chicken O/R Chicken 1 Private Selection Homestyle Slow Roasted Chicken Breast Ground Beef 1 Kroger Ground Sirloin 90/10 O/R Chicken 2 Sara Lee Fresh Ideas Oven Roasted Chicken Breast Ground Beef 2 Laura s Lean Ground Beef 92/8 Smoked Ham 1 Private Selection Smokehouse Hickory Ham Steak 1 Kroger Beef Top Sirloin Smoked Ham 2 Kroger Deli Think Sliced Smoked Ham Steak 2 Kroger Beef Bottom Round Steak Choice Culture Preparation and Inoculation of Test Portions Replicates of each matrix varied by food or surface type and were inoculated with Lactobacillus fermentum ATCC or Weissella viridescens ATCC at two target recovery levels on 3M Petrifilm AC Plates: a low level of 1 15 colony forming units (CFU)/plate and a medium level of CFU/plate. To prepare salad dressings and RTE salads for inoculation, the suspensions of L. fermentum and W. viridescens were prepared in deman, Rogasa, Sharpe broth (MRS) with 1.0% glucose. The L. fermentum inoculum was incubated aerobically for 24 ± 2 hours at 35 ± 2 C and the W. viridescens inoculum was incubated microaerophilically for 24 ± 2 hours at 35 ± 2 C. Twenty-five 1g sample portions were inoculated, mixed thoroughly, and held for 48 ± 6 hours at 10 C to allow the organism to equilibrate within the matrix. For the analysis of RTE meats, L. fermentum and W. viridescens inocula were prepared in MRS broth and incubated for 24 ± 2 hours at 35 ± 2 C. Twenty-five gram sample portions were inoculated, mixed thoroughly, and held in a CO 2 atmosphere to simulate packaging conditions for 48 ± 6 hours at 10 C. Following the equilibration step for all food matrices, 25 ± 1g test portions were diluted 1:10 with 0.1% peptone water. If necessary, subsequent dilutions were made in 90mL of 0.1% peptone water to achieve the target recovery levels on PLAB. Additionally, naturally contaminated brands of both RTE and raw meats were diluted in 0.1% peptone water until target recovery levels were achieved. Page 2

3 For the analysis of stainless steel environmental surfaces, sixteen 1" x 1" surface areas per organism were inoculated with a dilution of a L. fermentum or W. viridescens broth culture. The L. fermentum inoculum was prepared using Brain Heart Infusion Broth and incubated aerobically for 24 ± 2 hours at 35 ± 2 C. The W. viridescens inoculum was prepared in MRS and incubated microaerophilically for 24 ± 2 hours at 35 ± 2 C. The inoculated surfaces were left to dry at ambient temperature for 18 ± 2 hours prior to sampling. For each organism, eight surface areas were sampled using environmental swabs pre-moistened with Dey-Engley Neutralizing broth (D/E) and eight surface areas were sampled using environmental swabs pre-moistened with Letheen Broth (LB). The surface areas were sampled in both horizontal and vertical motions. After sampling, the D/E swabs were placed into a test tube containing 9mL of D/E and the LB swabs were placed into a test tube containing 9mL of LB. Subsequent dilutions of these samples were prepared in 9mL test tubes of either D/E or LB to achieve the target recovery levels on 3M Petrifilm Aerobic Count (AC) Plates. Aerobic Procedure for Lactic Acid Bacteria (PLAB) with 3M Petrifilm AC Plates Method For the PLAB procedure, a 2.0mL aliquot of sample was transferred to a test tube containing 2.0mL of Lactic Bacteria Broth. After vortexing the solution, a 1.0mL aliquot (0.5mL of sample and 0.5mL of Lactic Broth) was plated onto the 3M Petrifilm AC Plate. PLAB plates were assayed in quadruplicate and incubated aerobically at 35 ± 2 C for food samples and 25 ± 2 C for environmental samples for up to 72 ± 3 hours. Halos (acid zones) with and without associated colonies on PLAB plates were enumerated after 24 ± 2 hours, 48 ± 3 hours, and 72 ± 3 hours of incubation. The presence of colonies with associated gas bubbles were recorded after 72 ± 3 hours of incubation. For those RTE and raw meat samples that were naturally contaminated, isolated colonies recovered from the PLAB plates were identified by transferring to tryptic soy agar (TSA) and incubated for 24 ± 2 hours at 35 ± 2 C. Final organism identifications were achieved by the VITEK Anaerobe Identification (ANI) method. Compendial and ISO Reference Methods For the Compendial method procedure using All Purpose Tween agar plus 2% sucrose and bromocresol purple (APTS), 1.0mL sample aliquots were pour plated in duplicate and incubated aerobically at 25 ± 2 C for up to 72 ± 3 hours. In addition to APTS pour plates, salad dressing and stainless steel environmental samples were spread plated onto APTS using 0.25mL sample aliquots. For the ISO reference method procedure using MRS plus bromocresol purple, 1.0mL sample aliquots were pour plated in duplicate and incubated anaerobically at 35 ± 2 C. Two sets of MRS pour plates were prepared, one set incubated for 48 ± 3 hours and the other set incubated for 72 ± 3 hours. For each reference agar, colonies with and without halos were enumerated after 48 ± 3 hours and 72 ± 3 hours. After the 72 hour enumeration, a representative colony from each sample plate was transferred to MRS broth containing a Durham tube to detect gas production and was incubated at 35 ± 2 C for 5 to 7 days. Results and Discussion For the statistical analysis, a paired t-test for two sample means was calculated to analyze statistical differences between the PLAB counts and reference agar counts at varying time points. The PLAB and reference agar time points used in the statistical analyses are listed in Table B. The t-tests were calculated using counts across all brands and organisms for each inoculation level within the matrices. Two procedures were used to obtain the values used in the statistical comparison. For the first method (Method 1), one PLAB replicate from each brand and organism was compared to one reference agar replicate from each brand and organism. The PLAB counts were converted to a count per milliliter by multiplying the 0.5mL value by a factor of two. The count per milliliter values were converted to a Log 10 values and compared to single reference agar counts converted to Log 10 values in the t-test. For the second method (Method 2), all four PLAB replicates and both reference agar replicates were used in the t-test calculation. Two 3M Petrifilm AC Plate replicates were added together and the second two 3M Petrifilm AC Plate replicates were added together to produce two count per milliliter values. The two counts calculated values were converted to Log 10 values and averaged. The Page 3

4 reference agar counts were converted to Log 10 values and averaged. The averaged Log 10 values from the PLAB method were compared to averaged Log 10 values from each of the reference agars. Salad Dressings Table B. Method and Time Points Used in Statistical Analysis 48 Hour 3M Petrifilm AC Plate Counts vs. 48 Hour APTS Pour Plate Counts 48 Hour 3M Petrifilm AC Plate Counts vs. APTS Pour Plate Counts 48 Hour 3M Petrifilm AC Plate Counts vs. APTS Pour Plate Counts 3M Petrifilm AC Plate Counts vs. MRS Counts 3M Petrifilm AC Plate Counts vs. MRS Counts For the 24 hour time point, colonies with associated halos were observed on the low level W. viridescens PLAB plates and medium level W. viridescens PLAB plates had complete yellowing of the agar after 24 hours. The L. fermentum low and medium level PLAB plates had no colonies or acid zones at 24 hours. Statistically comparable log values were observed comparing 48 hour 3M Petrifilm Aerobic Count (AC) Plate counts vs. 48 hour APTS pour plate counts at both levels using both statistical procedures. A detailed list of t-test results for salad dressings is presented in Table 4 of Appendix 1. RTE Salads For the 24 hour time point, colonies with associated halos were observed on the low level W. viridescens PLAB plates. Medium level L. fermentum and W. viridescens PLAB plates were completely yellow after 48 hours. At the 48 and 72 hour time point, PLAB plates with colonies had associated halos and gas bubbles. Statistically comparable log values were observed comparing 72 hour 3M Petrifilm AC Plate counts vs. 72 hour MRS counts at the medium inoculation level using both statistical procedures. Statistically comparable log values were observed comparing 72 hour 3M Petrifilm AC Plate counts vs. 72 hour APTS pour plate counts at the medium inoculation level using t-test Method 2. A detailed list of t-test results for RTE salads is presented in Table 8 of Appendix 1. RTE Meats Colonies with associated halos were observed on PLAB plates from the naturally contaminated Private Selection Smokehouse Hickory Ham. All low level PLAB plates had colonies with associated halos and gas production at the 48 hour time point. All medium level PLAB plates were completely yellow at the 48 hour time point and had evidence of gas production. Statistically comparable log values were calculated for the medium level 48 hour 3M Petrifilm AC Plate counts vs. 48 hour APTS pour plate counts, 48 hour 3M Petrifilm AC Plate counts vs. 72 hour APTS pour plate counts, 72 hour 3M Petrifilm AC Plate counts vs. 72 hour APTS pour plate counts, and 72 hour 3M Petrifilm AC Plate counts vs. 72 hour MRS counts time points using both t-test methods. L. acidophilus at an acceptable confidence level, L. jensenii at a very good confidence level, and L. cellobiosus at an excellent confidence level were identified using the VITEK ANI method. A detailed list of t-test results for RTE meats is presented in Table 11 of Appendix 1. Raw Meats At the 24 hour time point, PLAB plates were completely yellow with no associated colonies. At the 48 hour time point, PLAB plates had colonies with associated gas production. APTS and MRS were overgrown with bacillus and an accurate enumeration of lactic organisms could not be completed. L. acidophilus, L. jensenii, and L. casei were identified using the VITEK ANI method at a low discrimination confidence level. A detailed list of PLAB counts for raw meats is presented in Table 12 of Appendix 1. Due to overgrowth of the competing bacillus, no statistical comparisons were conducted for the raw meats. Page 4

5 Stainless Steel Environmentals Colonies with associated acid zones and gas production were observed on LAB plates at the 48 hour time period and complete yellowing of the agars occurred at the 72 hour time point. Statistically comparable log values were calculated for the 48 hour 3M Petrifilm Aerobic Count (AC) Plate counts vs. 48 hour APTS pour plate counts comparison using both t-test methods. A detailed list of t-test results for stainless steel environmental surface swabs is presented in Table 17 of Appendix 1. Observations The method comparison results obtained in the evaluation demonstrate the reliability of the PLAB procedure as an easy-to-use method for the enumeration and detection of lactic acid organisms in a variety of foods and on stainless steel environmental surfaces. The method requires a simple plating procedure and eliminates the lengthy gas production confirmation procedure used in the reference methods. The PLAB method reduces the cost of sample preparation by eliminating the need for producing and sterilizing reference method agars and eliminates costly materials needed to create and maintain an anaerobic environment for MRS. The initial appearance of acid zones on PLAB is an accurate early indicator of the presence of lactic acid organisms as acid zones were always observed surrounding typical red colonies. Colony associated gas production was observed on each PLAB plate which is a distinct advantage over the reference procedures. The low inoculum level PLAB plates produced distinct zones and colonies sooner than the medium level PLAB plates. It was often observed that the medium level did not have distinct acid zones, instead the agar turned completely yellow and colonies were faint. When enumerating lactic organisms from matrices with a high volume of non-target bacteria, the PLAB plates were easier to accurately enumerate compared to APTS. APTS often had overgrowth of bacillus and other non- target bacteria, which made enumerating colonies with acid zones challenging. This gives PLAB an advantage as only true lactic acid producing organisms and no bacillus was observed on 3M Petrifilm AC Plates. Page 5 3M and Petrifilm are trademarks of 3M.

6 APPENDIX 1 Page 6

7 Table 1. Salad Dressing Results Lactobacillus fermentum Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (0.25mL) APTS (1.0mL) MRS (1.0mL) 24 Hour 48 Hour* * 48 Hour 48 Hour 48 Hour Replicate Dressing A B C D A B C D A B C D A B A B A B A B A B A B Caesar Caesar LoFat Caesar LoFat Caesar Ranch Ranch LoFat Ranch LoFat Ranch Catalina Catalina Blue Cheese Blue Cheese Blue Cheese Zesty Italian Zesty Italian Medium Level Low Level * Acid zones Caesar Caesar LoFat Caesar LoFat Caesar Ranch Ranch LoFat Ranch LoFat Ranch Catalina Catalina Blue Cheese Blue Cheese Blue Cheese Zesty Italian Zesty Italian Page 7

8 Table 2. Salad Dressing Results Weissella viridescens Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (0.25mL) APTS (1.0mL) MRS (1.0mL) 24 Hour 48 Hour* * 48 Hour 48 Hour 48 Hour Replicate Dressing A B C D A B C D A B C D A B A B A B A B A B A B Caesar Caesar LoFat Caesar LoFat Caesar Ranch Ranch LoFat Ranch LoFat Ranch Catalina Catalina Blue Cheese Blue Cheese Blue Cheese Zesty Italian Zesty Italian Medium Level Low Level * Acid zones Caesar Caesar LoFat Caesar LoFat Caesar Ranch Ranch LoFat Ranch LoFat Ranch Catalina Catalina Blue Cheese NA NA NA NA Blue Cheese Blue Cheese Zesty Italian Zesty Italian Page 8

9 Table 3. Salad Dressing Gas Production Results Lactobacillus fermentum Weissella viridescens Salad Dressing 3M Petrifilm Aerobic Count Plate APTS Spread Plate APTS Pour Plate MRS 3M Petrifilm Aerobic Count Plate APTS Spread Plate APTS Pour Plate MRS Caesar 1 Positive Positive Positive Positive Positive Positive Positive Positive Caesar 2 Positive Positive Positive Positive Positive Positive Positive Positive LoFat Caesar 1 Positive Positive Positive Positive Positive Positive Positive Positive LoFat Caesar 2 Positive Positive Positive Positive Positive Positive Positive Positive Ranch 1 Positive Positive Positive Positive Positive Positive Positive Positive Ranch 2 Positive Positive Positive Positive Positive Positive Positive Positive LoFat Ranch 1 Positive Positive Positive Positive Positive Positive Positive Positive LoFat Ranch 2 Positive Positive Positive Positive Positive Positive Positive Positive Catalina 1 Positive Positive Positive Positive Positive Positive Positive Positive Catalina 2 Positive Positive Positive Positive Positive Positive Positive Positive Blue Cheese 1 Positive Positive Positive Positive Positive Positive Positive Positive Blue Cheese 2 Positive Positive Positive Positive Positive Positive Positive Positive Blue Cheese 3 Positive Positive Positive Positive Positive Positive Positive Positive Zesty Italian 1 Positive Positive Positive Positive Positive Positive Positive Positive Zesty Italian 2 Positive Positive Positive Positive Positive Positive Positive Positive Table 4. Salad Dressing T-Test Results Comparison Method x 2 Replicates Method 2 Analysis of 4 Replicates 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 48 hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS Low Inoculum 72 hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS Medium Inoculum Page 9

10 Table 5. RTE Salad Results Lactobacillus fermentum Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (1.0mL) MRS (1.0mL) 24 Hour* 48 Hour* * 48 Hour 48 Hour Replicate Dressing A B C D A B C D A B C D A B A B A B A B Mustard Potato Mustard Potato Mustard Potato American Potato American Potato American Potato Macaroni Macaroni Macaroni Bean Bean Bean Coleslaw Coleslaw Coleslaw Medium Level Low Level * Acid zones Mustard Potato Mustard Potato Mustard Potato American Potato American Potato American Potato Macaroni Macaroni Macaroni Bean Bean Bean Coleslaw Coleslaw Coleslaw Page 10

11 Table 6. RTE Salad Results Weissella viridescens Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (1.0mL) MRS (1.0mL) 24 Hour* 48 Hour* * 48 Hour 48 Hour Replicate Dressing A B C D A B C D A B C D A B A B A B A B Mustard Potato Mustard Potato Mustard Potato American Potato American Potato American Potato Macaroni Macaroni Macaroni Bean Bean Bean Coleslaw Coleslaw Coleslaw Medium Level Low Level * Acid zones Mustard Potato Mustard Potato Mustard Potato American Potato American Potato American Potato Macaroni Macaroni Macaroni Bean Bean Bean Coleslaw Coleslaw Coleslaw Page 11

12 Table 7. RTE Salad Gas Production Results Lactobacillus fermentum Weissella viridescens RTE Salad 3M Petrifilm Aerobic Count Plate APTS Pour Plate MRS 3M Petrifilm Aerobic Count Plate APTS Pour Plate MRS Mustard Potato 1 Positive Positive Positive Positive Positive Positive Mustard Potato 2 Positive Positive Positive Positive Positive Positive Mustard Potato 3 Positive Positive Positive Positive Positive Positive American Potato 1 Positive Positive Positive Positive Positive Positive American Potato 2 Positive Positive Positive Positive Positive Positive American Potato 3 Positive Positive Positive Positive Positive Positive Macaroni 1 Positive Positive Positive Positive Positive Positive Macaroni 2 Positive Positive Positive Positive Positive Positive Macaroni 3 Positive Positive Positive Positive Positive Positive Bean 1 Positive Positive Positive Positive Positive Positive Bean 2 Positive Positive Positive Positive Positive Positive Bean 3 Positive Positive Positive Positive Positive Positive Coleslaw 1 Positive Positive Positive Positive Positive Positive Coleslaw 2 Positive Positive Positive Positive Positive Positive Coleslaw 3 Positive Positive Positive Positive Positive Positive Table 8. RTE Salad T-Test Results Comparison Method x 2 Replicates Method 2 Analysis of 4 Replicates 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 48 hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS Low Inoculum 72 hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS Medium Inoculum Page 12

13 Table 9. RTE Meat Results Meat Organism Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (1.0mL) MRS (1.0mL) 24 Hour* 48 Hour* * 48 Hour Replicate A B C D A B C D A B C D A B A B A B O/R Turkey 1 Lactobacillus O/R Turkey 2 Lactobacillus O/R Chicken 2 Lactobacillus O/R Turkey 1 Weissella O/R Turkey 2 Weissella O/R Chicken 2 Weissella Smoked Ham 1 Natural Contamination Smoked Ham 2 Natural Contamination Roast Beef 1 Natural Contamination Roast Beef 2 Natural Contamination O/R Chicken 1 Natural Contamination Medium Level Low Level * Acid zones O/R Turkey 1 Lactobacillus O/R Turkey 2 Lactobacillus O/R Chicken 2 Lactobacillus O/R Turkey 1 Weissella O/R Turkey 2 Weissella O/R Chicken 2 Weissella Smoked Ham 1 Natural Contamination Smoked Ham 2 Natural Contamination Roast Beef 1 Natural Contamination Roast Beef 2 Natural Contamination O/R Chicken 1 Natural Contamination Page 13

14 Table 10. RTE Meat Gas Production Results Lactobacillus fermentum Weissella viridescens RTE Meat 3M Petrifilm Aerobic Count Plate APTS Pour Plate MRS 3M Petrifilm Aerobic Count Plate APTS Pour Plate MRS O/R Turkey 1 Positive Positive Positive Positive Positive Positive O/R Turkey 2 Positive Positive Positive Positive Positive Positive O/R Chicken 2 Positive Positive Positive Positive Positive Positive RTE Meat Natural Contamination Smoked Ham 1 Positive Positive Positive Smoked Ham 2 Positive Positive Positive Roast Beef 1 Positive Positive Positive Roast Beef 2 Positive Positive Positive O/R Chicken 1 Positive Positive Positive Table 11. RTE Meat T-Test Results Comparison Method x 2 Replicates Method 2 Analysis of 4 Replicates 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 48 hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS Low Inoculum 72 hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS Medium Inoculum Page 14

15 Table 12. Raw Meat Results Meat Organism Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (1.0mL) MRS (1.0mL) 24 Hour* 48 Hour* * 48 Hour 48 Hour Replicate A B C D A B C D A B C D A B A B A B A B Ground Turkey 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Ground Turkey 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Chicken 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Chicken 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Ground Beef 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Ground Beef 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Steak 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Steak 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Medium Level Low Level Ground Turkey 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Ground Turkey 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Chicken 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Chicken 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Ground Beef 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Ground Beef 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Steak 1 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC Steak 2 NC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC * Acid zones NC= Natural Contamination TNTC= To Numerous to Count Table 13. Raw Meat Gas Production Results Natural Contamination Raw Meat 3M Petrifilm Aerobic Count Plate APTS Pour Plate MRS Ground Turkey 1 Positive Positive Positive Ground Turkey 2 Positive Positive Positive Chicken 1 Positive Positive Positive Chicken 2 Positive Positive Positive Ground Beef 1 Positive Positive Positive Ground Beef 2 Positive Positive Positive Steak 1 Positive Positive Positive Steak 2 Positive Positive Positive Page 15

16 Table 14. Stainless Steel Environmental Results Lactobacillus fermentum Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (0.25mL) APTS (1.0mL) MRS (1.0mL) 24 Hour 48 Hour* * 48 Hour 48 Hour 48 Hour Replicate Sample Diluent A B C D A B C D A B C D A B A B A B A B A B A B Swab 1 N Buffer Swab 2 N Buffer Swab 3 N Buffer Swab 4 N Buffer Swab 5 N Buffer Swab 6 N Buffer Swab 7 N Buffer Swab 8 N Buffer Medium Level Low Level Swab 1 N Buffer Swab 2 N Buffer Swab 3 N Buffer Swab 4 N Buffer Swab 5 N Buffer Swab 6 N Buffer Swab 7 N Buffer Swab 8 N Buffer Swab 9 Letheen Swab 10 Letheen Swab 11 Letheen Swab 12 Letheen Swab 13 Letheen Swab 14 Letheen Swab 15 Letheen Swab 16 Letheen Medium Level Low Level * Acid zones Swab 9 Letheen Swab 10 Letheen Swab 11 Letheen Swab 12 Letheen Swab 13 Letheen Swab 14 Letheen Swab 15 Letheen Swab 16 Letheen Page 16

17 Table 15. Stainless Steel Environmental Results Weissella viridescens Aerobic Procedure for Lactic Acid Bacteria with 3M Petrifilm Aerobic Count Plates (0.5mL) APTS (0.25mL) APTS (1.0mL) MRS (1.0mL) 24 Hour 48 Hour* * 48 Hour 48 Hour 48 Hour Replicate Sample Diluent A B C D A B C D A B C D A B A B A B A B A B A B Swab 1 N Buffer Swab 2 N Buffer Swab 3 N Buffer Swab 4 N Buffer Swab 5 N Buffer Swab 6 N Buffer Swab 7 N Buffer Swab 8 N Buffer Medium Level Low Level Swab 1 N Buffer Swab 2 N Buffer Swab 3 N Buffer Swab 4 N Buffer Swab 5 N Buffer Swab 6 N Buffer Swab 7 N Buffer Swab 8 N Buffer Swab 9 Letheen Swab 10 Letheen Swab 11 Letheen Swab 12 Letheen Swab 13 Letheen Swab 14 Letheen Swab 15 Letheen Swab 16 Letheen Medium Level Low Level * Acid zones Swab 9 Letheen Swab 10 Letheen Swab 11 Letheen Swab 12 Letheen Swab 13 Letheen Swab 14 Letheen Swab 15 Letheen Swab 16 Letheen Page 17

18 Table 16. Stainless Steel Environmental Gas Production Results Lactobacillus fermentum Weissella viridescens Environmentals 3M Petrifilm Aerobic Count Plate APTS Spread Plate APTS Pour Plate MRS 3M Petrifilm Aerobic Count Plate APTS Spread Plate APTS Pour Plate MRS 1 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 2 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 3 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 4 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 5 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 6 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 7 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 8 D/E Neutralizing Broth Positive Positive Positive Positive Positive Positive Positive Positive 1 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive 2 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive 3 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive 4 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive 5 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive 6 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive 7 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive 8 Letheen Broth Positive Positive Positive Positive Positive Positive Positive Positive Table 17. Stainless Steel Environmental T-Test Results Comparison Method x 2 Replicates Method 2 Analysis of 4 Replicates 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 3M Petrifilm Aerobic Count Plate Mean Ref Mean P-Value 48 hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS Low Inoculum 72 hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 48 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour APTS hour 3M Petrifilm Aerobic Count Plate vs. 72 hour MRS Medium Inoculum Page 18

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