SOLABIA SAS. 29 rue Delizy PANTIN. NF VALIDATION Validation of alternative analytical methods Application to food microbiology.

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1 ACCREDITATION No SCOPE AVAILABLE FROM SOLABIA SAS 29 rue Delizy PANTIN NF VALIDATION Validation of alternative analytical methods Application to food microbiology Summary report ISO validation of the method for enumeration of coagulase-positive Staphylococci in 24 h ±2 h Quantitative method This report consists of 128 pages including 12 appendices. This report may only be reproduced in whole. COFRAC accreditation certifies the Laboratory's expertise for those tests covered by the accreditation identified by the symbol. Version 0 10 February 2016 ADRIA DEVELOPPEMENT Creac h Gwen - F QUIMPER Cedex - Tél. (33) Fax (33) adria.developpement@adria.tm.fr - Website: NON-PROFIT ASSOCIATION - SIRET No EXISTENCE No VAT No. FR

2 1 INTRODUCTION 5 2 PRESENTATION OF THE METHODS Reference method Alternative method 5 3 METHOD COMPARATIVE STUDY Accuracy profile study Matrices used Raw results Statistical interpretation Conclusion Accuracy study Number and type of samples Artificial contaminants Raw results Statistical interpretation Specificity and selectivity study Protocols Results Conclusion Practicability 50 4 INTER-LABORATORY STUDY Study organisation Experimental parameter control Strain stability during transport Sample temperature upon receipt Sample temperature during transport Inoculum homogeneity Calculations, summary and interpretation of data Expert laboratory results Partner laboratory results 54 5 CONCLUSION 60 ADRIA Développement 2/ February 2016

3 Appendix 1 - ISO reference method: Horizontal method for the enumeration of coagulasepositive staphylococci (Staphylococcus aureus and other species) - Part 2: Technique using rabbit plasma fibrinogen agar medium 61 Appendix 2 - alternative method protocol 62 Appendix 3 - Accuracy profile study: raw results 63 Appendix 4 - Accuracy profile study: Statistical calculations 73 Appendix 5 - Accuracy study: artificial contamination of samples 79 Appendix 6 - Accuracy study: raw results 82 Appendix 7 - Accuracy study: summary 109 Appendix 8 - Accuracy study: statistical calculations - Spread method 112 Appendix 9 - Accuracy study: statistical calculations - Pour plate method 116 Appendix 10 - Accuracy study: statistical calculations - Spiral method 120 Appendix 11 - Specificity/Selectivity: raw results 124 Appendix 12 - Inoculum homogeneity 127 ADRIA Développement 3/ February 2016

4 Foreword All information required to validate the analysis guarantee is made available to SOLABIA. The results are summarised in tables and interpreted as per the ISO/FDIS (2015) standard. Manufacturer: Biokar Diagnostics Rue des Quarante Mines - BP BEAUVAIS Cedex Expert laboratory: ADRIA Développement ZA Creac h Gwen QUIMPER Cedex Method to validate: method for enumeration of coagulase-positive Staphylococci in 24 h ±2 h Validation standard: ISO/FDIS (2015): Microbiology of the food chain - Method validation Part 1: Vocabulary Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method Reference method : ISO Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 2: Technique using rabbit plasma fibrinogen agar medium Validation scope All human food products Validation body: AFNOR Certification Test conducted under accreditation ADRIA Développement 4/ February 2016

5 1 INTRODUCTION The method for the enumeration of coagulase-positive Staphylococci in 24 ±2 h was validated on 03 December 2015 for the following categories: meat and poultry products, dairy and seafood products (certificate no. BKR 23/10-12/15). In January 2016, an extension study was conducted for the Egg products and "Composite foods" categories, thus extending the scope of the alternative method to the analysis of all human food products. 2 PRESENTATION OF THE METHODS 2.1 Reference method The reference method use is the ISO standard - Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) - Part 2: Technique using rabbit plasma fibrinogen agar medium. The corresponding protocol is given in Appendix Alternative method The method provides enumeration in 24 ±2 h at 37 C, with no confirmation step. The incubation range can be extended to 48 h for spread inoculation and to 72 h for pour plate inoculation and when using the Spiral plater. These incubation times (minimum and maximum) were tested during initial validation. Various inoculation protocols (see Appendix 2) are available: - surface inoculation via manual spreading (pre-poured plates), - surface inoculation using the spiral plater (pre-poured plates), by depositing 100 µl of a log phase culture, validation being required for 50 and 100 µl, - pour plate inoculation (base kit + freeze-dried supplement). ADRIA Développement 5/ February 2016

6 3 METHOD COMPARATIVE STUDY 3.1 Accuracy profile study The accuracy profile study is a comparative study of the results obtained using the reference method and those obtained with the alternative method. This study is conducted using artificially contaminated samples. Only one type (of food) per category is tested Matrices used For each food category, one type was selected with a representative matrix. Two batches from this matrix were inoculated with a single strain at various loads. Two different batches of the same matrix per category were tested. The categories, types and matrices are described in table 1. Table 1 Initial validation Extension study Category Type Matrix Strain Meat products Dairy products Seafood Egg products Composite food Cooked delicatessens and ready-made meals Raw milk and raw milk-based dairy products Ready-made meals, terrines Raw dough, liquid egg portions Pizzas, quiche Cooked ham Raw milk Salmon terrine Pasteurised liquid egg portion Vegetable quiche Staphylococcus aureus Ad 161 Staphylococcus aureus Ad 905 Inoculation rate (log CFU/g) Spreading - Spiral Pour plate Staphylococcus aureus Ad 899 Staphylococcus aureus Ad Staphylococcus aureus Ad Raw results All of the results obtained per category are presented in Appendix Statistical interpretation Statistical calculations are given in Appendix 4. The following graphs are presented: - Figure 1: spread method, 22 h incubation, - Figure 2: pour plate method, 22 h incubation, - Figure 3: Spiral method, 22 h incubation. ADRIA Développement 6/ February 2016

7 - Figure 1 - Spread method, 22 h incubation, 0,600 (Food) Category (Food) Type Produits Meat products carnés Cooked delicatessens and ready meals Charcuterie cuite et plats cuisinés Cooked Charcuterie delicatessens cuite et plats and cuisinés ready meals 0,400 Bias 0,200 0,000 1,40 1,90 2,40 2,90 3,40 3,90 4,40 4,90 5,40-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Number Reference central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,95 0,090-0,183 0,363 YES YES ,11-0,110-0,383 0,163 YES YES ,99 0,010-0,263 0,283 YES YES ,96 0,030-0,243 0,303 YES YES ,99 0,010-0,263 0,283 YES YES ,96 0,030-0,243 0,303 YES YES ,04 0,040-0,233 0,313 YES YES ,18 0,000-0,273 0,273 YES YES ,04 0,220-0,053 0,493 YES YES ,09 0,038-0,170 0,246 YES YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,112 0,147 NO +/-0,500 (Food) Category (Food) Type Produits Dairy products laitiers Raw milk and dairy products made with raw milk Laits crus et produits laitiers au lait cru Bias Raw Laits milk crus and et dairy produits products laitiers made au with lait raw crumilk 0,600 0,500 0,400 0,300 0,200 0,100 0,000-0,1001,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50-0,200-0,300-0,400-0,500-0,600 Reference Median Bias β-eti AL = +/- 0.5 Sample Number Reference Central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,08 0,240 0,027 0,453 YES YES ,08 0,200-0,013 0,413 YES YES ,96 0,270 0,057 0,483 YES YES ,94 0,260 0,047 0,473 YES YES ,00 0,040-0,173 0,253 YES YES ,15 0,030-0,183 0,243 YES YES ,98 0,130-0,083 0,343 YES YES ,18-0,030-0,243 0,183 YES YES ,00 0,110-0,103 0,323 YES YES ,04 0,140-0,073 0,353 YES YES SD Repeatability Reference method Alternative method SD repeatability of reference method > Final AL 0,091 0,116 NO +/-0,500 ADRIA Développement 7/ February 2016

8 - Figure 1 - Spread method, 22 h incubation, (Food) Category (Food) Type 0,600 Produits de la mer Seafood Plats Ready cuisinés, meals, terrines Ready Plats meals, cuisinés, terrines terrines 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Number Reference Median Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,36 0,000-0,146 0,146 YES YES ,36 0,000-0,146 0,146 YES YES ,40 0,010-0,136 0,156 YES YES ,40 0,030-0,116 0,176 YES YES ,28 0,120-0,026 0,266 YES YES ,34 0,020-0,126 0,166 YES YES ,32 0,000-0,146 0,146 YES YES ,40-0,080-0,226 0,066 YES YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,115 0,079 NO +/-0,500 ADRIA Développement 8/ February 2016

9 Extension study (2015) - Figure 1 - Spread method, 22 h incubation, (Food) Category (Food) Type 0,600 Egg products Ovoproduits Raw pasta, liquid egg portions Pâtes crues, coule d'œuf Raw pasta, Pâtes liquid crues, egg coule portions d'œuf 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Name Reference central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,20 0,060-0,136 0,256 YES YES ,26-0,030-0,226 0,166 YES YES ,60 0,230 0,034 0,426 YES YES ,71 0,240 0,044 0,436 YES YES ,15 0,080-0,116 0,276 YES YES ,26 0,170-0,026 0,366 YES YES Reference Alternative SD repeatability of reference Final AL method method method > SD Repeatability 0,117 0,136 NO +/-0,500 (Food) Category (Food) Type Composite Aliments foods composites Pizzas, Pizzas, quiches quiches 0,600 Pizzas, quiches 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Name Reference Central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,40 0,000-0,176 0,176 YES YES ,53-0,020-0,196 0,156 YES YES ,82 0,060-0,116 0,236 YES YES ,90 0,280 0,104 0,456 YES YES ,20 0,290 0,114 0,466 YES YES ,32 0,260 0,084 0,436 YES YES Reference Alternative SD repeatability of reference Final AL method method method > SD Repeatability 0,045 0,122 NO +/-0,500 ADRIA Développement 9/ February 2016

10 - Figure 2 - Pour plate method, 22 h incubation, 0,600 (Food) Category (Food) Type Meat products Produits carnés Cooked delicatessens Charcuterie and ready cuite meals et plats cuisinés Cooked Charcuterie delicatessens cuite et plats and ready cuisinés meals 0,400 Bias 0,200 0,000 1,40 1,90 2,40 2,90 3,40 3,90 4,40 4,90 5,40-0,200 Série1 Série4 AL = +/ ,400-0,600 Reference Median Sample Number Reference central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,95 0,000-0,166 0,166 YES YES ,11 0,000-0,166 0,166 YES YES ,99-0,100-0,266 0,066 YES YES ,96-0,010-0,176 0,156 YES YES ,04 0,040-0,126 0,206 YES YES ,18-0,030-0,196 0,136 YES YES ,04-0,040-0,206 0,126 YES YES ,09-0,024-0,233 0,185 YES YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,111 0,088 NO +/-0,500 (Food) Category (Food) Type Dairy products Produits laitiers Raw milk and Laits dairy crus products et produits made with laitiers raw milk au lait cru Bias Raw Laits milk crus and et dairy produits products laitiers made with au raw lait milk cru 0,600 0,500 0,400 0,300 0,200 0,100 0,000-0,1001,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50-0,200-0,300-0,400-0,500-0,600 Reference Median Bias β-eti AL = +/- 0.5 Sample Number Reference Central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,08-0,040-0,225 0,145 YES YES ,08 0,100-0,085 0,285 YES YES ,96 0,010-0,175 0,195 YES YES ,94 0,140-0,045 0,325 YES YES ,00-0,020-0,205 0,165 YES YES ,15-0,110-0,295 0,075 YES YES ,98 0,130-0,055 0,315 YES YES ,18-0,030-0,215 0,155 YES YES SD Repeatability Reference method Alternative method SD repeatability of reference method > Final AL 0,091 0,100 NO +/-0,500 ADRIA Développement 10/ February 2016

11 - Figure 2 - Pour plate method, 22 h incubation, (Food) Category (Food) Type 0,600 Seafood Produits de la mer Ready meals, terrines Plats cuisinés, terrines Ready Plats meals, cuisinés, terrines terrines 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Number Reference Median Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,36 0,020-0,181 0,221 YES YES ,36 0,000-0,201 0,201 YES YES ,08 0,070-0,131 0,271 YES YES ,00 0,150-0,051 0,351 YES YES ,40 0,000-0,201 0,201 YES YES ,40-0,040-0,241 0,161 YES YES ,28 0,120-0,081 0,321 YES YES ,40-0,040-0,241 0,161 YES YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,115 0,109 NO +/-0,500 ADRIA Développement 11/ February 2016

12 Extension study (2015) - Figure 2 - Pour plate method, 22 h incubation, (Food) Category (Food) Type 0,600 Egg products Ovoproduits Raw pasta, liquid egg portions Pâtes crues, coule d'œuf Raw Pâtes pasta, crues, liquid coule egg portions d'œuf 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Name Reference central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,20 0,060-0,136 0,256 YES YES ,26-0,030-0,226 0,166 YES YES ,60 0,230 0,034 0,426 YES YES ,71 0,240 0,044 0,436 YES YES ,15 0,080-0,116 0,276 YES YES ,26 0,170-0,026 0,366 YES YES Reference Alternative SD repeatability of reference Final AL method method method > SD Repeatability 0,117 0,136 NO +/-0,500 (Food) Category (Food) Type Composite Aliments foods composites Pizzas, quiches Pizzas, quiches 0,600 Pizzas, quiches 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Name Reference Central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,40 0,000-0,176 0,176 YES YES ,53-0,020-0,196 0,156 YES YES ,82 0,060-0,116 0,236 YES YES ,90 0,280 0,104 0,456 YES YES ,20 0,290 0,114 0,466 YES YES ,32 0,260 0,084 0,436 YES YES Reference Alternative SD repeatability of reference Final AL method method method > SD Repeatability 0,045 0,122 NO +/-0,500 ADRIA Développement 12/ February 2016

13 - Figure 3 - Spiral method, 22 h incubation, 0,600 (Food) Category (Food) Type Meat products Produits carnés Cooked delicatessens and ready meals Charcuterie cuite et plats cuisinés Cooked Charcuterie delicatessens cuite et plats and cuisinés ready meals 0,400 Bias 0,200 0,000 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Number Reference central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,99-0,140-0,340 0,060 YES YES ,96 0,220 0,020 0,420 YES YES ,04 0,140-0,060 0,340 YES YES ,18-0,030-0,230 0,170 YES YES ,04 0,140-0,060 0,340 YES YES ,18-0,030-0,230 0,170 YES YES ,04 0,110-0,090 0,310 YES YES ,09 0,048-0,161 0,257 YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,111 0,107 NO +/-0,500 (Food) Category (Food) Type Dairy products Produits laitiers Raw Laits milk and crus dairy et products produits made laitiers with raw au milk lait cru Bias Laits Raw crus milk and et produits dairy products laitiers made au with lait raw crumilk 0,600 0,500 0,400 0,300 0,200 0,100 0,000-0,1001,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50-0,200-0,300-0,400-0,500-0,600 Reference Median Bias β-eti AL = +/- 0.5 Sample Number Reference Central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,96-0,010-0,177 0,157 YES YES ,94 0,100-0,067 0,267 YES YES ,00 0,080-0,087 0,247 YES YES ,15 0,000-0,167 0,167 YES YES ,98 0,130-0,037 0,297 YES YES ,18-0,100-0,267 0,067 YES YES ,00 0,110-0,057 0,277 YES YES ,04 0,000-0,167 0,167 YES YES SD Repeatability Reference method Alternative method SD repeatability of reference method > Final AL 0,085 0,090 NO +/-0,500 ADRIA Développement 13/ February 2016

14 - Figure 3 - Spiral method, 22 h incubation, (Food) Category (Food) Type Seafood Produits de la mer Ready meals, terrines Plats cuisinés, terrines 0,600 Plats Ready cuisinés, meals, terrines terrines 0,400 Bias 0,200 0,000 2,90 3,40 3,90 4,40 4,90 5,40 5,90-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Number Reference Median Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,00 0,230-0,012 0,472 YES YES ,08 0,150-0,092 0,392 YES YES ,40 0,080-0,162 0,322 YES YES ,40 0,110-0,132 0,352 YES YES ,28 0,080-0,162 0,322 YES YES ,34 0,000-0,242 0,242 YES YES ,32 0,080-0,162 0,322 YES YES ,40 0,000-0,242 0,242 YES YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,109 0,130 NO +/-0,500 ADRIA Développement 14/ February 2016

15 Extension study (2015) - Figure 3 - Spiral method, 22 h incubation, (Food) Category (Food) Type 0,600 Egg products Ovoproduits Pâtes Raw pasta, crues, liquid coule egg portions d'œuf Raw Pâtes pasta, crues, liquid coule egg portions d'œuf 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Name Reference central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,76 0,140 0,007 0,273 YES YES ,89 0,110-0,023 0,243 YES YES ,60-0,020-0,153 0,113 YES YES ,71 0,050-0,083 0,183 YES YES ,15-0,040-0,173 0,093 YES YES ,26-0,150-0,283-0,017 YES YES Reference Alternative SD repeatability of reference Final AL method method method > SD Repeatability 0,113 0,092 NO +/-0,500 (Food) Category (Food) Type Composite Aliments foods composites Pizzas, quiches Pizzas, quiches 0,600 Pizzas, quiches 0,400 Bias 0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200 Bias β-eti AL = +/ ,400-0,600 Reference Median Sample Name Reference Central value Bias Lower β-eti Upper β-eti β-eti compared to AL=±0.5 Acceptable β-eti compared to final AL Acceptable ,92 0,230 0,047 0,413 YES YES ,04 0,110-0,073 0,293 YES YES ,82-0,060-0,243 0,123 YES YES ,90-0,020-0,203 0,163 YES YES ,20 0,060-0,123 0,243 YES YES ,32 0,080-0,103 0,263 YES YES Reference Alternative SD repeatability of reference Final AL method method method > SD Repeatability 0,044 0,127 NO +/-0, Conclusion In all cases, the alternative method is considered to be equivalent to the reference method, the bias always falling within the acceptability limits set at ±0.5 log CFU/g. ADRIA Développement 15/ February 2016

16 3.2 Accuracy study The accuracy study is a comparative study of the result obtained using the reference method and those obtained with the alternative method. This study is conducted using naturally and/or artificially contaminated samples. Various food categories, types and products will be tested for this purpose Number and type of samples The number of samples analysed and processed per category and analytical protocol is given in table 2. ADRIA Développement 16/ February 2016

17 Initial validation Extension study Categories 1 Meat products poultry products 2 Dairy products 3 Seafood 4 Egg products 5 Composite foods a Type Table 2 - Number and type of samples Raw meats (minced steak, carpaccio, seasoned meats) Number of samples analysed Spreading Number of results processed 22 h 48h 72 h Pour plate Spiral Spreadin g Inclusion b Raw delicatessens (smoked duck filet, ham, poultry sausages, dried sausages) c Cooked delicatessens and ready-made meals Total a Raw milk and raw milk-based dairy products b Pasteurised milk and pasteurised milk-based products c Dairy desserts Total a Raw peeled shrimps, cocktail b Smoked and marinated fish c Ready-made meals, terrines Total a Raw dough, liquid egg portions b Desserts, pastries c Fresh pasta Total a Salads, ready meals, sandwiches b Pizza, quiches, pastry shells c Ready-made meals Total Spiral TOTAL ADRIA Développement 17/ February 2016

18 Samples not used for statistical processing are presented in tables 3 to 5 for the three enumeration methods. Table 3 - Samples not used for statistical processing - Spreading method Initial validation Spl. no. Product Reference method ISO Alternative method Spreading method 22h 48h Category 3817 Chicken thigh < * 1.00* 1 a 3878 Turkey paupiette 1.30* < * 1 a 4222 Beef carpaccio with pistou sauce * 1.48* 1 a 4223 Beef carpaccio with basil * < a 4303 Blanquette 1.00* 1.00* 1.00* 1 a 4304 Red meat a 4338 Neck skin a 4339 Neck skin * 1.48* 1 a 3816 Raw sausage <1.00 <1.00 < b 3880 Sausages seasoned with herbs <1.00 <1.00 < b 4772 Perche sausage unreadable unreadable unreadable 1 b 4773 Rosette < b 3777 Wings BBQ 1.00* 1.30* 1.30* 1 c 3779 Wings BBQ 1.00* 1.30* 1.30* 1 c 3780 Wings BBQ 1.30* c 3781 Wings BBQ 1.30* 1.48* 1.00* 1 c 3782 Wings BBQ * 1.30* 1 c 4768 Chicken curry < c 3773 Reblochon made with raw milk <3.00 <3.00 < a 3775 Saint Nectaire made with raw milk <1.00 <1.00 < a 3815 Reblochon made with raw milk 1.48* a 4040 Raw milk 1.00* 1.30* 1.00* 2 a 4336 Goat's cheese 1.30* a 3873 Pasteurised milk 2.48* b 3872 Black forest 1.30* 1.00* 1.00* 2 c 4228 Rice pudding * 3.30* 2 c 4652 Swordfish steak <1.00 <1.00 < a 4302 Spring roll * 1.00* 3 c Type Test conducted under accreditation ADRIA Développement 18/ February 2016

19 Extension study Spl. no. Product Reference method ISO Alternative method Spreading method 22h 48h Category 7447 Puff pastry with butter <1.00 <1.00 < a 7448 Puff pastry with butter <1.00 <1.00 < a 7442 Flan <1.00 <1.00 < b 7443 Apple tart <1.00 <1.00 < b 7444 Apricot tart <1.00 <1.00 < b 7681 Mixed vegetables <1.00 <1.00 < a 7682 Grated carrots <1.00 <1.00 < a 7684 Ham and cheese croissants <1.00 <1.00 < b 7683 Couscous <1.00 <1.00 < c 7685 Hacao with Xiu Mai chicken <1.00 <1.00 < c 8088 Couscous >5.00 >5.00 > c Type *: < 4 colonies per plate Unreadable: plates unreadable due to the presence of significant secondary flora Test conducted under accreditation ADRIA Développement 19/ February 2016

20 Table 4 - Samples not used for statistical processing - Pour plate method Initial validation Extension study Spl. no. Product Reference method ISO Alternative method Pour plate method 22h 72h Category 3817 Chicken thigh < a 3878 Turkey paupiette 1.30* 1.00* 1.00* 1 a 4223 Beef carpaccio with basil * 3.48* 1 a 4303 Blanquette 1.00* 1.00* 1.00* 1 a 3816 Raw sausage <1.00 <1.00 < b 3880 Sausages seasoned with herbs <1.00 <1.00 < b 4772 Perche sausage unreadable unreadable unreadable 1 b 3777 Wings BBQ 1.00* 1.00* 1.00* 1 c 3779 Wings BBQ 1.00* c 3780 Wings BBQ 1.30* 1.48* c 3781 Wings BBQ 1.30* 1.30* 1.30* 1 c 3782 Wings BBQ 1.60 < * 1 c 3773 Reblochon made with raw milk <3.00 <3.00 < a 3775 Saint Nectaire made with raw milk <1.00 <1.00 < a 3815 Reblochon made with raw milk 1.48* a 3876 Saint Nectaire made with raw milk * 2 a 4040 Raw milk 1.00* <1.00 < a 4336 Goat's cheese 1.30* 1.48* a 3873 Pasteurised milk 2.48* <2.00 < b 3872 Black forest 1.30* 1.00* 1.30* 2 c 4228 Rice pudding * 3.30* 2 c 4652 Swordfish steak <1.00 <1.00 < a 3868 Scallop terrine * c 7447 Puff pastry with butter <1.00 <1.00 < a 7448 Puff pastry with butter <1.00 <1.00 < a 3778 Custard cream 1.78 <1.00 < b 7442 Flan <1.00 <1.00 < b 7443 Apple tart <1.00 <1.00 < b 7444 Apricot tart <1.00 <1.00 < b 7681 Mixed vegetables <1.00 <1.00 < a 7682 Grated carrots <1.00 <1.00 < a 7684 Ham and cheese croissants <1.00 <1.00 < b 7683 Couscous <1.00 <1.00 < c 7685 Hacao with Xiu Mai chicken <1.00 <1.00 < c 8088 Couscous >5.00 >5.00 > c *: < 4 colonies per plate Unreadable: plates unreadable due to the presence of significant secondary flora Type Test conducted under accreditation ADRIA Développement 20/ February 2016

21 Table 5 - Samples not used for statistical processing - Spiral method Initial validation No. Spl. Product Alternative method ISO Alternative method Spiral method 22h 72h Category 3774 Duck manchons / 1 a 3817 Chicken thigh <1.00 <2.00 < a 3877 Turkey paupiette 1.90 <2.00 < a 3878 Turkey paupiette 1.30* <2.00 < a 3879 Turkey paupiette * 2.00* 1 a 4222 Beef carpaccio with pistou sauce 2.00 <2.00 < a 4303 Blanquette 1.00* <2.00 < a 4304 Red meat * 2.00* 1 a 4337 Neck skin * 2.30* 1 a 4338 Neck skin * 2.00* 1 a 4339 Neck skin 1.95 <2.00 < a 3816 Raw sausage <1.00 <2.00 < b 3880 Sausages seasoned with herbs <1.00 <2.00 < b 4651 Raw ham * 2.48* 1 b 4772 Perche sausage unreadable unreadable unreadable 1 b 4773 Rosette * < b 3777 Wings BBQ 1.00* <2.00 < c 3779 Wings BBQ 1.00* 2.30* 2.30* 1 c 3780 Wings BBQ 1.30* <2.00 < c 3781 Wings BBQ 1.30* 2.30* 2.00* 1 c 3782 Wings BBQ 1.60 <2.00 < c 4768 Chicken curry < c 4771 Cooked ham < c 3771 Unripened cheese made with raw cow's milk 2.54 <2.00 < a 3773 Reblochon made with raw milk <3.00 <2.00 < a 3775 Saint Nectaire made with raw milk <1.00 <2.00 < a 3815 Reblochon made with raw milk 1.48* 2.30* < a 4040 Raw milk 1.00* 2.00* 2.00* 2 a 4042 Tomme made with raw milk * 2.48* 2 a 4336 Goat's cheese 1.30* <2.00 < a 3873 Pasteurised milk 2.48* 2.00* b 4541 Buttermilk * 2.00* 2 b 4543 Pasteurised milk * 2.30* 2 b 3872 Black forest 1.30* < * 2 c 4226 Tiramisu * 2.30* 2 c 4041 Raw salmon fillet * 2.00* 3 a 4652 Swordfish steak <1.00 <2.00 < a Type Test conducted under accreditation ADRIA Développement 21/ February 2016

22 Extension study No. Spl. Product Alternative method ISO Alternative method Spiral method 22h 72h Category 3869 Salmon duo salad 1.90 <2.00 < c 3871 Crayfish salad * 2.48* 3 c 4302 Spring roll 2.08 <2.00 < c 6655 Puff pastry * 2.48* 4 a 7447 Puff pastry with butter <1.00 <2.00 < a 7448 Puff pastry with butter <1.00 <2.00 < a 3778 Custard cream 1.78 <2.00 < b 7442 Flan <1.00 <2.00 < b 7443 Apple tart <1.00 <2.00 < b 7444 Apricot tart <1.00 <2.00 < b 7681 Mixed vegetables <1.00 <2.00 < a 7682 Grated carrots <1.00 <2.00 < a 6659 Cheese and tomato pizza * 2.30* 5 b 7684 Ham and cheese croissants <1.00 <2.00 < b 7683 Couscous <1.00 <2.00 < c 7685 Hacao with Xiu Mai chicken <1.00 <2.00 < c Type *: < 4 colonies per plate Artificial contaminants 92 samples were artificially contaminated with stressed strains, or by the seeding protocol. The strains inoculated, the injury protocol and injury measurement are given in Appendix samples gave results that could be processed by the spread and pour plate methods, along with 79 by the Spiral method for plates incubated for 22 h at 37 C. Naturally contaminated samples thus represent 20.0% for the spread method, 21.6% for the pour plate method and 12.2% for the Spiral method Raw results The raw results are given in Appendix 6. An overview is given in Appendix 7. Single samples were analysed by each of the methods such that 15 usable samples were obtained for each category. ADRIA Développement 22/ February 2016

23 Data were grouped into three categories: - results usable by both methods, - results with fewer than 4 colonies per plate by one or other of the methods (highlighted by an asterisk (*) in the raw results). These results were quantified such as to represent the actual result as accurately as possible. - results below or above the limit of quantification by one or other of the methods Statistical interpretation Spreading method The calculations are given in Appendix 8. The results pair (reference method / alternative method), both per category and for all categories, are given in Figures 4 to 9 for the 22 h incubation period and in Figures 10 to 15 for the 48 h incubation period. ADRIA Développement 23/ February 2016

24 Figure 4 Alternative Méthode alternative method (log ufc/g) (log Cat 11: : Meat Produits products carnés-etalement Spread 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode de method référence (log (log CFU/g) ufc/g) Type a Type b Type c valeurs Corrected corigées values <4 <4 colonies/boite / plate Figure 5 Alternative Méthode method alternative (log CFU/g) ufc/g) Cat 2: Cat Dairy 2 : Produits products laitiers-etalement Spread 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log CFU/g) (log ufc/g) Type a Type b Type c valeurs Corrected corigées values <4 <4 colonies/boite / plate Figure 6 Alternative Méthode alternative method (log ufc/g) CFU/g) Cat Cat 3: 3 Seafood : Produits de Spread la mer-etalement 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log CFU/g) (log ufc/g) Type a Type a Type b Type b Type c valeurs Corrected corrigées values <4 <4 colonies/boite / plate ADRIA Développement 24/ February 2016

25 Figure 7 Figure 8 Alternative Méthode alternative method (log (log ufc/g) CFU/g) Alternative Méthode method alternative (log (log ufc/g) CFU/g) Cat 4: Cat Egg 4 : products Ovoproduits -Spread Etalement 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log (log CFU/g) ufc/g) Cat 5 : 5: Aliments Ready prêt to à cook/eat cuire/ consommer foods - Etalement Spread 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log (log CFU/g) ufc/g) Type a Type b Type c Valeurs Corrected corrigées values Type Type a a Type b b Type c c Valeurs Corrected corrigées values Figure 9 All categories Toutes catégories-etalement Spread 22h 22h Alternative Méthode alternative method (log ufc/g) CFU/g) 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method référence (log CFU/g) ufc/g) Cat1 Cat2 Cat 3 Cat 4 Cat 5 Valeurs Corrected corrigées values < <4 4 colonies / plate /boîte y=x ADRIA Développement 25/ February 2016

26 Figure 10 Alternative Méthode alternative method (log (log ufc/g) CFU/g) Cat Cat 1: 1 : Meat Produits products carnés-etalement Spread 48h 48h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Méthode de référence(log ufc/g) Reference method (log CFU/g) Type a Type b Type c Type c Corrected values valeurs corrigées <4 colonies / plate <4 colonies/boite Figure 11 Alternative Méthode alternative method (log (log ufc/g) CFU/g) Cat 22: : Produits Dairy products laitiers-etalement Spread 48h 48h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence(log (log CFU/g) ufc/g) type Type a a Type b Type cc valeurs Corrected corrigées values <4 <4 colonies/boite / plate Figure 12 Alternative method (log CFU/g) Méthode alternative (log ufc/g) Cat 33: : Produits Seafood de la Spread mer-etalement 48h 48h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence(log CFU/g) ufc/g) Type a Type b Type cc valeurs Corrected corrigées values <4 <4 colonies/boite / plate ADRIA Développement 26/ February 2016

27 Figure 13 Figure 14 Alternative Méthode method alternative (log ufc/g) CFU/g) Alternative Méthode method alternative (log ufc/g) CFU/g) Cat 4: Egg Cat 4 products : Ovoproduits Spread - Etalement 48h Reference Méthode de référence method (log (log ufc/g) CFU/g) Cat Cat 5: Ready 5 : Aliments to cook/eat prêt à cuire foods /consommer Spread 48h 48h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log (log CFU/g) ufc/g) Type a Type a Type b Type b Type Type c c Valeurs Corrected corrigées values Type a a Type Type b b Type Type c c Valeurs corrigées Corrected values Figure 15 Alternative method (log CFU/g) Méthode alternative (log ufc/g) Toutes All categories catégories-etalement Spread 48h 48h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Méthode référence (log ufc/g) Reference method (log CFU/g) Cat1 Cat2 Cat 3 Cat 4 Cat 5 Valeurs Corrected corrigées values < <4 4 colonies / plate /boîte y=x A summary of calculated values is given in table 6. ADRIA Développement 27/ February 2016

28 Table 6 - Calculated values - Spread method Incubation period 22 h 48 h Category n DD S D 95% lower 95% upper limit limit / / / / / / / / / / All categories / / / / / / / / / / All categories The Bland-Altman plots are given in Figures 16 (for the 22 h incubation period) and 17 (for the 48 h incubation period). Figure 16 Figure 17 Difference Différence Bland-Altman -Toutes catégories-etalement 22H Bland-Altman - All categories Spread 22h 1,30 0,80 Cat 2/4777 0,30-0,20-0,70 Cat 4/7450-1,20 0,00 2,00 4,00 6,00 8,00 Mean Moyenne log log CFU/g UFC/g Bland-Altman Bland-Altman - All -Toutes categories catégories-etalement Spread 48h 48H Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Valeurs Corrected corrigées values <4 <4 colonies/boîte colonies/plate Limite Lower basse limit Limite Upper haute limit Linéaire Linear Difference Différence 2,00 1,50 1,00 Cat 4 /6656 0,50 0,00-0,50-1,00-1,50-2,00 0,00 2,00 4,00 6,00 8,00 Mean Moyenne log CFU/g log UFC/g Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Valeurs corrigées Corrected values <4 <4 colonies colonies/plate / boîte Limite Lower basse limit Limite Upper haute limit Linéaire Linear Samples for which the difference in result obtained by the reference method and that obtained by the alternative method is greater or less than the limits, are entered into tables 7 and 8 with associated comments. ADRIA Développement 28/ February 2016

29 Table 7 - Observed discrepancies! Spread method, 22 h incubation SOLABIA Corrected values < 4 colonies per plate Category Type Spl. no. Reference Alternative method method Mean Difference Comment 1 a Limit of quantification 2 a Lower count by the reference method 2 c Lower count by the reference method 2 a Lower count by the reference method 1 a Limit of quantification 1 a Lower count by the alternative method 1 a Lower count by the alternative method 2 a Lower count by the alternative method 2 c Lower count by the alternative method 3 a Lower count by the alternative method 3 c Lower count by the alternative method 4 a Lower count by the alternative method 4 c Lower count by the alternative method Table 8 - Observed discrepancies! Spread method, 48 h incubation Category Type Spl. no. Reference Alternative method method Mean Difference Comment 1 a Limit of quantification 2 a Lower count by the reference method 2 c Lower count by the reference method 1 c Lower count by the alternative method 1 a Lower count by the alternative method 1 a Lower count by the alternative method 1 a Limit of quantification 1 b Limit of quantification 1 c Limit of quantification 2 c Lower count by the alternative method 3 c Lower count by the alternative method 4 c Lower count by the alternative method ADRIA Développement 29/ February 2016

30 Conclusion For a 22 h incubation period, a lower count by the alternative method was observed in 8 cases and by the reference method in 3 cases. The differences observed, however, were low for 5 samples as they were of , -0.53, -0.60, and log CFU/g. For two samples, there was a problem with the limit of quantification, for the ISO method in one case (sample 3817) and for the alternative method in the other case (sample 3878). For a 48 h incubation period, the alternative method gave 6 results below the ISO method, while the ISO method gave 2 results below the alternative method. Overall, the two methods equivalent, whatever the incubation period of the alternative method Pour plate method The calculations are given in Appendix 9. The results pair (reference method / alternative method), both per category and for all categories, are given in Figures 18 to 23 for the 22 h incubation period and in Figures 24 to 29 for the 72 h incubation period. ADRIA Développement 30/ February 2016

31 Figure 18 Alternative Méthode alternative method (log (log ufc/g) CFU/g) Cat Cat 1: 1 Meat : Produits products carnés-inclusion Pour plate 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode de method référence (log CFU/g) ufc/g) Type a Type b Type c Corrected values valeurs corigées <4 colonies / plate <4 colonies/boite Figure 19 Alternative Méthode alternative method (log (log ufc/g) CFU/g) Cat 2: Cat Dairy 2 : Produits products laitiers-inclusion Pour plate - 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode de method référence (log (log CFU/g) ufc/g) Type a Type a Type b Type b Type c valeurs Corrected corigées values <4 <4 colonies/boite / plate Figure 20 Alternative Méthode alternative method (log (log ufc/g) CFU/g) Cat Cat 3: Seafood 3 : Produits Pour de la mer-inclusion plate - 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log (log CFU/g) ufc/g) Type a Type b Type c valeurs Corrected corigées values <4 colonies / plate <4 colonies/boite ADRIA Développement 31/ February 2016

32 Figure 21 Alternative Méthode method alternative (log ufc/g) CFU/g) Cat 4: Egg Cat 4 products : Ovoproduit -Pour Inclusion plate 22h- 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log CFU/g) (log ufc/g) Type a a Type bb Type Type c c Valeurs Corrected corrigées values Figure 22 Alternative Méthode method alternative (log ufc/g) CFU/g) Cat Cat 5: 5 : Ready Aliments to prêt cook/eat à cuire/ consommer foods - Inclusion Pour plate 22h - 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Méthode de référence ufc/g) Reference method (log CFU/g) Type a a Type b b Type Type c c Valeurs Corrected corrigées values Figure 23 Alternative Méthode alternative method (log (log ufc/g) CFU/g) All categories Toutes catégories-inclusion - 22h 22h 10,00 8,00 Cat1 Cat2 6,00 Cat 3 Cat 4 4,00 Cat 5 2,00 Valeurs Corrected corrigées values < <4 4 colonies / plate /boîte 0,00 y=x 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method référence (log CFU/g) ufc/g) ADRIA Développement 32/ February 2016

33 Figure 24 Figure 25 Alternative Méthode alternative method (log ufc/g) CFU/g) Alternative Méthode method alternative (log CFU/g) ufc/g) Cat 1: Cat Meat 1 : Produits products carnés-inclusion Pour plate 72h 72h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Méthode de référence (log ufc/g) Reference method (log CFU/g) Cat 2: Cat Dairy 2 : Produits products laitiers-inclusion Pour plate 72h- Reference Méthode method de référence (log CFU/g) (log ufc/g) Type a Type a Type b Type b Type c valeurs Corrected corrigées values <4 <4 colonies:boite colonies/plate 10,00 8,00 6,00 Type a Type b 4,00 Type c 2,00 valeurs Corrected corrigées values 0,00 <4 <4 colonies/boite colonies/plate 0,00 2,00 4,00 6,00 8,00 10,00 Figure 26 Alternative Méthode method alternative (log ufc/g) CFU/g) Cat 3: Cat Seafood 3 : Produits Pour de la plate mer-inclusion - 72h 72h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log CFU/g) (log ufc/g) Type aa Type bb Type cc valeurs Corrected corrigées values Figure 27 10,00 Cat 4 : Ovoproduit - Inclusion 72h Alternative Méthode method alternative (log (log CFU/g) ufc/g) 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log CFU/g) (log ufc/g) Type Type a a type Type b b Type Type c c Valeurs Corrected corrigées values ADRIA Développement 33/ February 2016

34 Figure 28 Alternative Méthode method alternative (log (log CFU/g) ufc/g) Cat 55: : Aliments Ready prêt to à cook/eat cuire /consommer foods - Inclusion Pour 72h plate - 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Méthode de référence (log ufc/g) Reference method (log CFU/g) Type aa Type bb Type cc Valeurs Corrected corrigées values Figure 29 All categories Toutes catégories-inclusion Pour plate - 72h 72h Alternative Méthode method alternative (log CFU/g) ufc/g) 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method référence (log (log CFU/g) ufc/g) Cat1 Cat2 Cat 3 Cat 4 Cat 5 Valeurs corrigées Corrected values < 4 colonies /boîte <4 colonies / plate y=x A summary of calculated values is given in table 9. Table 9 - Calculated values - Spread method Incubation period 22 h 48 h Category n DD S D 95% lower 95% upper limit limit / / / / / / / / / / All categories / / / / ADRIA Développement 34/ February 2016

35 / / / / / / All categories The Bland-Altman plots are given in Figures 30 (for the 22 h incubation period) and 31 (for the 72 h incubation period). Figure 30 Figure 31 Difference Différence Difference Différence Bland-Altman -Toutes - All categories catégories-inclusion Pour plate 22H - 22h 2,00 1,50 1,00 Cat 3 /3871 0,50 0,00-0,50 Cat 4 /7445-0,67-1,00-1,50-2,00 0,00 2,00 4,00 6,00 8,00 Mean Moyenne log log CFU/g UFC/g Bland-Altman --Toutes All categories catégories-inclusion Pour plate - 72h 72H 2,00 1,50 1,00 0,50 Cat 3 /3871 0,00-0,50 Cat 4 / ,00-1,50-2,00 0,00 2,00 4,00 6,00 8,00 Mean Moyenne log CFU/g log UFC/g Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Valeurs corrigées Corrected values <4 colonies/boîte <4 colonies/plate Limite basse Lower limit Limite haute Upper limit Linéaire Linear Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Valeurs corrigées Corrected values <4 colonies / boîte <4 colonies/plate Limite basse Lower limit Limite haute Upper limit Linéaire Linear Samples for which the difference in result obtained by the reference method and that obtained by the alternative method is greater or less than the limits, are entered into tables 10 and 11 with associated comments. ADRIA Développement 35/ February 2016

36 Table 10 - Observed discrepancies! Pour plate method, 22 h incubation Corrected values < 4 colonies per plate Category Type Spl. no. Reference method Alternative method Mean Difference Comment 1 a Limit of quantification 1 c Lower count by the reference method 2 a Lower count by the reference method 3 b Lower count by the reference method 3 c Lower count by the reference method 1 c Limit of quantification 1 c Lower count by the alternative method 2 c Limit of quantification 2 c Lower count by the alternative method 2 b Limit of quantification 3 c Lower count by the alternative method 4 b Lower count by the alternative method Table 11 - Observed discrepancies! Pour plate method, 72 h incubation Category Type Spl. no. Reference Alternative method method Mean Difference Comment 1 a Limit of quantification 1 a Lower count by the reference method 1 c Lower count by the reference method 2 a Lower count by the reference method 3 b Lower count by the reference method 3 c Lower count by the reference method 1 c Lower count by the alternative method 2 a Limit of quantification 2 b Limit of quantification 2 c Lower count by the alternative method 3 c Lower count by the alternative method 3 c Lower count by the alternative method 4 c Lower count by the alternative method 4 b Lower count by the alternative method ADRIA Développement 36/ February 2016

37 Conclusion For a 22 h incubation period, the reference method proved deficient in 4 cases and the alternative method in 4 cases, with count differences ranging from 0.33 to 0.78 log CFU/g and from to log CFU/g. For a 72 h incubation period, the reference method proved deficient in 5 cases and the alternative method in 6 cases, with count differences ranging from 0.33 to 0.70 log CFU/g and from to log CFU/g. Overall, the two methods equivalent, whatever the incubation period of the plates Spiral method The calculations are given in Appendix 10. The results pair (reference method / alternative method), both per category and for all categories, are given in Figures 32 to 37 for the 22 h incubation period and in Figures 38 to 43 for the 72 h incubation period. ADRIA Développement 37/ February 2016

38 Figure 32 Figure 33 Alternative Méthode alternative method (log CFU/g) ufc/g) Alternative Méthode alternative method (log (log ufc/g) CFU/g) Cat 1: Cat Meat 1 : Produits products carnés-spiral 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode de method référence (log (log CFU/g) ufc/g) Cat 2: Dairy Cat 2 products : Produits laitiers-spiral - 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log CFU/g) (log ufc/g) Type aa Type b Type Type cc Corrected values valeurs corigées <4 colonies/plate <4 colonies/boite Type aa Type bb Type cc valeurs Corrected corigées values <4 <4 colonies/boite colonies/plate Figure 34 Méthode Alternative alternative method (log (log ufc/g) CFU/g) Cat Cat 3: Seafood 3 : Produits Spiral de la - mer-spiral 22h 22h 10,00 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Reference Méthode method de référence (log CFU/g) (log ufc/g) Type Type aa Type bb Type c c valeurs Corrected corigées values <4 <4 colonies/boite colonies/plate ADRIA Développement 38/ February 2016