Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
Page 1 of 92
MicroVal Study 2017LR76
Validation of the 3M™ Petrifilm™ ™ Rapid E. coli / Coliform Count Plate for the Enumeration of E. coli and Coliform in a Broad Range of Foods
following ISO 16140-2:2016 Guidelines: Method Comparison Study Report
Version 2: MLS + ILS December 14th, 2018
Contact Details: Q Laboratories 1400 Harrison Ave. Cincinnati, OH 45214 Tel: 513-471-1300 www.qlaboratories.com
Report issued and authorized by: Q Laboratories Benjamin Bastin, Microbiology R&D Supervisor
2/92
The MCS report is prepared in accordance with ISO 16140-1:2016 [1] and
ISO 16140-2:2016 [2] and the most recent version of the MicroVal Technical
Committee for interpretation on ISO 16140-2, version 1.0.
Company: 3M Food Safety
Expert Laboratory: Q Laboratories
Method/Kit name: 3M™ Petrifilm™ Rapid E. coli / Coliform Count Plate
Validation standard: ISO 16140-2:2016 Microbiology of food chain – Method
Validation – Part 2: Protocol for the validation of alternative (proprietary) methods
against a reference method.
Reference methods:
1. ISO 4832:2006, Microbiology of food and animal feeding stuffs – Horizontal
method for the enumeration of coliforms – Colony-count technique
2. ISO 16649-2:2001, Microbiology of food and animal feeding stuffs –
Horizontal method for the enumeration of β-glucuronidase-positive
Escherichia coli – Part 2: Colony count technique.at 44 °C using 5-bromo-4-
chloro-3-indolyl β-D-glucuronide
Scope of validation: Broad Range of Foods and Select Environmental Surfaces.
Food Categories tested: Meat Products, Dairy and Egg Products, Fish and Seafood
Products, Vegetable Products, Ready to Eat and Ready to Reheat, Animal Feed and
Samples from the Industrial Production Environment
Certification organization: Lloyd's Register
List of abbreviations
- AL Acceptability Limit
- AP Accuracy Profile
- Art. Cont. Artificial contamination
- CFU Colony Forming Units
- CL Confidence Limit (usually 95%)
- EL Expert Laboratory
- �̅� Average difference
- g Gram
- h Hour
- ILS Interlaboratory Study
- Incl/Excl Inclusivity and Exclusivity
- MCS Method Comparison Study
- min minute
- ml Millilitre
3/92
- MR (MicroVal) Method Reviewer
- MVTC MicroVal Technical Committee
- n number of samples
- na not applicable
- RT Relative Trueness
- SD Standard Deviation of Differences
- 10-1 dilution 10-fold dilution of original food
- 10-2 dilution 100-fold dilution of original food
- VRBG/L Violet Red Bile Glucose Agar with Lactose
- TBX Tryptone Bile X-Glucuronide Medium
- TNTC Too numerous to count
4/92
Table of Contents
1 Introduction 5
2 Method Protocols 6
2.1 Reference Method – ISO 4832 6
2.2 Reference Method – ISO 16649-2:2001 6
2.3 Alternative method 6
3 Methods Comparison Study 6
3.1 Sample Preparation 6
3.2 Trueness Study 7
3.2.1 Calculations and Interpretation 9
3.2.2 Conclusion of the Truness Study 28
3.3 Accuracy Profile Study 28
3.3.1 Calculation and Data Interpretation 30
3.3.2 Conclusion of the Accuracy Profile Study 31
3.4 Inclusivity and Inclusivity and Exclusivity Study 70
4 Interlaboratory Study 74
4.1 Organization 74
4.2 Calculations and interpretation of data 75
5 References 85
6 ANNEX A: Flow Diagram of the ISO 4832 Reference Method 86
7 ANNEX B: Flow Diagram of the ISO 16649-2:2001 Reference Method 87
8 ANNEX C: Flow Diagram of the 3M Petrifilm Rapid E. coli and Coliform Count Plate Alternative Method 88
9 ANNEX D: Kit Insert for 3M Petrifilm Rapid E. coli / Coliform Count Plate 88
See the attached document. 88
10 ANNEX E: Summary of Artificial Contamination 89
5/92
1 Introduction
The 3M™ Petrifilm™Rapid E. coli / Coliform Count Plate was validated according to ISO 16140-2:2016 for the enumeration of coliforms and E. coli in a broad range of foods and environmental surfaces. All testing was conducted by Q Laboratories, Inc.
Below is a summary of the 3M Petrifilm Rapid E. coli / Coliform Count Plate Method.
For each food type, 10 g test portions were diluted at a 1:10 ratio using the appropriate diluent. Each environmental sponge was diluted with 90 mL of diluent. Paired 10 g test portions were evaluated for the alternative and reference methods for all food types in the scope.
1.1. After dilution, 1.0 mL of sample was dispensed onto the 3MPetrifilm Rapid E. coli / Coliform Count Plate plate. Plates were incubated at 30 ± 1°C for dairy products and 37 ± 1°C and 42 ± 1°C for all other matrices. All colonies were enumerated at 18 hours.
1.2. For the purposes of the MicroVal validation, up to 5 atypical coliform colonies were confirmed through transfer into tubes of Brilliant Green Lactose Bile (BGLB) Broth and incubating at 24 ± 2 hours at 37 ± 1 ºC. Production of gas indicated the presence of coliform.
Reference methods: ISO 4832:2006 [3] and ISO 16649-2:2001 [4]. See Annex A-E for flow charts and kit inserts of the methods.
Scope of the validation study is: Broad range of food matrices and select environmental surfaces.
Samples were prepared in accordance with ISO 6887: parts 1 – 5. [5]
Categories included:
− Meat Products (Combined category raw/RTC meats and poultry)
− Dairy and Egg Products
− Fish and Seafood Products
− Vegetable Products
− Ready to Eat and Ready to Reheat (Combined category RTE/RTRH meats and poultry)
− Animal Feed
− Samples from the Industrial Production Environment
Criteria evaluated during the study were:
− Method Comparison Study (MCS)
• Relative Trueness study
• Relative Accuracy study
• Inclusivity and exclusivity study
− Interlaboratory Study (ILS)
6/92
2 Method Protocols
The MCS was carried out using 10 gram portions of sample material for all food matrices and environmental sponges for all samples from the industrial production environment. In both the trueness and accuracy studies, 3M Petrifilm Rapid E. coli / Coliform Count Plate was evaluated at 18 hours of incubation. A paired design was utilized for all foods, as the reference and the alternative
method shared the same diluent.
2.1 Reference Method – ISO 4832
See the flow diagram in Annex A.
Sample preparations used in the reference method were done according to ISO
6887-series.
2.2 Reference Method – ISO 16649-2:2001
See the flow diagram in Annex B.
Sample preparations used in the reference method were done according to ISO 6887-series.
2.3 Alternative method
See the flow diagram of the alternative method in Annex C and the 3M Petrifilm
Rapid E. coli / Coliform Count Plate kit insert in Annex D for a detailed description of
the method.
The 3M Petrifilm Rapid E. coli / Coliform Count Plate test is a selective and differential sample-ready-culture medium system which contains proprietary nutrients, a cold-water-soluble gelling agent, 5-bromo-4-chloro-3-indoyl-D-glucuronide (BCIG), an indicator of glucuronidase activity, and a tetrazolium indicator that facilitates colony enumneration.
The food samples were prepared using 10 g test portions or an environmental surface sponge diluted with 90 mL of the appropriate diluent. Colonies were enumerated after 18 hours of incubation at specified temperatures (37 ± 1oC for coliform and E. coli; 42 ± 1oC for E. coli); (30 ± 1°C for dairy coliform and E. coli).
3 Methods Comparison Study
3.1 Sample Preparation
The Method Comparison Study was carried out using 10 g test portions or sponges for environmental industrial production samples. The samples were prepared for analysis and diluted in accordance with ISO 6887 (all parts). A 1:10 dilution was performed for all food types. All analysis were conducted using paired samples
7/92
3.2 Trueness Study
The selected types per category tested are listed in Table 1.
In accordance with ISO 16140-2, for each category, a minimum of 15 samples were tested. Within each type, a minimum of 5 samples (producing interpretable results) that cover a representative variety of products were evaluated. All samples covered a wide range of contamination that could be observed in the food items (i.e. 10 CFU/g to 105 CFU/g).
Table 1. List of Categories and Types of Items to be Tested within the Trueness Study
Category Food Type Number of
Samples1
Test Portion
Size
Meat Products
(Combined
category raw/RTC
meats and poultry)
Raw Meat 5 10 g
Breaded Meats
(i.e. Frozen Wings) 5 10 g
Cured and Smoked Products (raw) 5 10 g
Dairy and Egg
Products
Pasteurized Dairy Products 5 10 g
Raw Dairy Products 5 10 g
Desserts and Egg Products 5 10 g
Fish and Seafood Products
Raw Products 5 10 g
Marinated Smoked Items 5 10 g
Cooked Products 5 10 g
Vegetable Products
Raw Vegetables 5 10 g
Pre-Cooked Vegetables 5 10 g
Processed Vegetables 5 10 g
Ready to Eat and Ready to Reheat
Cold Ready Meals 5 10 g
Pasta 5 10 g
Mayonnaise based deli salads with
processed ingredients 5 10 g
Animal Feed
Finished Products for Domestic 5 10 g
Finished Products for Livestock 5 10 g
Ingredients for domestic/livestock 5 10 g
Samples from the
Industrial
Production
Environment2
Surface Samples 15 Environmental
Sponge
1Total number of samples = minimum of 15 paired samples/category; 2 Limited claims for surface samples only
Samples were evaluated for natural contamination of the target organisms. Thirty (30%) of total samples evaluted were naturally contaminated with coliform and E. coli. For positive samples used in the evaluation, 63% of samples were obtained from natural contamination. The following spiking protocol indicates procedures to inoculate a wide range of food items. Not all spiking protocols were required for the validation.
8/92
1.1. For high water activity products, a liquid inoculum was heated at 50 °C for 10 minutes prior to inoculation.
1.2. For low water activity products, a lyophilized culture was used. 1.3. The stressed cultures was used to inoculate the relevant food matrix and
would be analyzed immediately. The level of injury was evaluated by plating on a non-selective agar (Tryptic Soy Agar) and a selective agar (TBX for Escherichia coli and VRBL for Coliform). It was proposed to obtain between a 50% (0.3log) and a 70% (0.5log) injured cells.
The following seeding protocol indicates procedures to inoculate a wide range of food items. Not all seeding protocols were required for the validation.
1.1. Overnight cultures of Escherichia coli and coliform were inoculated at the target contamination level into the food matrix and then stored under relevant conditions for the food matrix being analyzed prior to analysis:
1.2. For refrigerated products, samples were stored at 5 °C for 48 to 72 hours. 1.3. For frozen foods, samples were stored at -20 °C for a minimum of 2 weeks. 1.4. For shelf stable products, samples were stored at room temperature (22-25
°C) for a minimum of 2 weeks.
Table 2 provides a list of strains that were used for artificial inoculations. These cultures originated from comparable sample types as the ones to be inoculated.
Table 2. List of Proposed Inoculation Organisms for the Trueness Study
Organisms Source Origin
Citrobacter freundii QL1 100813-2A Sliced deli turkey meat
Cronobacter condimenti DSM2 27966 Infant Formula
Enterobacter amnigenus ATCC3 51816 Milk, Minnesota
Hafnia alvei ATCC 51815 Milk, Minnesota
Klebsiella pneumonia QL 11007-7 Raw hamburger
Rahnella aqualitis ATCC 55046 Soil
Serratia marcescens ATCC 8100 Milk, Cork Ireland
Escherichia coli QL 16160-4 Chicken Nuggets
Escherichia coli QL 11010-2 Bottled Water
Escherichia coli QL 16154-7 Flour
Escherichia coli QL 16154-2 Raw Ground Beef
Escherichia coli QL16160-2 Chicken Carcass Rinsate
Escherichia coli QL 16160-10 Raw Pork
Escherichia coli QL 16160-2 Mixed Frozen Vegetables
Escherichia coli QL 16154-3 Beef Trim
Escherichia coli QL 16154-13 Potable Water
Escherichia coli QL 16154-2 Raw Ground Beef
Escherichia coli QL 16154-7 Flour 1 QL-Q Laboratories Culture Collection , 2 DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 3 ATCC-American Type Culture Collection The inoculation of samples was at the range usually associated with the test organisms and within the capabilities of the test methods. Enumeration methods covered the range 10 CFU/g to 105 CFU/g.
9/92
3.2.1 Calculations and Interpretation
All results were tabulated, calculated and interpreted according to ISO 16140-2. The obtained results for each food item per category and all food items in all categories were plotted. The line of identity was drawn (y=x) . The data obtained was analzyed using the scatter plot and the line of identity. The figures below display a summary of the data. The raw data from the relative trueness study are presented in Annex F.
Figure 1A: Meat Products – Coliform 37 ± 1°C
Figure 1B: Dairy and Egg Products – Coliform 30 ± 1°C & 37 ± 1°C
10/92
Figure 1C: Fish and Seafood Products – Coliform 37 ± 1°C
Figure 1D: Vegetable Products – Coliform 37 ± 1°C
Figure 1E: Ready to Eat and Ready to Reheat Products – Coliform 37 ± 1°C
11/92
Figure 1F: Animal Feed Products – Coliform 37 ± 1°C
Figure 1G: Surface Samples – Coliform 37 ± 1°C
Figure 1H: All Categories – Coliform 30 ± 1°C & 37 ± 1°C1
130°C for dairy products and 37°C for all other matrices
12/92
Figure 1I: Meat Products – E. coli 37 ± 1°C
Figure 1J: Meat Products – E. coli 42 ± 1°C
Figure 1K: Dairy and Egg Products – E. coli 30 ± 1°C & 37 ± 1°C
13/92
Figure 1L: Dairy and Egg Products – E. coli 30 ± 1°C & 42 ± 1°C
Figure 1M: Fish and Seafood Products – E. coli 37 ± 1°C
Figure 1N: Fish and Seafood Products – E. coli 42 ± 1°C
14/92
Figure 1O: Vegetable Products – E. coli 37 ± 1°C
Figure 1P: Vegetable Products – E. coli 42 ± 1°C
Figure 1Q: Ready to Eat and Ready to Reheat Products – E. coli 37 ± 1°C
15/92
Figure 1R: Ready to Eat and Ready to Reheat Products – E. coli 42 ± 1°C
Figure 1S: Animal Feed Products – E. coli 37 ± 1°C
Figure 1T: Animal Feed Products – E. coli 42 ± 1°C
16/92
Figure 1U: Surface Samples – E. coli 37 ± 1°C
Figure 1V: Surface Samples – E. coli 42 ± 1°C
Figure 1W: All Categories – E. coli 30 ± 1°C & 37 ± 1°C1
130°C for dairy products and 37°C for all other matrices
17/92
Figure 1X: All Categories – E. coli 30 ± 1°C & 42 ± 1°C1
130°C for dairy products and 42°C for all other matrices
According to ISO 16140-2:2012, section 6.1.2.3, the results of the scatter plots and the line of identity were interpreted based on a visual observation on the amount of bias and extreme results. The data appeared to be acceptable per category and as a whole. The obtained results were analyzed using the Bland-Altman method as detailed in section 6.1.2.3 in ISO 16140-2. Table 3. Summary of the Calculated Differences and Confidence Limits
Organism Food
Category Temperature n
95%
Lower
Limit
95%
Upper
Limit
Coliform
Meat
Products 37 ± 1°C 15 0.0238 0.0355 -0.0523 0.0999
Dairy and
Egg
Products
30 ± 1°C &
37 ± 1°C 15 0.0463 0.0528 -0.0669 0.1595
Fish and
Seafood
Products
37 ± 1°C 15 0.0113 0.0371 -0.0683 0.0909
Vegetable
Products 37 ± 1°C 15 0.0126 0.0504 -0.0955 0.1206
Ready to
Eat and
Ready to
Reheat
37 ± 1°C 15 0.0051 0.0342 -0.0682 0.0784
Animal Feed
Products 37 ± 1°C 15
-0.0031
0.0238 -0.0540 0.0479
Surface
Samples 37 ± 1°C 15
-0.0031
0.0348 -0.0777 0.0715
All
Categories
30 ± 1°C &
37 ± 1°C 105 0.0133 0.0416 -0.0692 0.0957
D Ds
18/92
Organism Food
Category Temperature n
95% Lower Limit
95% Upper Limit
E. coli
Meat
Products
37 ± 1°C 15 -
0.0714 0.0446 -0.1670 0.0242
42 ± 1°C 15 0.0001 0.0927 -0.1987 0.1990
Dairy and
Egg
Products
30 ± 1°C &
37 ± 1°C 15
-
0.0126 0.0649 -0.1518 0.1267
30 ± 1°C &
42 ± 1°C 15
-
0.0073 0.0623 -0.1409 0.1263
Fish and
Seafood
Products
37 ± 1°C 15 -
0.1019 0.1284 -0.3774 0.1736
42 ± 1°C 15 -
0.0365 0.0892 -0.2277 0.1547
Vegetable
Products
37 ± 1°C 15 -
0.0834 0.1324 -0.3673 0.2005
42 ± 1°C 15 -
0.0359 0.1761 -0.4137 0.3419
Ready to
Eat and
Ready to
Reheat
37 ± 1°C 15 -
0.0831 0.1204 -0.3412 0.1751
42 ± 1°C 15 -
0.0305 0.0851 -0.2131 0.1521
Animal Feed
Products
37 ± 1°C 15 -
0.0726 0.1220 -0.3344 0.1892
42 ± 1°C 15 0.0193 0.1451 -0.2919 0.3306
Surface
Samples
37 ± 1°C 15 -
0.1236 0.1392 -0.4222 0.1750
42 ± 1°C 15 -
0.0001 0.1731 -0.3715 0.3712
All
Categories
30 ± 1°C &
37 ± 1°C 105
-
0.0784 0.1140 -0.3045 0.1477
30 ± 1°C &
42 ± 1°C 105
-
0.0053 0.1244 -0.2520 0.2413
The results of the difference and scatter plot were interpreted, based on a visual observation on the amount of bias and extreme results. It was expected that not more than 1 in 20 data values will lie outside the confidence limits. Any disagreements with the expectation were recorded.
The symbols used ensure the different types of product in each category are clearly identified. The average of each pair of data values and the difference between these values were calculated for each sample and for each category.
D Ds
19/92
Figure 2A: Bland-Altman for Meat Products – Coliform 37 ± 1°C
Figure 2B: Bland-Altman for Dairy and Egg Products – Coliform 30 ± 1°C & 37 ± 1°C
Figure 2C: Bland-Altman for Fish and Seafood Products – Coliform 37 ± 1°C
20/92
Figure 2D: Bland-Altman for Vegetable Products – Coliform 37 ± 1°C
Figure 2E: Bland-Altman for Ready to Eat and Ready to Reheat Products – Coliform 37 ± 1°C
Figure 2F: Bland-Altman for Animal Feed Products – Coliform 37 ± 1°C
21/92
Figure 2G: Bland-Altman for Surface Samples – Coliform 37 ± 1°C
Figure 2H: Bland-Altman for All Categories – Coliform 30 ± 1°C & 37 ± 1°C1
130°C for dairy products and 37°C for all other matrices
Figure 2I: Bland-Altman for Meat Products – E. coli 37 ± 1°C
22/92
Figure 2J: Bland-Altman for Meat Products – E. coli 42 ± 1°C
Figure 2K: Bland-Altman for Dairy and Egg Products – E. coli 30 ± 1°C & 37 ± 1°C
Figure 2L: Bland-Altman for Dairy and Egg Products – E. coli 30 ± 1°C & 42 ± 1°C
23/92
Figure 2M: Bland-Altman for Fish and Seafood Products – E. coli 37 ± 1°C
Figure 2N: Bland-Altman for Fish and Seafood Products – E. coli 42 ± 1°C
Figure 2O: Bland-Altman for Vegetable Products – E. coli 37 ± 1°C
24/92
Figure 2P: Bland-Altman for Vegetable Products – E. coli 42 ± 1°C
Figure 2Q: Bland-Altman for Ready to Eat and Ready to Reheat Products – E. coli 37 ± 1°C
Figure 2R: Bland-Altman for Ready to Eat and Ready to Reheat Products – E. coli 42 ± 1°C
25/92
Figure 2S: Bland-Altman for Animal Feed Products – E. coli 37 ± 1°C
Figure 2T: Bland-Altman for Animal Feed Products – E. coli 42 ± 1°C
Figure 2U: Bland-Altman for Surface Samples – E. coli 37 ± 1°C
26/92
Figure 2V: Bland-Altman for Surface Samples – E. coli 42 ± 1°C
Figure 2W: Bland-Altman for All Categories – E. coli 30 ± 1°C & 37 ± 1°C1
130°C for dairy products and 37°C for all other matrices
Figure 2X: Bland-Altman for All Categories – E. coli 30 ± 1°C & 42 ± 1°C1
130°C for dairy products and 42°C for all other matrices
27/92
For all Categories, there were five out of 105 food items which lie outside the CLs for Coliform, ten out of 105 food items for E. coli incubated at 37 ± 1°C and thirteen out of 105 food items for E. coli incubated at 42 ± 1°C.
The 28 total food items which are outside of the CLs are shown in Table 4. There were no identifiable trends in these data as they covered both target organisms at all three temperatures in 7 different food types and being associated with both natural and artificial contamination. For all of the results that were outside of the confidence limits, there were zero results that were greater than a 0.5 Log difference indicating that none of the outliers would be considered statistically signficantly different.
Table 4. Results falling outside of confidence limits
Food
Category
Food
Type
Sample
Code Food Item
Organism &
Temperature
Spiking/
Seeding
Protocol
Difference
(alternative-
reference)
Dairy & Egg
Products
Pasteurized
Dairy Products 5 Ice Cream
Coliform
30 ± 1°C
Natural
Contamination 0.1139
Dairy & Egg
Products
Raw Dairy
Products 5 Camembert
Coliform
30 ± 1°C
Natural
Contamination 0.1022
Dairy & Egg
Products
Desserts &
Egg Products 5 Cream Puffs
Coliform
37 ± 1°C
5 ± 1°C for 48-
72 hours 0.1139
Vegetable
Products
Raw
Vegetables 3 Raddish
Coliform
37 ± 1°C
Natural
Contamination -0.0746
Surface
Samples 4 Air Vent 1
Coliform
37 ± 1°C
Natural
Contamination -0.0792
Fish &
Seafood
Products
Raw Products 2 Pollock E. coli
37 ± 1°C
5 ± 1°C for 48-
72 hours -0.3979
Fish &
Seafood
Products
Marinated
Smoked Items 3
Butter & Herb
Cod
E. coli
37 ± 1°C
Natural
Contamination -0.4058
Vegetable
Products
Raw
Vegetables 4 Red Bell Pepper
E. coli
37 ± 1°C
Natural
Contamination -0.3680
Vegetable
Products
Pre-Cooked
Vegetables 4 Frozen Onions
E. coli
37 ± 1°C
5 ± 1°C for 48-
72 hours -0.3388
RTE & RTRH
Foods
Cold Ready
Meals 1
Chicken Caesar
Wrap
E. coli
37 ± 1°C
Natural
Contamination -0.3680
RTE & RTRH
Foods Pasta 3
Pasta with
Broccoli and
Cheddar Sauce
E. coli
37 ± 1°C
Natural
Contamination -0.3590
Animal Feed
Finished
Products for
Livestock
2 Chicken Feed E. coli
37 ± 1°C
Natural
Contamination -0.3590
Animal Feed
Finished
Products for
Livestock
4 Purina Goat
Grower Feed
E. coli
37 ± 1°C
Natural
Contamination -0.3332
Surface
Samples 6 Air Vent
E. coli
37 ± 1°C
Natural
Contamination -0.3632
Surface
Samples 14 Cement Table
E. coli
37 ± 1°C
Natural
Contamination -0.3736
28/92
Meat Products
Cured and
Smoked
Products (raw)
1 Hot Pork
Sausage
E. coli
42 ± 1°C
Natural
Contamination 0.3010
Food
Category
Food
Type
Sample
Code Food Item
Organism &
Temperature
Spiking/
Seeding
Protocol
Difference
(alternative-
reference)
Fish &
Seafood
Products
Marinated
Smoked Items 3
Butter & Herb
Cod
E. coli
42 ± 1°C
Natural
Contamination -0.3332
Vegetable
Products
Raw
Vegetables 1 Broccoli
E. coli
42 ± 1°C
Natural
Contamination 0.3332
Vegetable
Products
Raw
Vegetables 4 Red Bell Pepper
E. coli
42 ± 1°C
Natural
Contamination -0.3332
Vegetable
Products
Pre-Cooked
Vegetables 4 Frozen Onions
E. coli
42 ± 1°C
5°C for 48-72
hours -0.3010
RTE & RTRH
Foods
Cold Ready
Meals 1
Chicken Caesar
Wrap
E. coli
42 ± 1°C
Natural
Contamination -0.3010
Animal Feed
Finished
Products for
Domestic
3 Iams E. coli
42 ± 1°C
Natural
Contamination 0.3010
Animal Feed
Finished
Products for
Livestock
1 Cow Mana E. coli
42 ± 1°C
Natural
Contamination 0.3010
Animal Feed
Finished
Products for
Livestock
4 Purina Goat
Grower Feed
E. coli
42 ± 1°C
Natural
Contamination 0.3010
Surface
Sponge 6 Air Vent 3
E. coli
42 ± 1°C
5 ± 1°C for 48-
72 hours -0.3310
Surface
Sponge 7 Pans 1-5
E. coli
42 ± 1°C
5 ± 1°C for 48-
72 hours 0.3010
Surface
Sponge 9 Holding Tank
E. coli
42 ± 1°C
5 ± 1°C for 48-
72 hours 0.3332
Surface
Sponge 14 Cement Table
E. coli
42 ± 1°C
5 ± 1°C for 48-
72 hours -0.3010
3.2.2 Conclusion of the Truness Study
Based on the data obtained in the evaluation, the relative trueness study of the alternative method is considered equivalent to the reference method.
3.3 Accuracy Profile Study
The accuracy profile study is a comparative study between the results obtained by the reference method and the results of the alternative method. The study was conducted using artificially contaminated samples. Two different food types per category were tested.
Number of Samples
For each category being examined, 2 food types were tested at three levels of contamination (low, medium and high). These levels covered the entire range of
29/92
contamination of the selected food type. Artificial contamination was performed using the seeding protocols specified in Table 5 below. For each contamination level, a bulk lot was co-inoculated with both E. coli and coliform and split into 2 samples. From each sample, 5 replicate test portions were evaluated.
Table 5: Summary of Food Categories and Food Items
Food
Category Food Type Food Items
Replicates/
Test Portion
Size1
Inoculating
Organism
(Seeding
protocol)
Target Contamination
Levels
Meat
Products
(Combined
category
raw/RTC
meats and
poultry)
Raw Fresh
Meats2/
Breaded Meats
Minced Meat
&
Raw Frozen
Chicken Wings
15 rep per food item/ 10 g
Klebsiella pneumoniae
& E. coli Liquid
10-100 CFU/g
100-1,000 CFU/g
10,000-100,000 CFU/g
Dairy and
Egg
Products
Raw dairy
products2/Egg
Products (heat
processed) with
additives
Raw milk/whole
liquid egg
15 rep per food item/
10 mL
Hafnia alvei &
E. coli Liquid
10-100 CFU/mL
100-1,000 CFU/mL
10,000-100,000 CFU/mL
Fish and Seafood Products
Raw
products2/smoked
products
Tuna Sushi/
smoked salmon
15 rep per food item/ 10 g
E. coli Liquid/stressed
10-100 CFU/g
100-1,000 CFU/g
10,000-100,000 CFU/g
Vegetable Products
Raw vegetable
products/Heat
processed
vegetable juices2
Bunched Spinach/
pasteurized carrot
juice
15 rep per food item/ 10 g
Klebsiella oxytoca
& E. coli
Liquid/stressed
10-100 CFU/g
100-1,000 CFU/g
10,000-100,000 CFU/g
Ready to Eat
and Ready
to Reheat
Cold Ready
Meals/Mayonnaise
based deli salads
with raw
ingredients2
Ready-made
Sandwiches/
raw vegetable
salad with
dressings.
15 rep per food item/ 10 g
Citrobacter freundii
& E. coli Liquid
1-10 CFU/g
100-1,000 CFU/g
10,000-100,000 CFU/g
Animal Feed
Finished Products
for Livestock/Plant
Origin Ingredients2
Chicken
Feed/Soybean
Meal
15 rep per food item/ 10 g
Enterobacter cloacae
& E. coli
lyophilized
10-100 CFU/g
100-1,000 CFU/g
10,000-100,000 CFU/g
Samples
from the
Industrial
Production
Environment
Surface
Samples2,3
Stainless
Environmental
sponges/Sealed
Concrete
Environmental
sponges
15 rep per food item/
Sampling Device
Enterobacter gergoviae
& E. coli Liquid
10-100 CFU/ Sampling Device
100-1,000 CFU/ Sampling Device
10,000-100,000 CFU/ Sampling Device
30/92
1Total number of samples = minimum of 210 (includes paired samples replicates for 3M™ Petrifilm™ and ISO 4832/16649-2) 2 Food type used for frozen plate enumeration evaluation 3Only environmental surface samples will be claimed for this category.
Each matrix was artificially contaminated with a different coliform and E. coli strain. Each inoculum was prepared by transferring a single colony from trypticase soy agar with 5% sheep blood (SBA) into BPW (ISO) and incubating the culture at 35 ± 2oC for 24 ± 2 hours. Following incubation, the culture was diluted to a target level using BPW (ISO) as the diluent. For each inoculated food matrix, bulk portions were spiked and blended in large sterile stainless steel containers. Sterile spatulas were used to mix the bulk portions to ensure the inoculum was evenly distributed throughout the matrix. The minced meat, raw milk, whole liquid eggs, tuna sushi, smoked salmon, bunched spinach, pasteurized carrot juice, ready-made sandwiches, and raw vegetable salad with dressings were held for 48-72 hours at refrigerated temperature (2-8⁰C) prior to analysis to allow time for the organism to equilibrate within the sample. Raw frozen chicken wings were held for two weeks at -20 °C. For chicken feed and soybean meal, a lyophilized inoculum was used to inoculate a bulk lot of each matrix and was then homogenized and held at ambient temperature (20-25⁰C) for two weeks. For surface samples, the inoculatd surface was held at ambient temperature (20-25⁰C) for 16 to 18 hours prior to sampling. Test portions were statistically evaluated according to ISO 16140-2:2016 and MicroVal. The accuracy profile (AP) was used to check the requirement that the alternative method produces a result for a sample that differs from the value produced by the reference method by less than a certain acceptability criterion. Prior to inoculation of the whole liquid eggs and pasteurized carrot juice the broth culture inoculum was heat stressed in a waterbath for 10 ± 1 minute at 50 ± 1°C. The degree of injury was estimated by plating an aliquot of diluted culture onto Violet Red Bile (VRB) agar and NA. The agars were incubated at 35 ± 1°C for 24 ± 2 hours and the colonies enumerated. The degree of injury was estimated as
Where nselect = number of colonies on selective agar and nnonselect = number of colonies on non-selective agar. For one food type of each category, two sets of plates were prepared from the samples. After incubation, one set of plates was placed at -15oC for one week and one set was enumerated immediately. After the frozen storage period, the plates were removed and enumerated. Additionally, from the set of plates stored at -15oC, up to 5 atypical colonies were picked and struck to non-selective agar to demonstrate that further identification from the plates is possible. 3.3.1 Calculation and Data Interpretation
For the Accuracy Profile calculations, the Excel® spreadsheet provided on the ISO website (http://standards.iso.org/iso/16140) was used.
100)1( xn
n
nonselect
select−
31/92
The accuracy profile was used to check the requirement that the alternative method produces a result for a sample that differs from the value produced by the reference method by less than a certain acceptability criterion. According to the study design, three different levels of contamination were examined. For each sample of every category, the statistical values were calculated in accordance of ISO 16140-2:2016, section 6.1.3.3.
The raw data is provided in Annex H. The statistical analysis and the accuracy profiles are displayed below in Figures 3A-3AL.
3.3.2 Conclusion of the Accuracy Profile Study
The data generated for each category meet the acceptance criteria outlined in ISO 16140-2:2016, section 6.1.3.3. Based on the statistical analysis, the alternative method is considered equivalent to the reference method when immediately enumerated after incubation and can be stored at -15°C for one week. For two food items, raw milk and environmental surface sponges, 1 sample replicate was identified as an outlier causing the AP to fall outside the acceptable limits but within the standard deviation. Since the criteria for additional analysis was not met (sample within standard deviation) no additional statistical analysis was performed. Each of the two outliers just slightly falls outside the CI interval. When removing this single replicate, and replacing it with the means of the other four data points in the concentration level, the method’s performance falls within the acceptable CI’s.
32/92
Figure 3A: Accuracy Statistical Analysis of Raw Fresh Meats – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 0.000 -0.160 0.160 YES YES
1B 1.78 0.000 -0.160 0.160 YES YES
2A 2.78 -0.115 -0.276 0.045 YES YES
2B 2.76 -0.048 -0.209 0.112 YES YES
3A 4.81 0.057 -0.103 0.217 YES YES
3B 4.83 -0.033 -0.193 0.127 YES YES
Reference
method
Alternative
method
SD Repeatability 0.114 0.111 +/- 0.500
(Food) Category Meat Products
(Food) Type Raw Fresh Meats - Coliform 37
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Fresh Meats - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.000 -0.189 0.189 YES YES
1B 1.60 0.000 -0.189 0.189 YES YES
2A 2.41 -0.073 -0.262 0.117 YES YES
2B 2.46 0.000 -0.189 0.189 YES YES
3A 4.58 0.011 -0.178 0.200 YES YES
3B 4.64 -0.138 -0.327 0.051 YES YES
Reference
method
Alternative
method
SD Repeatability 0.124 0.131 +/- 0.500
(Food) Category
(Food) Type
Meat Products
Raw Fresh Meats - E. coli 37 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Fresh Meats - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
33/92
Figure 3B: Accuracy Statistical Analysis of Raw Fresh Meats – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.000 -0.188 0.188 YES YES
1B 1.60 0.000 -0.188 0.188 YES YES
2A 2.41 -0.035 -0.222 0.153 YES YES
2B 2.46 0.015 -0.173 0.202 YES YES
3A 4.58 0.033 -0.155 0.221 YES YES
3B 4.64 -0.087 -0.275 0.101 YES YES
Reference
method
Alternative
method
SD Repeatability 0.124 0.130 +/- 0.500
(Food) Category
(Food) Type
Meat Products
Raw Fresh Meats - E. coli 42 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Fresh Meats - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
34/92
Figure 3C: Accuracy Statistical Analysis of Raw Fresh Meats of Frozen Plates – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 0.000 -0.147 0.147 YES YES
1B 1.78 0.000 -0.147 0.147 YES YES
2A 2.78 -0.097 -0.244 0.050 YES YES
2B 2.76 -0.008 -0.155 0.140 YES YES
3A 4.81 0.063 -0.084 0.210 YES YES
3B 4.83 -0.033 -0.180 0.114 YES YES
Reference
method
Alternative
method
SD Repeatability 0.114 0.102 +/- 0.500
(Food) Category Meat Products
(Food) Type Raw Fresh Meats - Coliform 37
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Fresh Meats - Coliform 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
35/92
Figure 3D: Accuracy Statistical Analysis of Raw Fresh Meats of Frozen Plates – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.279 0.085 YES YES
1B 1.60 0.097 -0.085 0.279 YES YES
2A 2.41 0.000 -0.182 0.182 YES YES
2B 2.46 0.015 -0.167 0.197 YES YES
3A 4.58 0.054 -0.128 0.236 YES YES
3B 4.64 -0.075 -0.257 0.107 YES YES
Reference
method
Alternative
method
SD Repeatability 0.124 0.126 +/- 0.500
(Food) Category Meat Products
(Food) Type Raw Fresh Meats - E. coli 42 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Fresh Meats - E. coli 42 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.277 0.084 YES YES
1B 1.60 0.097 -0.084 0.277 YES YES
2A 2.41 0.000 -0.180 0.180 YES YES
2B 2.46 0.015 -0.166 0.195 YES YES
3A 4.58 0.054 -0.127 0.234 YES YES
3B 4.64 -0.075 -0.256 0.105 YES YES
Reference
method
Alternative
method
SD Repeatability 0.124 0.125 +/- 0.500
(Food) Category
(Food) Type
Meat Products
Raw Fresh Meats - E. coli 37 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Fresh Meats - E. coli 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
36/92
Figure 3E: Accuracy Statistical Analysis of Breaded Meats – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 -0.058 -0.269 0.153 YES YES
1B 1.85 0.058 -0.153 0.269 YES YES
2A 2.90 -0.104 -0.315 0.107 YES YES
2B 2.92 0.035 -0.176 0.246 YES YES
3A 4.83 0.081 -0.129 0.292 YES YES
3B 4.87 0.012 -0.199 0.222 YES YES
Reference
method
Alternative
method
SD Repeatability 0.083 0.146 +/- 0.500
Meat Products(Food) Category
SD repeatability of reference
method <= 0.125
YES
(Food) Type Breaded Meats - Coliform 37 ±
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Breaded Meats - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.264 0.071 YES YES
1B 1.70 -0.097 -0.264 0.071 YES YES
2A 2.59 -0.060 -0.227 0.108 YES YES
2B 2.61 -0.022 -0.189 0.146 YES YES
3A 4.70 -0.131 -0.298 0.037 YES YES
3B 4.66 0.053 -0.114 0.221 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.116 +/- 0.500
(Food) Type
Meat Products
Breaded Meats - E. coli 37 ±
(Food) Category
YES
SD repeatability of reference
method <= 0.125Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Breaded Meats - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
37/92
Figure 3F: Accuracy Statistical Analysis of Breaded Meats – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.276 0.082 YES YES
1B 1.70 0.000 -0.179 0.179 YES YES
2A 2.59 -0.035 -0.214 0.144 YES YES
2B 2.61 0.010 -0.169 0.189 YES YES
3A 4.70 -0.108 -0.287 0.071 YES YES
3B 4.66 0.062 -0.117 0.241 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.124 +/- 0.500
(Food) Type
Meat Products
Breaded Meats - E. coli 42 ±
(Food) Category
YES
SD repeatability of reference
method <= 0.125Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
sReference Median
Breaded Meats - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
38/92
Figure 3G: Accuracy Statistical Analysis of Raw Dairy Products – 30 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 2.80 -0.021 -0.177 0.135 YES YES
1B 2.90 -0.040 -0.196 0.116 YES YES
2A 3.66 0.062 -0.094 0.217 YES YES
2B 3.76 -0.056 -0.212 0.100 YES YES
3A 4.41 0.000 -0.156 0.156 YES YES
3B 4.45 -0.032 -0.188 0.124 YES YES
Reference
method
Alternative
method
SD Repeatability 0.095 0.108 +/- 0.500YES
SD repeatability of reference
method <= 0.125Final AL
Dairy and Egg Products
Raw Dairy Products - Coliform
(Food) Category
(Food) Type
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Dairy Products - Coliform 30 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 2.66 0.027 -0.205 0.260 YES YES
1B 2.70 0.017 -0.215 0.249 YES YES
2A 3.56 0.000 -0.232 0.232 YES YES
2B 3.53 0.000 -0.232 0.232 YES YES
3A 4.11 -0.269 -0.501 -0.036 NO NO
3B 4.04 0.163 -0.070 0.395 YES YES
Reference
method
Alternative
method
SD Repeatability 0.091 0.161 +/- 0.500
(Food) Category Dairy and Egg Products
(Food) Type Raw Dairy Products - E. coli 30
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Dairy Products - E. coli 30 ± 1°C
Bias
β-ETI
AL = +/- 0.5
39/92
Figure 3H: Accuracy Statistical Analysis of Raw Dairy Products of Frozen Plates – 30 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 2.80 0.000 -0.140 0.140 YES YES
1B 2.90 0.005 -0.135 0.145 YES YES
2A 3.66 0.009 -0.131 0.149 YES YES
2B 3.76 -0.015 -0.155 0.125 YES YES
3A 4.41 0.000 -0.140 0.140 YES YES
3B 4.45 0.015 -0.125 0.155 YES YES
Reference
method
Alternative
method
SD Repeatability 0.095 0.097 +/- 0.500
Dairy and Egg Products(Food) Category
SD repeatability of reference
method <= 0.125
YES
(Food) Type Raw Dairy Products - Coliform
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Dairy Products - Coliform 30 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 2.66 -0.040 -0.234 0.155 YES YES
1B 2.70 -0.056 -0.250 0.139 YES YES
2A 3.56 -0.038 -0.233 0.157 YES YES
2B 3.53 0.025 -0.170 0.220 YES YES
3A 4.11 -0.269 -0.464 -0.074 YES YES
3B 4.04 -0.228 -0.423 -0.034 YES YES
Reference
method
Alternative
method
SD Repeatability 0.092 0.135 +/- 0.500
(Food) Type
Dairy and Egg Products
Raw Dairy Products - E. coli 30
(Food) Category
YES
SD repeatability of reference
method <= 0.125Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00
Bia
s
Reference Median
Raw Dairy Products - E. coli 30 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
40/92
Figure 3I: Accuracy Statistical Analysis of Egg Products – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 -0.125 -0.294 0.044 YES YES
1B 1.78 0.125 -0.044 0.294 YES YES
2A 2.77 0.014 -0.154 0.183 YES YES
2B 2.83 -0.027 -0.196 0.142 YES YES
3A 4.89 0.022 -0.147 0.191 YES YES
3B 4.89 0.022 -0.147 0.191 YES YES
Reference
method
Alternative
method
(Food) Type
(Food) Category Dairy and Egg Products
Final AL
Egg Products - Coliform 37 ±
SD repeatability of reference
method <= 0.125
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Egg Products - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.218 0.024 YES YES
1B 1.60 0.097 -0.024 0.218 YES YES
2A 2.61 0.120 -0.002 0.241 YES YES
2B 2.66 -0.040 -0.161 0.082 YES YES
3A 4.69 -0.077 -0.199 0.044 YES YES
3B 4.73 -0.201 -0.322 -0.080 YES YES
Reference
method
Alternative
method
SD Repeatability 0.088 0.084 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
(Food) Category Dairy and Egg Products
Final AL
(Food) Type Egg Products - E. coli 37 ± 1°C
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Egg Products - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
41/92
Figure 3J: Accuracy Statistical Analysis of Egg Products – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.000 -0.118 0.118 YES YES
1B 1.60 0.097 -0.021 0.215 YES YES
2A 2.61 0.111 -0.007 0.230 YES YES
2B 2.66 0.000 -0.118 0.118 YES YES
3A 4.69 -0.057 -0.175 0.062 YES YES
3B 4.73 0.016 -0.103 0.134 YES YES
Reference
method
Alternative
method
SD Repeatability 0.088 0.082 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
(Food) Category Dairy and Egg Products
Final AL
(Food) Type Egg Products - E. coli 42 ± 1°C
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
sReference Median
Egg Products - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
42/92
Figure 3K: Accuracy Statistical Analysis of Raw Fish and Seafood Products – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 0.155 0.037 0.273 YES YES
1B 1.95 0.000 -0.118 0.118 YES YES
2A 2.90 -0.022 -0.141 0.096 YES YES
2B 2.91 0.036 -0.082 0.154 YES YES
3A 4.85 -0.006 -0.125 0.112 YES YES
3B 4.90 -0.034 -0.152 0.085 YES YES
Reference
method
Alternative
method
SD Repeatability 0.083 0.082 +/- 0.500
(Food) Category Fish And Seafood Products
(Food) Type Raw Products - Coliform 37 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Products - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.247 0.053 YES YES
1B 1.70 0.000 -0.150 0.150 YES YES
2A 2.56 0.000 -0.150 0.150 YES YES
2B 2.64 -0.064 -0.214 0.086 YES YES
3A 4.58 -0.048 -0.198 0.102 YES YES
3B 4.58 -0.012 -0.162 0.139 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.104 +/- 0.500
(Food) Category
(Food) Type
Fish And Seafood Products
Raw Products - E. coli 37 ± 1°C
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Products - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
43/92
Figure 3L: Accuracy Statistical Analysis of Raw Fish and Seafood Products – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.000 -0.326 0.326 YES YES
1B 1.70 0.000 -0.326 0.326 YES YES
2A 2.56 0.023 -0.303 0.350 YES YES
2B 2.64 -0.041 -0.368 0.285 YES YES
3A 4.58 -0.023 -0.350 0.303 YES YES
3B 4.58 0.011 -0.315 0.338 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.226 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
Final AL
(Food) Category
(Food) Type
Fish And Seafood Products
Raw Products - E. coli 42 ± 1°C
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Products - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
44/92
Figure 3M: Accuracy Statistical Analysis of Raw Fish and Seafood Products of Frozen Plates– 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 0.109 0.005 0.213 YES YES
1B 1.95 -0.051 -0.155 0.053 YES YES
2A 2.90 -0.005 -0.109 0.098 YES YES
2B 2.91 0.011 -0.093 0.115 YES YES
3A 4.85 -0.013 -0.117 0.091 YES YES
3B 4.90 0.011 -0.093 0.115 YES YES
Reference
method
Alternative
method
SD Repeatability 0.083 0.072 +/- 0.500YES
SD repeatability of reference
method <= 0.125Final AL
Fish And Seafood Products
Raw Products - Coliform 37 ±
(Food) Category
(Food) Type
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Products - Coliform 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.000 -0.110 0.110 YES YES
1B 1.70 0.000 -0.110 0.110 YES YES
2A 2.56 -0.038 -0.148 0.072 YES YES
2B 2.64 -0.075 -0.185 0.034 YES YES
3A 4.58 -0.048 -0.158 0.061 YES YES
3B 4.58 -0.023 -0.133 0.086 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.076 +/- 0.500
(Food) Category Fish And Seafood Products
(Food) Type Raw Products - E. coli 37 ± 1°C
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00
Bia
s
Reference Median
Raw Products - E. coli 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
45/92
Figure 3N: Accuracy Statistical Analysis of Raw Fish and Seafood Products of Frozen Plates– 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.079 -0.036 0.195 YES YES
1B 1.70 0.079 -0.036 0.195 YES YES
2A 2.56 0.067 -0.049 0.182 YES YES
2B 2.64 -0.052 -0.168 0.063 YES YES
3A 4.58 -0.012 -0.127 0.104 YES YES
3B 4.58 0.011 -0.104 0.127 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.080 +/- 0.500
(Food) Category
(Food) Type
Fish And Seafood Products
Raw Products - E. coli 42 ± 1°C
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Products - E. coli 42 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
46/92
Figure 3O: Accuracy Statistical Analysis of Smoked Fish and Seafood Products – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 -0.058 -0.189 0.073 YES YES
1B 1.90 0.000 -0.131 0.131 YES YES
2A 2.84 0.085 -0.046 0.217 YES YES
2B 2.89 -0.006 -0.137 0.126 YES YES
3A 4.88 -0.042 -0.173 0.089 YES YES
3B 4.78 0.014 -0.117 0.146 YES YES
Reference
method
Alternative
method
SD Repeatability 0.083 0.091 +/- 0.500
Fish And Seafood Products(Food) Category
SD repeatability of reference
method <= 0.125
YES
(Food) Type Smoked Products - Coliform 37
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Smoked Products - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.241 0.047 YES YES
1B 1.70 -0.097 -0.241 0.047 YES YES
2A 2.56 -0.038 -0.182 0.107 YES YES
2B 2.48 0.000 -0.144 0.144 YES YES
3A 4.56 -0.094 -0.238 0.050 YES YES
3B 4.49 0.000 -0.144 0.144 YES YES
Reference
method
Alternative
method
SD Repeatability 0.085 0.100 +/- 0.500
(Food) Type
Fish And Seafood Products
Smoked Products - E. coli 37 ±
(Food) Category
YES
SD repeatability of reference
method <= 0.125Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Smoked Products - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
47/92
Figure 3P: Accuracy Statistical Analysis of Smoked Fish and Seafood Products – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.000 -0.144 0.144 YES YES
1B 1.70 -0.097 -0.241 0.047 YES YES
2A 2.56 0.000 -0.144 0.144 YES YES
2B 2.48 0.014 -0.130 0.159 YES YES
3A 4.56 -0.065 -0.209 0.079 YES YES
3B 4.49 0.027 -0.117 0.172 YES YES
Reference
method
Alternative
method
SD Repeatability 0.085 0.100 +/- 0.500
SD repeatability of reference
method <= 0.125Final AL
(Food) Category
YES
(Food) Type
Fish And Seafood Products
Smoked Products - E. coli 42 ±
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Smoked Products - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
48/92
3Q: Accuracy Statistical Analysis of Raw Vegetable Products – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 2.80 -0.007 -0.104 0.090 YES YES
1B 2.76 -0.008 -0.104 0.089 YES YES
2A 3.59 0.000 -0.097 0.097 YES YES
2B 3.61 -0.045 -0.141 0.052 YES YES
3A 4.45 -0.016 -0.113 0.081 YES YES
3B 4.45 -0.016 -0.113 0.081 YES YES
Reference
method
Alternative
method
SD Repeatability 0.054 0.067 +/- 0.500YES
SD repeatability of reference
method <= 0.125Final AL
Vegetable Products
Raw Vegetable Products -
(Food) Category
(Food) Type
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Vegetable Products - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 2.43 -0.070 -0.254 0.115 YES YES
1B 2.46 -0.047 -0.232 0.137 YES YES
2A 3.36 -0.061 -0.245 0.124 YES YES
2B 3.38 -0.176 -0.361 0.009 YES YES
3A 4.15 -0.105 -0.290 0.080 YES YES
3B 4.08 0.000 -0.185 0.185 YES YES
Reference
method
Alternative
method
SD Repeatability 0.088 0.128 +/- 0.500
(Food) Category Vegetable Products
(Food) Type Raw Vegetable Products - E.
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Raw Vegetable Products - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
49/92
3R: Accuracy Statistical Analysis of Raw Vegetable Products – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 2.43 -0.016 -0.162 0.129 YES YES
1B 2.46 -0.031 -0.177 0.115 YES YES
2A 3.36 -0.019 -0.165 0.127 YES YES
2B 3.38 -0.125 -0.271 0.021 YES YES
3A 4.15 -0.067 -0.213 0.079 YES YES
3B 4.08 0.035 -0.111 0.181 YES YES
Reference
method
Alternative
method
SD Repeatability 0.089 0.101 +/- 0.500
(Food) Category Vegetable Products
(Food) Type Raw Vegetable Products - E.
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
sReference Median
Raw Vegetable Products - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
50/92
Figure 3S: Accuracy Statistical Analysis of Heat Processed Vegetable Juices – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 0.051 -0.067 0.170 YES YES
1B 1.95 -0.109 -0.228 0.009 YES YES
2A 2.91 0.005 -0.113 0.124 YES YES
2B 2.92 -0.038 -0.156 0.081 YES YES
3A 4.91 0.016 -0.103 0.134 YES YES
3B 4.89 -0.006 -0.124 0.113 YES YES
Reference
method
Alternative
method
SD Repeatability 0.071 0.082 +/- 0.500YES
(Food) Type
(Food) Category Vegetable Products
Final AL
Heat Processed Vegetable
SD repeatability of reference
method <= 0.125
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Heat Processed Vegetable Juices - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 -0.176 -0.351 -0.001 YES YES
1B 1.70 -0.097 -0.272 0.078 YES YES
2A 2.72 0.000 -0.175 0.175 YES YES
2B 2.75 -0.135 -0.310 0.039 YES YES
3A 4.56 0.012 -0.163 0.187 YES YES
3B 4.56 0.023 -0.151 0.198 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.121 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
(Food) Category Vegetable Products
Final AL
(Food) Type Heat Processed Vegetable
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Heat Processed Vegetable Juices - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
51/92
Figure 3T: Accuracy Statistical Analysis of Heat Processed Vegetable Juices – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 -0.079 -0.252 0.094 YES YES
1B 1.70 0.079 -0.094 0.252 YES YES
2A 2.72 0.032 -0.141 0.205 YES YES
2B 2.75 -0.115 -0.288 0.059 YES YES
3A 4.56 0.023 -0.150 0.197 YES YES
3B 4.56 0.046 -0.127 0.219 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.120 +/- 0.500
(Food) Category Vegetable Products
Final AL
(Food) Type Heat Processed Vegetable
SD repeatability of reference
method <= 0.125
YES
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Heat Processed Vegetable Juices - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
52/92
Figure 3U: Accuracy Statistical Analysis of Heat Processed Vegetable Juices of Frozen Plates– 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 0.000 -0.104 0.104 YES YES
1B 1.95 -0.051 -0.155 0.053 YES YES
2A 2.91 -0.005 -0.109 0.099 YES YES
2B 2.92 0.010 -0.094 0.114 YES YES
3A 4.91 0.016 -0.088 0.120 YES YES
3B 4.89 -0.023 -0.127 0.081 YES YES
Reference
method
Alternative
method
SD Repeatability 0.071 0.072 +/- 0.500YES
(Food) Type
(Food) Category Vegetable Products
Final AL
Heat Processed Vegetable
SD repeatability of reference
method <= 0.125
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Heat Processed Vegetable Juices - Coliform 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 -0.079 -0.209 0.051 YES YES
1B 1.70 -0.097 -0.227 0.033 YES YES
2A 2.72 -0.017 -0.147 0.113 YES YES
2B 2.75 -0.049 -0.179 0.081 YES YES
3A 4.56 -0.025 -0.155 0.105 YES YES
3B 4.56 0.012 -0.118 0.142 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.090 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
(Food) Category Vegetable Products
Final AL
(Food) Type Heat Processed Vegetable
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Heat Processed Vegetable Juices - E. coli 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
53/92
Figure 3V: Accuracy Statistical Analysis of Heat Processed Vegetable Juices of Frozen Plates– 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 0.000 -0.115 0.115 YES YES
1B 1.70 0.079 -0.036 0.195 YES YES
2A 2.72 0.016 -0.099 0.132 YES YES
2B 2.75 -0.024 -0.139 0.092 YES YES
3A 4.56 0.023 -0.092 0.139 YES YES
3B 4.56 0.035 -0.081 0.150 YES YES
Reference
method
Alternative
method
SD Repeatability 0.101 0.080 +/- 0.500
Vegetable Products(Food) Category
SD repeatability of reference
method <= 0.125
YES
(Food) Type Heat Processed Vegetable
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Heat Processed Vegetable Juices - E. coli 42 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
54/92
Figure 3W: Accuracy Statistical Analysis of Cold Ready Meals – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 0.000 -0.101 0.101 YES YES
1B 1.90 0.000 -0.101 0.101 YES YES
2A 2.86 0.017 -0.084 0.119 YES YES
2B 2.90 -0.006 -0.107 0.096 YES YES
3A 4.87 -0.030 -0.131 0.071 YES YES
3B 4.89 -0.041 -0.142 0.060 YES YES
Reference
method
Alternative
method
SD Repeatability 0.068 0.070 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
Final AL
(Food) Category RTE/RTRH
(Food) Type Cold Ready Meals - Coliform 37
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Cold Ready Meals - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 -0.176 -0.320 -0.032 YES YES
1B 1.70 -0.097 -0.241 0.047 YES YES
2A 2.48 -0.015 -0.159 0.130 YES YES
2B 2.43 0.087 -0.057 0.232 YES YES
3A 4.56 -0.051 -0.196 0.093 YES YES
3B 4.60 -0.111 -0.255 0.034 YES YES
Reference
method
Alternative
method
SD Repeatability 0.111 0.100 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
Final AL
(Food) Type Cold Ready Meals - E. coli 37 ±
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Cold Ready Meals - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
55/92
Figure 3X: Accuracy Statistical Analysis of Cold Ready Meals – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.78 -0.079 -0.232 0.074 YES YES
1B 1.70 0.000 -0.153 0.153 YES YES
2A 2.48 0.028 -0.125 0.181 YES YES
2B 2.43 0.125 -0.028 0.278 YES YES
3A 4.56 0.000 -0.153 0.153 YES YES
3B 4.60 -0.071 -0.224 0.082 YES YES
Reference
method
Alternative
method
SD Repeatability 0.111 0.106 +/- 0.500
(Food) Category
(Food) Type
RTE/RTRH
Cold Ready Meals - E. coli 42 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
sReference Median
Cold Ready Meals - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
56/92
Figure 3Y: Accuracy Statistical Analysis of Mayonnaise based Deli Salad with Raw Ingredients – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 -0.058 -0.175 0.059 YES YES
1B 1.90 0.000 -0.117 0.117 YES YES
2A 2.93 -0.015 -0.132 0.102 YES YES
2B 2.95 -0.063 -0.180 0.054 YES YES
3A 4.87 -0.037 -0.154 0.080 YES YES
3B 4.86 0.000 -0.117 0.117 YES YES
Reference
method
Alternative
method
SD Repeatability 0.074 0.081 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
(Food) Type Mayonnaise based deli salads
Final AL
RTE/RTRH(Food) Category
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Mayonnaise based deli salads with raw ingredients -Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.000 -0.159 0.159 YES YES
1B 1.60 0.000 -0.159 0.159 YES YES
2A 2.66 -0.019 -0.178 0.139 YES YES
2B 2.68 -0.038 -0.197 0.121 YES YES
3A 4.48 -0.015 -0.174 0.144 YES YES
3B 4.51 -0.058 -0.217 0.101 YES YES
Reference
method
Alternative
method
SD Repeatability 0.123 0.110 +/- 0.500
SD repeatability of reference
method <= 0.125Final AL
(Food) Category
YES
(Food) Type
RTE/RTRH
Mayonnaise based deli salads
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Mayonnaise based deli salads with raw ingredients - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
57/92
Figure 3Z: Accuracy Statistical Analysis of Mayonnaise based Deli Salad with Raw Ingredients – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.000 -0.165 0.165 YES YES
1B 1.60 0.000 -0.165 0.165 YES YES
2A 2.66 0.018 -0.146 0.183 YES YES
2B 2.68 -0.018 -0.183 0.146 YES YES
3A 4.48 0.000 -0.165 0.165 YES YES
3B 4.51 -0.074 -0.238 0.091 YES YES
Reference
method
Alternative
method
SD Repeatability 0.123 0.114 +/- 0.500
(Food) Type
RTE/RTRH
Mayonnaise based deli salads
(Food) Category
YES
SD repeatability of reference
method <= 0.125Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Mayonnaise based deli salads with raw ingredients - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
58/92
Figure 3AA: Accuracy Statistical Analysis of Mayonnaise based Deli Salad with Raw Ingredients of Frozen Plates– 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 0.000 -0.098 0.098 YES YES
1B 1.90 -0.058 -0.156 0.040 YES YES
2A 2.93 -0.010 -0.108 0.088 YES YES
2B 2.95 -0.010 -0.108 0.088 YES YES
3A 4.87 -0.018 -0.116 0.080 YES YES
3B 4.86 0.006 -0.092 0.104 YES YES
Reference
method
Alternative
method
SD Repeatability 0.074 0.068 +/- 0.500
(Food) Category RTE/RTRH
(Food) Type Mayonnaise based deli salads
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Mayonnaise based deli salads with raw ingredients -Coliform 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.000 -0.169 0.169 YES YES
1B 1.60 0.000 -0.169 0.169 YES YES
2A 2.66 -0.019 -0.188 0.150 YES YES
2B 2.68 -0.048 -0.217 0.121 YES YES
3A 4.48 -0.046 -0.215 0.123 YES YES
3B 4.51 -0.090 -0.259 0.079 YES YES
Reference
method
Alternative
method
SD Repeatability 0.123 0.117 +/- 0.500
(Food) Category
(Food) Type
RTE/RTRH
Mayonnaise based deli salads
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Mayonnaise based deli salads with raw ingredients - E. coli 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
59/92
Figure 3AB: Accuracy Statistical Analysis of Mayonnaise based Deli Salad with Raw Ingredients of Frozen Plates– 42 ± 1°C 3M
Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.097 -0.058 0.251 YES YES
1B 1.60 0.176 0.022 0.331 YES YES
2A 2.66 0.027 -0.127 0.182 YES YES
2B 2.68 0.009 -0.146 0.163 YES YES
3A 4.48 0.000 -0.154 0.154 YES YES
3B 4.51 -0.028 -0.182 0.126 YES YES
Reference
method
Alternative
method
SD Repeatability 0.123 0.107 +/- 0.500
(Food) Type
RTE/RTRH
Mayonnaise based deli salads
(Food) Category
YES
SD repeatability of reference
method <= 0.125Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Mayonnaise based deli salads with raw ingredients - E. coli 42 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
60/92
Figure 3AC: Accuracy Statistical Analysis of Finished Products for Livestock – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.90 0.000 -0.101 0.101 YES YES
1B 1.85 0.058 -0.043 0.159 YES YES
2A 2.91 0.011 -0.090 0.112 YES YES
2B 2.94 -0.053 -0.154 0.048 YES YES
3A 4.91 0.026 -0.075 0.127 YES YES
3B 4.89 0.053 -0.048 0.154 YES YES
Reference
method
Alternative
method
SD Repeatability 0.069 0.070 +/- 0.500
Animal Feed
Finished Products for Livestock -
(Food) Category
(Food) Type
SD repeatability of reference
method <= 0.125Final AL
YES
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Finished Products for Livestock - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 -0.243 -0.353 -0.133 YES YES
1B 1.78 -0.176 -0.286 -0.066 YES YES
2A 2.71 -0.017 -0.127 0.092 YES YES
2B 2.69 0.026 -0.084 0.136 YES YES
3A 4.59 -0.100 -0.209 0.010 YES YES
3B 4.61 -0.094 -0.204 0.015 YES YES
Reference
method
Alternative
method
SD Repeatability 0.108 0.076 +/- 0.500
(Food) Category Animal Feed
(Food) Type Finished Products for Livestock -
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Finished Products for Livestock - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
61/92
Figure 3AD: Accuracy Statistical Analysis of Finished Products for Livestock – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 -0.243 -0.376 -0.110 YES YES
1B 1.78 -0.176 -0.309 -0.043 YES YES
2A 2.71 0.000 -0.133 0.133 YES YES
2B 2.69 0.042 -0.091 0.175 YES YES
3A 4.59 -0.060 -0.192 0.073 YES YES
3B 4.61 0.000 -0.133 0.133 YES YES
Reference
method
Alternative
method
SD Repeatability 0.108 0.092 +/- 0.500
(Food) Category Animal Feed
(Food) Type Finished Products for Livestock -
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Finished Products for Livestock - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
62/92
Figure 3AE: Accuracy Statistical Analysis of Plant Origin Ingredients – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 0.000 -0.127 0.127 YES YES
1B 1.90 0.000 -0.127 0.127 YES YES
2A 2.92 -0.038 -0.165 0.089 YES YES
2B 2.91 0.016 -0.111 0.143 YES YES
3A 4.90 -0.059 -0.186 0.068 YES YES
3B 4.88 0.006 -0.121 0.133 YES YES
Reference
method
Alternative
method
SD Repeatability 0.081 0.088 +/- 0.500
(Food) Category Animal Feed
Final AL
Plant Origin Ingredients -
SD repeatability of reference
method <= 0.125
YES
(Food) Type
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Plant Origin Ingredients - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.000 -0.114 0.114 YES YES
1B 1.78 -0.176 -0.290 -0.062 YES YES
2A 2.71 -0.064 -0.178 0.050 YES YES
2B 2.69 0.009 -0.105 0.123 YES YES
3A 4.62 -0.092 -0.206 0.022 YES YES
3B 4.58 -0.075 -0.189 0.039 YES YES
Reference
method
Alternative
method
SD Repeatability 0.115 0.079 +/- 0.500
(Food) Category Animal Feed
Final AL
(Food) Type Plant Origin Ingredients - E. coli
SD repeatability of reference
method <= 0.125
YES
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Plant Origin Ingredients - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
63/92
Figure 3AF: Accuracy Statistical Analysis of Plant Origin Ingredients – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.000 -0.133 0.133 YES YES
1B 1.78 -0.176 -0.309 -0.043 YES YES
2A 2.71 -0.017 -0.150 0.115 YES YES
2B 2.69 0.026 -0.107 0.159 YES YES
3A 4.62 -0.043 -0.176 0.089 YES YES
3B 4.58 -0.048 -0.181 0.085 YES YES
Reference
method
Alternative
method
SD Repeatability 0.115 0.092 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
(Food) Category Animal Feed
Final AL
(Food) Type Plant Origin Ingredients - E. coli
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
sReference Median
Plant Origin Ingredients - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
64/92
Figure 3AG: Accuracy Statistical Analysis of Plant Origin Ingredients of Frozen Plates – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 0.058 -0.046 0.162 YES YES
1B 1.90 -0.058 -0.162 0.046 YES YES
2A 2.92 -0.011 -0.115 0.093 YES YES
2B 2.91 0.005 -0.099 0.109 YES YES
3A 4.90 -0.011 -0.115 0.093 YES YES
3B 4.88 0.000 -0.104 0.104 YES YES
Reference
method
Alternative
method
SD Repeatability 0.081 0.072 +/- 0.500
Animal Feed(Food) Category
SD repeatability of reference
method <= 0.125
YES
(Food) Type Plant Origin Ingredients -
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Plant Origin Ingredients - Coliform 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.000 -0.152 0.152 YES YES
1B 1.78 -0.079 -0.231 0.072 YES YES
2A 2.71 -0.026 -0.178 0.125 YES YES
2B 2.69 -0.027 -0.179 0.124 YES YES
3A 4.62 -0.105 -0.256 0.047 YES YES
3B 4.58 -0.075 -0.226 0.077 YES YES
Reference
method
Alternative
method
SD Repeatability 0.115 0.105 +/- 0.500
(Food) Type
Animal Feed
Plant Origin Ingredients - E. coli
(Food) Category
YES
SD repeatability of reference
method <= 0.125Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Plant Origin Ingredients - E. coli 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
65/92
Figure 3AH: Accuracy Statistical Analysis of Plant Origin Ingredients of Frozen Plates – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.60 0.097 -0.053 0.247 YES YES
1B 1.78 0.000 -0.150 0.150 YES YES
2A 2.71 0.017 -0.133 0.167 YES YES
2B 2.69 0.009 -0.141 0.159 YES YES
3A 4.62 -0.032 -0.182 0.118 YES YES
3B 4.58 -0.048 -0.198 0.102 YES YES
Reference
method
Alternative
method
SD Repeatability 0.115 0.104 +/- 0.500YES
SD repeatability of reference
method <= 0.125Final AL
Animal Feed
Plant Origin Ingredients - E. coli
(Food) Category
(Food) Type
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Plant Origin Ingredients - E. coli 42 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
66/92
3AI: Accuracy Statistical Analysis of Surface Samples – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 0.000 -0.195 0.195 YES YES
1B 2.72 -0.026 -0.221 0.169 YES YES
2A 2.92 -0.027 -0.222 0.168 YES YES
2B 3.62 -0.043 -0.238 0.151 YES YES
3A 4.46 -0.161 -0.356 0.034 YES YES
3B 4.86 -0.072 -0.267 0.123 YES YES
Reference
method
Alternative
method
SD Repeatability 0.080 0.135 +/- 0.500
SD repeatability of reference
method <= 0.125
YES
Final AL
(Food) Category
(Food) Type
Samples from the Industrial
Surface Samples - Coliform 37 ±
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Surface Samples - Coliform 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.332 0.138 YES YES
1B 2.36 -0.248 -0.483 -0.012 YES YES
2A 2.56 -0.065 -0.300 0.170 YES YES
2B 2.88 0.265 0.030 0.501 NO NO
3A 4.00 0.150 -0.085 0.386 YES YES
3B 4.49 -0.111 -0.346 0.124 YES YES
Reference
method
Alternative
method
SD Repeatability 0.093 0.163 +/- 0.500
SD repeatability of reference
method <= 0.125Final AL
(Food) Category
YES
(Food) Type
Samples from the Industrial
Surface Samples - E. coli 37 ±
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Surface Samples - E. coli 37 ± 1°C
Bias
β-ETI
AL = +/- 0.5
67/92
3AJ: Accuracy Statistical Analysis of Surface Samples – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.324 0.130 YES YES
1B 2.36 -0.158 -0.384 0.069 YES YES
2A 2.56 -0.025 -0.251 0.202 YES YES
2B 2.91 -0.096 -0.322 0.131 YES YES
3A 3.99 0.155 -0.072 0.382 YES YES
3B 4.49 -0.044 -0.271 0.182 YES YES
Reference
method
Alternative
method
SD Repeatability 0.085 0.157 +/- 0.500
(Food) Category Samples from the Industrial
(Food) Type Surface Samples - E. coli 42 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
sReference Median
Surface Samples - E. coli 42 ± 1°C
Bias
β-ETI
AL = +/- 0.5
68/92
Figure 3AK: Accuracy Statistical Analysis of Surface Samples of Frozen Plates – 37 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
é
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.85 0.000 -0.105 0.105 YES YES
1B 2.72 0.008 -0.097 0.114 YES YES
2A 2.92 -0.061 -0.166 0.044 YES YES
2B 3.62 -0.010 -0.116 0.095 YES YES
3A 4.46 -0.082 -0.188 0.023 YES YES
3B 4.86 -0.018 -0.124 0.087 YES YES
Reference
method
Alternative
method
SD Repeatability 0.080 0.073 +/- 0.500
Samples from the Industrial
Surface Samples - Coliform 37 ±
(Food) Category
(Food) Type
SD repeatability of reference
method <= 0.125Final AL
YES
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Surface Samples - Coliform 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 -0.097 -0.221 0.027 YES YES
1B 2.36 -0.186 -0.310 -0.061 YES YES
2A 2.56 -0.094 -0.218 0.030 YES YES
2B 2.88 -0.103 -0.227 0.021 YES YES
3A 4.00 -0.217 -0.342 -0.093 YES YES
3B 4.49 -0.130 -0.254 -0.005 YES YES
Reference
method
Alternative
method
SD Repeatability 0.093 0.086 +/- 0.500
(Food) Category Samples from the Industrial
(Food) Type Surface Samples - E. coli 37 ±
SD repeatability of reference
method <= 0.125
YES
Final AL
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Surface Samples - E. coli 37 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
69/92
Figure 3AL: Accuracy Statistical Analysis of Surface Samples of Frozen Plates – 42 ± 1°C 3M Petrifilm Rapid E. coli / Coliform Count Plate
Sample NameReference
Central valueBias Lower β-ETI Upper β-ETI
β-ETI
compared to
AL=±0.5
Acceptable
β-ETI
compared to
final AL
Acceptable
1A 1.70 0.000 -0.175 0.175 YES YES
1B 2.36 -0.040 -0.214 0.135 YES YES
2A 2.56 -0.025 -0.200 0.150 YES YES
2B 2.91 0.296 0.121 0.470 YES YES
3A 3.99 -0.146 -0.321 0.029 YES YES
3B 4.49 -0.029 -0.204 0.146 YES YES
Reference
method
Alternative
method
SD Repeatability 0.085 0.121 +/- 0.500
SD repeatability of reference
method <= 0.125Final AL
(Food) Category
YES
(Food) Type
Samples from the Industrial
Surface Samples - E. coli 42 ±
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Bia
s
Reference Median
Surface Samples - E. coli 42 ± 1°C (Frozen Plates)
Bias
β-ETI
AL = +/- 0.5
70/92
3.4 Inclusivity and Inclusivity and Exclusivity Study
The inclusivity study involved pure target strains to be detected by the alternative method. The exclusivity study involved pure non-target strains, which can be potentially cross-reactive, but are not expected to be detected by the alternative method. See Table 6-9 for the list of strains. 50 target strains of coliform and E. coli and 30 non-target strains of coliform and E. coli were tested. All isolates were enriched in non-selective broth for 18-24 hours at 37 ± 1ºC. Ten fold serial dilutions were conducted in order to achieve a final concentration of ~50 cells/mL. A 1.0 mL aliquot from this dilution was plated onto three sets of plates. Individual plates were incubated for 18 hours at 30 ± 1ºC, 37 ± 1ºC and 42 ± 1ºC.
Table 6: E. coli Inclusivity Strains
Number Genus Species Source Origin
1 Escherichia coli ATCC1 8739 Feces
2 Escherichia coli NBRC2 15034 Clinical Specimen
3 Escherichia coli ATCC 25922 Not Available
4 Escherichia coli ATCC 11229 feces
5 Escherichia coli ATCC 10536 Not Available
6 Escherichia coli ATCC 11303 Not Available
7 Escherichia coli ATCC 11775 food, Minnesota
8 Escherichia coli Q Labs 11010-2 Bottled Water
9 Escherichia coli ATCC 14948 Not Available
10 Escherichia coli ATCC 23848 Not Available
11 Escherichia coli ATCC 33849 Not Available
12 Escherichia coli ATCC 35218 canine, Tennessee
13 Escherichia coli ATCC 35421 Not Available
14 Escherichia coli Q Labs 41411.1 Bottled water
15 Escherichia coli Q Labs 41411.2 Bottled water
16 Escherichia coli Q Labs16154-1 Beef Carcass Sponge
17 Escherichia coli Q Labs 16154-2 Raw Ground Beef
18 Escherichia coli Q Labs 16154-3 Beef Trim
19 Escherichia coli Q Labs 16154-5 Flavored Seasoning
20 Escherichia coli Q Labs 16154-7 Flour
21 Escherichia coli Q Labs 16154-12 Chicken Carcass Rinse
22 Escherichia coli Q Labs 16154-13 Potable Water
23 Escherichia coli Q Labs 16154-15 Raw Chicken Breast
24 Escherichia coli Q Labs 16160-2 Mixed Frozen Vegetables
25 Escherichia coli Q Labs 16160-3 Frozen Greens
26 Escherichia coli Q Labs 16160-4 Chicken Nuggets
27 Escherichia coli Q Labs 16160-6 Flavored Seasoning
28 Escherichia coli Q Labs16160-8 Chicken Carcass Rinse
29 Escherichia coli Q Labs 16160-9 Fresh Baby Spinach
30 Escherichia coli Q Labs 16160-10 Raw Pork
31 Escherichia coli Q Labs 16160-11 Raw Beef Trimmings
32 Escherichia coli Q Labs 16160-13 Irrigation Rinse Water
33 Escherichia coli Q Labs 16160-16 Sprouts
34 Escherichia coli Q Labs 16160-17 Fresh Spinach
35 Escherichia coli Q Labs 16160-18 Frozen Mixed Vegetables
36 Escherichia coli 3M4 REC1 TBP5
37 Escherichia coli 3M REC5 TBP
71/92
Number Genus Species Source Origin
38 Escherichia coli 3M REC7 TBP
39 Escherichia coli 3M REC8 TBP
40 Escherichia coli 3M REC9 TBP
41 Escherichia coli 3M REC10 TBP
42 Escherichia coli 3M REC11 TBP
43 Escherichia coli 3M REC12 TBP
44 Escherichia coli 3M REC13 TBP
45 Escherichia coli 3M REC14 TBP
46 Escherichia coli 3M REC15 TBP
47 Escherichia coli 3M REC16 TBP
48 Escherichia coli 3M REC17 TBP
49 Escherichia coli 3M REC18 TBP
50 Escherichia coli 3M REC19 TBP 1ATCC-American Type Culture Collection, 2NBRC-Nite Biological Resource Center, 3QL- Q Laboratories Culture Collection, 43M – 3M Food Safety, 5. TBP- To be provided by 3M Food Safety
Table 7: E. coli Exclusivity Strains
Number Genus Species Source Origin
1 Acinetobacter baumanii ATCC1 19606 Urine
2 Aeromonas viridans QL2 17041-8 Raw Milk Isolate
3 Alcaligenes faecalis ATCC 8750 Not Available
4 Bordetella bronchiseptica ATCC 10580 Lung of dog
5 Brochothrix thermosphacta ATCC 11509 Animal-derived foodstuff
6 Carnobacterium maltaromaticum ATCC 43224 Vacuum Packed Beef
7 Citrobacter freundii QL 100813-2A Deli Meat
8 Citrobacter farmeri ATCC 51613 Clinical Isolate
9 Cronobacter condimenti QL 17031.1 Infant Formula
10 Cronobacter sakazakii QL 11007-9 Rice Flour
11 Enterococcus faecium ATCC 516 Clinical isolate
12 Haemophilus influenzae ATCC 19418 Not Available
13 Hafnia alvei ATCC 51815 Milk
14 Kurthia gibsonii ATCC 43195 RTE meat
15 Klebsiella oxytoca ATCC 43165 Clinical
16 Klebsiella pneumoniae QL 1100-7 Raw meat
17 Kluyvera Intermedia ATCC 33110 Surface Water
18 Microbacterium testaceum ATCC 15829 Paddy
19 Morganella morganii ATCC 25829 Human isolate
20 Pantoea agglomerans ATCC 19552 sewage
21 Proteus mirabilis QL 11007-6 Animal specimen
22 Providencia stuartii QL 11007-5 Clinical isolate
23 Pseudomonas extremorientialis QL 17041-1 Raw Milk Isolate
24 Pseudomonas fluorescens QL 17041-3 Raw Milk Isolate
25 Rahnella aquatilis QL 14181-2A Soy protein
26 Salmonella Typhimurium QL 011414-2 Raw material
27 Serratia marcescens QL 11007-1 Bottled water
28 Streptococcus pyogenes ATCC 19615 Pharynx of child following sore
throat.
29 Sphingomonas paucimobilis ATCC 29837 Hospital Respirator
30 Vibrio vulnificus QL 021111A Seafood Product 1ATCC-American Type Culture Collection, 2QL-Q Laboratories Culture Collection.
72/92
Table 8: Total Coliform Inclusivity Strains
Number Genus Species Source Origin
1 Citrobacter amalonaticus ATCC1 25405 feces
2 Citrobacter koseri ATCC 27156 Not Available
3 Citrobacter koseri ATCC BAA-895 clinical specimen - human (infant)
4 Citrobacter braakii ATCC 43162 clinical isolate, California
5 Citrobacter farmeri ATCC 51633 human feces, North Carolina
6 Citrobacter freundii Q Labs2 100813-2A sliced deli meat (turkey)
7 Citrobacter freundii Q Labs 11007-10 Clinical Hospital
8 Citrobacter freundii ATCC 43864 Not Available
9 Citrobacter youngae ATCC 11102 Not Available
10 Enterobacter aerogenes Q Labs 100813-1A Raw vegetable
11 Enterobacter aerogenes ATCC 13048 sputum
12 Enterobacter aerogenes ATCC 35029 Not Available
13 Enterobacter amnigenus ATCC 51816 milk, Minnesota
14 Enterobacter cancerogenus Q Labs 11010-2 Bottled Water
15 Enterobacter cancerogenus Q Labs 11010-1 Bottled Water
16 Enterobacter cloacae Q Labs 100813-3A Soy protein
17 Enterobacter cloacae ATCC 13047 Spinal fluid
18 Enterobacter cloacae NBRC3 13535 Spinal fluid
19 Enterobacter cloacae NBRC 13536 Not Available
20 Enterobacter gergoviae ATCC 33028 Urine, France
21 Hafnia alvei ATCC 51815 Milk
22 Klebsiella oxytoca ATCC 51817 Milk
23 Klebsiella oxytoca ATCC 11686 TBP
24 Klebsiella oxytoca NCTC4 49334 Not Available
25 Klebsiella oxytoca ATCC 700324 Not Available
26 Klebsiella pneumoniae ATCC 11296 Not Available
27 Klebsiella pneumoniae ATCC 4352 Dairy Products (Cow's Milk)
28 Klebsiella pneumoniae ATCC 13440 TBP5
29 Klebsiella pneumoniae Q Labs 11007-7 Raw hamburger
30 Kluyvera intermedia ATCC 33110 Surface Water
31 Pantoea agglomerans ATCC 19552 sewage
32 Rahnella aquatilis ATCC 55046 soil
33 Serratia liquefaciens ATCC 27592 Milk
34 Serratia marcescens Q Labs 11007-1 Bottled Water
35 Serratia marcescens ATCC 14756 Human Isolate
36 Serratia marcescens ATCC 13880 Human Isolate
37 Escherichia coli Q Labs 16160-1 Mixed Frozen Vegetables
38 Escherichia coli Q Labs 16160-2 Frozen Greens
39 Escherichia coli Q Labs 16160-3 Chicken Nuggets
40 Escherichia coli Q Labs 16160-4 Beef Carcass Sponge
41 Escherichia coli Q Labs 16160-5 Flavored Seasoning
42 Escherichia coli Q Labs 16160-6 Flavored Seasoning
43 Escherichia coli Q Labs 16160-7 Chicken Carcass Rinse
44 Escherichia coli Q Labs 16160-8 Fresh Baby Spinach
45 Escherichia coli Q Labs 16160-9 Raw Pork
46 Escherichia coli Q Labs 16160-10 Raw Beef Trimmings
47 Escherichia coli Q Labs 16160-11 Raw Beef Trimmings
48 Escherichia coli Q Labs 16160-12 Irrigation Rinse Water
49 Escherichia coli Q Labs 16160-13 Beef Carcass Sponge
50 Escherichia coli Q Labs 16160-14 Chicken Carcass Rinse
1 ATCC-American Type Culture Collection, 2QL- Q Laboratories Culture Collection, 3NBRC-Nite Biological Resource Center,
4NCTC-National Collection Type Cultures, 5TBP – To be provided by 3M
73/92
Table 9: Total Coliform Exclusivity Strains
Number Genus species Source Origin
1 Acinetobacter baumanii ATCC1 19606 Urine
2 Aeromonas viridans QL2 17041-8 Raw Milk Isolate
3 Alcaligenes faecalis ATCC 8750 Not Available
4 Bacillus cereus ATCC 6464 Soil
5 Bacillus subtilis ATCC 6633 Not Available
6 Bordetella bronchiseptica ATCC 10580 Lung of dog
7 Brochothrix thermosphacta ATCC 11509 Animal-derived foodstuff
8 Enterococcus durans ATCC 19432 Not Available
9 Enterococcus faecalis ATCC 29212 Urine
10 Enterococcus faecium ATCC 51559 Clinical isolate
11 Enterococcus hirae ATCC 8043 Not Available
12 Haemophilus influenzae ATCC 19418 Not Available
13 Kurthia gibsonii ATCC 43195 meat
14 Kurthia zopfii ATCC 10538 Not Available
15 Leuconostoc mesenteroides ATCC 8293 fermenting olives
16 Listeria innocua ATCC 33090 cow brain
17 Listeria ivanovii
ATCC BAA-139
washing water
18 Listeria monocytogenes ATCC 7644 Clinical Isolate
19 Listeria seeligeri ATCC 11289 Human feces
20 Listeria welshimeri ATCC 43549 soil
21 Micrococcus luteus ATCC 10240 air
22 Pseudomonas aeruginosa ATCC 27853 Clinical isolate
23 Pseudomonas aeruginosa ATCC 35554 Not Available
24 Pseudomonas fluorescens QL 17041-3 Raw Milk Isolate
25 Staphylococcus hominis ATCC 27844 Human skin
26 Staphylococcus aureus ATCC 6538 human lesion
27 Streptococcus pneumoniae ATCC 6302 Not Available
28 Streptococcus pyogenes ATCC 19615 Pharynx of child following sore
throat.
29 Vibrio parahaemolyticus ATCC 17802 Shirasu food poisoning
30 Vibrio vulnificus QL 021111A Seafood Product 31 Edwardsiella tarda ATCC 15947 Feces, human 32 Morganella morganii ATCC 25829 Human 33 Proteus hauseri ATCC 13315 Human Feces 34 Proteus vulgaris ATCC 6380 Clinical Isolate 35 Providencia rettgeri ATCC 14505 Not Available 36 Providencia stuartii QL 11007-5 Environmental Isolate
37 Salmonella Choleraeusis ATCC 53000
X-ray induced mutant of an equine isolate
38 Salmonella Newport ATCC 6962 food poisoning fatality
39 Salmonella Arizonae QL 11007-4 Veterinary Isolate 40 Yersinia enterocolitica ATCC 49397 clinical specimen
1ATCC-American Type Culture Collection, 2QL – Q Laboratories Culture Collection
Data Intrepreation for Inclusivity and Exclusivity Study
For the inclusivity and exclusivity study, all 100 inclusivity E. coli and total coliform organisms were correctly identified by the 3M Petrifilm Rapid E. coli / Coliform Count Plate method and all 70 exclusivity E. coli and total coliform organisms were correctly excluded by the 3M Petrifilm Rapid E. coli / Coliform Count Plate method, resulting in 100.00% Postive Agreement (PA) and a 0.00% Negative Deviation (ND).
74/92
The strains provided by 3M were strictly used in the Inclusivity portion of the study and not used in the development of the product.
4 Interlaboratory Study
The interlaboratory study (ILS) was organized and data evaluated according to the details provided in ISO 16140-2:2016.
4.1 Organization
The ILS was performed using 9 collaborators from 8 organizations representing 5 different countries. The food item used in the evaluation was dry dog kibble. The food item was obtained from a local retailer and screened for the presence of naturally occurring E. coli and coliform by the ISO 16649-2 and ISO 4832 reference methods. No natural contamination was observed so four separate levels of contamination were targeted for the evaluation: uninoculated, 0 CFU/g; low, 10-100 CFU/g; medium, 100-1,000 CFU/g; and high 1,000-10,000 CFU/g. To obtain the required contamination levels, bulk lots of the matrix were artificially contaminated with a lyophilized culture of Escherichia coli [Q Laboratories (QL) isolate 11007-8 (origin – beef hide)] and Klebsiella pneumonia [Q Laboratories (QL) isolate 11007-7 (origin – raw hamburger)] at each target contamination level. Both organisms were included in order to determine the target total coliform contamination level. Two replicate samples from each of the four contamination levels were analyzed by both the candidate and reference methods in a paired study design. Preparation of the Inocula and Test Portions The E. coli and K. pneumonia isolates used in this evaluation was lyophilized prior to inoculation. The cultures were first propagated onto Tryptic Soy Agar with 5% Sheep Blood (SBA) from a Q Laboratories frozen stock culture stored at -70°C. To prepare the cultures for lyophilization, a single, well isolated colony from SBA was transferred into BWP (ISO) and incubated at 37 ± 2 oC for 18-24 hours. The cultures were diluted in a sterile cryoprotectant, reconstituted 10% non-fat dry milk (NFDM), and freeze dried for 48-72 hours. A bulk lot of the test matrix was inoculated with each culture at a high level expected to yield all positive results. An aliquot of the high-level inoculum was further mixed with uninoculated powdered infant formula to produce the low-level inoculum. After inoculation, the matrix was held for a minimum of 2 weeks at ambient temperature (20 - 25oC). The inoculated test product was packaged into separate 25 g samples in sterile Whirl-Pak® bags and shipped to the collaborators.
Test Portion Distribution All samples were labeled with a randomized, blind-coded 3 digit number affixed to the sample container. Nine (9) participants from 8 separate locations participated. Test portions were shipped in leak-proof insulated containers via overnight delivery according to the Category B Dangerous Goods shipment regulations set forth by
75/92
International Air Transport Association (IATA). Test portions were shipped at ambient temperatures (20-25oC). Upon receipt, samples were held at ambient temperature until analysis was initiated. In addition to each of the test portions, collaborators received a test portion for the matrix labeled as Aerobic Plate Count (APC), to determine total background count in the matrix. The APC background screen samples were prepared from the bulk lot of test matrix, prior to inoculation. Additionally, a temperature probe was included in the shipment. Participants were instructed to submit the data from the temperature probe upon receipt of the shipment.
Table 6: Sample receipt data for ILS samples
Collaborator Temperature
(°C)
1 20.7
2 21.5
3 21.4
4 23.2
5 20.9
6 24.8
7 23.6
8 24.5
9 20.2
4.2 Calculations and interpretation of data The data from the collaborative trial were calculated and interpreted according to
section 6.2.3 of ISO 16140-2:2016 using the freely available Excel® spreadsheet
(http://standards.iso.org/iso/16140).
The log transformed data for the coliform and E. coli results of the alternative method
and reference method are presented in Tables 7 and 8, respectively. The Accuracy
profile graph is shown in Figure 9A and 9B.
76/92
Table 7: Summary of the results of the ILS: 3M Petrifilm REC- Coliform 37 ± 1oC
Collaborators Level Reference method Alternative method
ISO 4832 3M Petrifilm REC
1 Uninoculated <1.000 <1.000 <1.000 <1.000
2 Uninoculated <1.000 <1.000 <1.000 <1.000
3 Uninoculated <1.000 <1.000 <1.000 <1.000
4 Uninoculated <1.000 <1.000 <1.000 <1.000
5 Uninoculated <1.000 <1.000 <1.000 <1.000
6 Uninoculated <1.000 <1.000 <1.000 <1.000
7 Uninoculated <1.000 <1.000 <1.000 <1.000
8 Uninoculated <1.000 <1.000 <1.000 <1.000
9 Uninoculated <1.000 <1.000 <1.000 <1.000
1 Low 1.708 1.613 2.004 1.785
2 Low 1.613 1.041 1.908 1.322
3 Low 1.708 1.708 1.613 1.041
4 Low 1.322 1.785 1.041 1.041
5 Low 1.785 1.613 1.785 1.851
6 Low 1.908 1.908 1.613 1.908
7 Low 1.785 2.004 1.322 1.959
8 Low 2.004 1.613 1.491 1.785
9 Low 1.491 1.851 1.613 2.004
1 Medium 2.179 2.004 2.344 2.233
2 Medium 2.603 2.654 2.545 2.400
3 Medium 2.449 2.592 2.520 2.682
4 Medium 2.603 2.741 2.344 2.569
5 Medium 2.673 2.700 2.400 2.520
6 Medium 2.400 2.303 2.581 2.624
7 Medium 2.779 2.930 2.741 2.876
8 Medium 2.581 2.700 2.820 2.846
9 Medium 2.634 2.493 2.700 2.741
1 High 3.179 3.344 3.004 3.083
2 High 3.303 3.258 3.179 3.344
3 High 3.581 3.545 3.382 3.400
4 High 3.464 3.400 3.258 3.281
5 High 3.233 3.400 3.433 3.592
6 High 3.258 3.179 3.400 3.507
7 High 3.233 3.433 3.507 3.344
8 High 3.344 3.479 3.464 3.464
9 High 3.400 3.303 3.533 3.281
77/92
Table 8: Summary of the results of the ILS: 3M Petrifilm REC – E. coli 37 ± 1oC
Collaborators Level Reference method Alternative method
ISO 16449-2 3M Petrifilm REC
1 Uninoculated <1.000 <1.000 <1.000 <1.000
2 Uninoculated <1.000 <1.000 <1.000 <1.000
3 Uninoculated <1.000 <1.000 <1.000 <1.000
4 Uninoculated <1.000 <1.000 <1.000 <1.000
5 Uninoculated <1.000 <1.000 <1.000 <1.000
6 Uninoculated <1.000 <1.000 <1.000 <1.000
7 Uninoculated <1.000 <1.000 <1.000 <1.000
8 Uninoculated <1.000 <1.000 <1.000 <1.000
9 Uninoculated <1.000 <1.000 <1.000 <1.000
1 Low 1.613 1.322 1.613 1.708
2 Low 1.491 1.041 1.908 1.322
3 Low 1.708 1.322 1.491 1.041
4 Low 1.322 1.613 1.041 1.041
5 Low 1.785 1.613 1.613 1.041
6 Low 1.785 1.708 1.491 1.491
7 Low 1.322 1.708 1.322 1.785
8 Low 1.851 1.613 1.041 1.613
9 Low 1.322 1.613 1.491 1.491
1 Medium 2.149 2.004 2.083 2.303
2 Medium 2.364 2.344 2.258 2.004
3 Medium 2.303 2.179 2.045 1.959
4 Medium 2.581 2.417 2.149 2.417
5 Medium 2.520 2.464 2.449 2.179
6 Medium 2.149 2.258 2.303 2.303
7 Medium 2.281 2.520 2.417 2.520
8 Medium 2.233 2.258 2.479 2.507
9 Medium 2.303 2.004 2.464 2.400
1 High 3.083 3.303 3.004 2.908
2 High 3.258 2.959 3.004 3.179
3 High 3.470 3.479 3.281 3.083
4 High 3.303 3.382 3.004 3.083
5 High 3.507 3.083 3.179 3.004
6 High 3.004 2.785 3.149 3.004
7 High 3.207 3.400 3.479 3.258
8 High 3.045 3.083 3.233 3.149
9 High 3.004 3.281 3.303 3.207
78/92
Table 9: Summary of the results of the ILS: 3M Petrifilm REC – E. coli 42 ± 1oC
Collaborators Level Reference method Alternative method
ISO 16449-2 3M Petrifilm REC
1 Uninoculated <1.000 <1.000 <1.000 <1.000
2 Uninoculated <1.000 <1.000 <1.000 <1.000
3 Uninoculated <1.000 <1.000 <1.000 <1.000
4 Uninoculated <1.000 <1.000 <1.000 <1.000
5 Uninoculated <1.000 <1.000 <1.000 <1.000
6 Uninoculated <1.000 <1.000 <1.000 <1.000
7 Uninoculated <1.000 <1.000 <1.000 <1.000
8 Uninoculated <1.000 <1.000 <1.000 <1.000
9 Uninoculated <1.000 <1.000 <1.000 <1.000
1 Low 1.613 1.322 1.322 1.041
2 Low 1.491 1.041 1.708 1.041
3 Low 1.708 1.322 1.322 1.041
4 Low 1.322 1.613 1.491 1.041
5 Low 1.785 1.613 1.491 1.491
6 Low 1.785 1.708 1.491 1.708
7 Low 1.322 1.708 1.041 1.613
8 Low 1.851 1.613 1.322 1.708
9 Low 1.322 1.613 1.613 1.708
1 Medium 2.149 2.004 2.303 2.233
2 Medium 2.364 2.344 2.520 2.303
3 Medium 2.303 2.179 2.083 2.258
4 Medium 2.581 2.417 2.233 2.479
5 Medium 2.520 2.464 2.449 2.233
6 Medium 2.149 2.258 2.303 2.004
7 Medium 2.281 2.520 2.382 2.479
8 Medium 2.233 2.258 2.464 2.520
9 Medium 2.303 2.004 2.507 2.400
1 High 3.083 3.303 3.045 2.908
2 High 3.258 2.959 3.004 3.149
3 High 3.470 3.479 3.083 2.908
4 High 3.303 3.382 3.083 3.281
5 High 3.507 3.083 3.233 3.233
6 High 3.004 2.785 3.303 3.149
7 High 3.207 3.400 3.281 3.045
8 High 3.045 3.083 3.083 3.449
9 High 3.004 3.281 3.149 3.004
79/92
Figure 9A: Accuracy profile of the alternative method (Coliform 37 ± 1oC) in the ILS
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
1,5 2,0 2,5 3,0 3,5
Accura
cy (
diffe
rence o
f L
og)
Levels Log(cfu/g)
Bias
Relative Lower TI limit (beta = 80%)
Relative Upper TI limit (beta = 80%)
80/92
Figure 9B: Accuracy profile of the alternative method (E. coli 37 ± 1oC ) in the ILS
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
1,5 2,0 2,5 3,0 3,5
Accura
cy (
diffe
rence o
f L
og)
Levels Log(cfu/g)
Bias
Relative Lower TI limit (beta = 80%)
Relative Upper TI limit (beta = 80%)
81/92
Figure 9C: Accuracy profile of the alternative method (E. coli 42 ± 1oC ) in the ILS
The ILS statistical analysis is presented in Tables 10 -12. Based on data presented in these tables, the repeatibility standard deviations (Sr) and between-labs standard devaiations (SL) indicate similar results between the alternative and reference method. The reproducibility standard deviation (SR) indicated slightly better results with the alternative method than the reference method.
According to the ISO 16140-2:2016 standard, if any of the values of the β-ETI fall outside of the ±0.5log AL then a further calculation is required. None of the β-ETI values fell outside the range, indicating no additonal calculations were required. Based on the data obtained within these studies, the alternative method is considered equivalent to the reference methods.
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
1,5 2,0 2,5 3,0 3,5
Accura
cy (
diffe
rence o
f L
og)
Levels Log(cfu/g)
Bias
Relative Lower TI limit (beta = 80%)
Relative Upper TI limit (beta = 80%)
82/92
Table 10: Statistical analysis of the ILS data (3M Petrifilm REC – Coliform 37 ± 1oC)
Accuracy profile 0.5
Study Name
Date
Coordinator TRUE
Tolerance probability (beta) 80% 80% 80%
Acceptability limit in log (lambda) 0.68 0.68 0.68
Alternative method Reference method
Levels Low Medium High Low Medium HighTarget value 1.682 2.555 3.350
Number of participants (K) 9 9 9 9 9 9
Average for alternative method 1.601 2.581 3.357 1.682 2.555 3.350
Repeatability standard deviation (sr) 0.293 0.091 0.095 0.233 0.088 0.087
Between-labs standard deviation (sL) 0.194 0.170 0.129 0.084 0.213 0.085
Reproducibility standard deviation (sR) 0.351 0.193 0.160 0.247 0.231 0.122
Corrected number of dof 15.018 9.981 11.299 16.494 9.270 13.112
Coverage factor 1.388 1.439 1.422
Interpolated Student t 1.341 1.372 1.361
Tolerance interval standard deviation 0.3636 0.2021 0.1677
Lower TI limit 1.114 2.304 3.128
Upper TI limit 2.089 2.859 3.585
Bias -0.080 0.026 0.007
Relative Lower TI limit (beta = 80%) -0.568 -0.251 -0.222 TRUE
Relative Upper TI limit (beta = 80%) 0.407 0.303 0.235 FALSE
Lower Acceptability Limit -0.68 -0.68 -0.68
Upper Acceptability Limit 0.68 0.68 0.68
New acceptability limits may be based on reference method pooled variancePooled repro standard dev of reference 0.207
3M Petrifilm Rapid E.coli/Coliform Count Plate
Oct-18
Q Laboratories
Select ALL blue lines to draw the accuracy profile as illustrated in the worksheet "Graph Profile"
Application of clause 6.2.3 Step 8: If any of the values for the β-ETI fall outside the
acceptability limits, calculate the pooled average reproducibility standard deviation of the reference
method.Step 9: Calculate new acceptability limits as a function
of this standard deviation.
83/92
Table 11: Statistical analysis of the ILS data (3M Petrifilm REC – E. coli 37 ± 1oC)
Accuracy profile 0.5
Study Name
Date
Coordinator TRUE
Tolerance probability (beta) 80% 80% 80%
Acceptability limit in log (lambda) 0.64 0.64 0.64
Alternative method Reference method
Levels Low Medium High Low Medium HighTarget value 1.505 2.307 3.179
Number of participants (K) 9 9 9 9 9 9
Average for alternative method 1.399 2.289 3.136 1.505 2.307 3.179
Repeatability standard deviation (sr) 0.294 0.125 0.107 0.205 0.089 0.136
Between-labs standard deviation (sL) 0.000 0.137 0.098 0.106 0.125 0.135
Reproducibility standard deviation (sR) 0.294 0.186 0.145 0.231 0.154 0.191
Corrected number of dof 16.941 12.449 13.380 15.835 11.155 12.980
Coverage factor 1.370 1.410 1.402
Interpolated Student t 1.334 1.354 1.348
Tolerance interval standard deviation 0.3017 0.1938 0.1509
Lower TI limit 0.996 2.026 2.933
Upper TI limit 1.801 2.551 3.340
Bias -0.107 -0.018 -0.043
Relative Lower TI limit (beta = 80%) -0.509 -0.280 -0.246 TRUE
Relative Upper TI limit (beta = 80%) 0.296 0.244 0.160 FALSE
Lower Acceptability Limit -0.64 -0.64 -0.64
Upper Acceptability Limit 0.64 0.64 0.64
New acceptability limits may be based on reference method pooled variancePooled repro standard dev of reference 0.194
3M Petrifilm Rapid E.coli/Coliform Count Plate
Oct-18
Q Laboratories
Select ALL blue lines to drawthe accuracy profile as illustrated in the worksheet "Graph Profile"
Application of clause 6.2.3 Step 8: If any of the values for the β-ETI fall outside
the acceptability limits, calculate the pooled average reproducibility standard deviation of the reference
method.Step 9: Calculate new acceptability limits as a
function of this standard deviation.
84/92
Table 12: Statistical analysis of the ILS data (3M Petrifilm REC – E. coli 42 ± 1oC)
Accuracy profile 0.5
Study Name
Date
Coordinator TRUE
Tolerance probability (beta) 80% 80% 80%
Acceptability limit in log (lambda) 0.64 0.64 0.64
Alternative method Reference method
Levels Low Medium High Low Medium HighTarget value 1.505 2.307 3.179
Number of participants (K) 9 9 9 9 9 9
Average for alternative method 1.378 2.340 3.130 1.505 2.307 3.179
Repeatability standard deviation (sr) 0.286 0.130 0.139 0.205 0.089 0.136
Between-labs standard deviation (sL) 0.000 0.080 0.040 0.106 0.125 0.135
Reproducibility standard deviation (sR) 0.286 0.153 0.145 0.231 0.154 0.191
Corrected number of dof 16.941 15.298 16.686 15.835 11.155 12.980
Coverage factor 1.370 1.386 1.374
Interpolated Student t 1.334 1.339 1.334
Tolerance interval standard deviation 0.2943 0.1583 0.1492
Lower TI limit 0.986 2.128 2.930
Upper TI limit 1.771 2.552 3.329
Bias -0.127 0.033 -0.050
Relative Lower TI limit (beta = 80%) -0.519 -0.179 -0.249 TRUE
Relative Upper TI limit (beta = 80%) 0.266 0.245 0.149 FALSE
Lower Acceptability Limit -0.64 -0.64 -0.64
Upper Acceptability Limit 0.64 0.64 0.64
New acceptability limits may be based on reference method pooled variancePooled repro standard dev of reference 0.194
3M Petrifilm Rapid E.coli/Coliform Count Plate
Oct-18
Q Laboratories
Select ALL blue lines to draw the accuracy profile as illustrated in the worksheet "Graph Profile"
Application of clause 6.2.3 Step 8: If any of the values for the β-ETI fall outside the
acceptability limits, calculate the pooled average reproducibility standard deviation of the reference
method.Step 9: Calculate new acceptability limits as a function
of this standard deviation.
85/92
5 References
1. International Standard: ISO 16140-1 – Microbiology of the food chain – Method validation. Part 1: Vocabulary, First Edition, 2016-06-15.
2. International Standard: ISO 16140-2 – Microbiology of the food chain – Method validation. Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method. First Edition, 2016-06-15.
3. International Standard: ISO 4832:2006 – Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of coliforms – Colony-count technique. Third Edition, 2006-02.
4. International Standard: ISO 16649-2:2001 – Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli - Part 2: Colony-count technique at 44°C using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide. First Edition, 2001-04.
5. International Standard: ISO 6887 (All parts) – Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination. First Editions.
86/92
6 ANNEX A: Flow Diagram of the ISO 4832 Reference Method
87/92
7 ANNEX B: Flow Diagram of the ISO 16649-2:2001 Reference Method
88/92
8 ANNEX C: Flow Diagram of the 3M Petrifilm Rapid E. coli and Coliform Count Plate Alternative Method
9 ANNEX D: Kit Insert for 3M Petrifilm Rapid E. coli / Coliform Count Plate
See the attached document.
89/92
10 ANNEX E: Summary of Artificial Contamination
Trueness Study
Category Type Sample # Item
Target
Inoculum
Level
Inoculating
Organisms Origin Stressing Hold time
Lo
g1
0
Inju
ry
Meat Products Breaded Meat
QL017 Chicken Nuggets 1 x 103 Klebsiella pneumonia
QL 11007-7 & E.
coli QL 16160-4
Meat &
Chicken
Nuggets
Heat
50 ºC for
10 mins
0.62 QL018 Spicy Chicken
Nuggets 1 x 104
QL019 BBQ Chicken Wings 1 x 105
Dairy and Egg
Products
Pasteurized Dairy
Products
QL035 Butter 1 x 103 Cronobacter
condimenti
DSM 27966 & E.
coli
QL 11010-2
Infant Formula
& Bottled
Water
5⁰C for 48-72
hours QL036 Ricotta 1 x 103
QL037 Whipping Cream 1 x 103
Desserts & Egg
Products
QL056 Rice Pudding 1 x 102 Hafnia alvei
ATCC 51815 & E.
coli
QL 16154-7
Milk,
Minnesota &
Flour
5⁰C for 48-72
hours
QL057 Cream Puffs 1 x 102
Fish and Seafood Products
Raw Products
QL067 Cod 1 x 105
Serratia marcescens
ATCC 8100 & E. coli
QL 16154-2
Milk, Cork
Ireland & Raw
Ground Beef
5⁰C for 48-72
hours
QL068 Pollock 1 x 105
QL069 Shrimp 1 x 105
QL071 Catfish 1 x 103
QL072 Trout 1 x 104
Marinated Smoked
Items
QL081 Lemon Pepper
Scallops 1 x 103
Klebsiella pneumonia
QL 11007-7 & E.
coli QL 16160-8
Meat &
Chicken
Carcass Rinse
Heat
50ºC for
10 mins
0.56
QL082 Smoky Bourbon
Salmon 1 x 103
90/92
Cooked Products
QL091 Cooked Shrimp 1 x 103 Citrobacter freundii
QL 100813-2A &
E. coli QL 16160-10
Sliced deli
turkey meat &
Raw Pork
Heat
50ºC for
10 mins
0.67 QL093 Lobster Cakes 1 x 102
QL094 Salmon Burgers 1 x 102
Vegetable
Products
Pre-Cooked
Vegetables
QL111 Frozen Lima Beans 1 x 103
Serratia marcescens
ATCC 8100 & E. coli
QL 16160-2
Milk, Cork
Ireland &
Mixed Frozen
Vegetables
-20⁰C for 2
weeks
QL112 Frozen Green Beans 1 x 103
QL113 Frozen Carrots 1 x 103
QL114 Frozen Onions 1 x 103
QL115 Frozen Corn 1 x 103
Ready to Eat and Ready to
Reheat
Cold Ready Meals
QL130 Roast Beef Pinwheel 1 x 104
Citrobacter freundii
QL 100813-2A &
E. coli QL 16154-3
Sliced deli
turkey meat &
Beef Trim
5⁰C for 48-72
hours QL131
Ham and Turkey
Sandwich 1 x 104
QL132 Uncrustable PB + J 1 x 104
Pasta
QL144 Macaroni & Cheese 1 x 103
Klebsiella pneumonia
QL 11007-7 & E. coli QL 16154-13
Meat &
Potable Water
Heat
50ºC for
10 mins
0.59
QL145 Lasagna 1 x 103
QL146 Cheese Ravioli 1 x 102
QL147 Lentil Pasta with
Cheddar Sauce 1 x 102
Animal Feed
Products
Finished Products
for Domestics QL160
Temptations Cat
Treats 1 x 103
Serratia marcescens
ATCC 8100 & E. coli
QL 16154-2
(Lyophilized)
Milk, Cork
Ireland & Raw
Ground Beef
Ambient for 2
weeks
Ingredients for
Domestic/Livesto
ck
QL176 Soybean Meal 1 x 102
Rahnella aqualitis
ATCC 55046 & E.
coli QL 16154-7
(Lyophilized)
Soil & Flour Ambient for
2 weeks
QL177 Rolled Oats 1 x 102
QL178 Yellow Cornmeal 1 x 103
QL179 Pearled Barley 1 x 103
QL181 Long Grain White
Rice 1 x 103
91/92
Accuracy Study
Category Type Item
Target
Inoculum
Level
Inoculating
Organism Origin Spiking Seeding
%
Injury
Meat Products
(Combined
category raw/RTC
meats and poultry)
Dairy and Egg
Products
Raw Fresh
Meats Minced Meat
~1.0 x 102 Klebsiella pneumoniae
ATCC 13882 & E. coli QL 16154-2
Water & Raw
Ground Beef
5°C 48 to 72
Hours ~1.0 x 103
~1.0 x 105
Breaded Meats Raw Frozen Chicken
Wings
~1.0 x 102 Klebsiella pneumonia ATCC 11296 &
E. coli QL 16154-12
Water & Chicken
Carcass Rinsate -20°C 2 weeks ~1.0 x 103
~1.0 x 105
Dairy and Egg Products
Raw Dairy
Products Raw Cow’s Milk
~1.0 x 102 Hafnia alvei ATCC 51815 &
E. coli QL 16160-4
Milk & Chicken
Nuggets
5°C 48 to 72 Hours
~1.0 x 103
~1.0 x 105
Egg Products
(heat processed)
with additives
Whole Liquid Eggs
~1.0 x 102 Hafnia alvei QL 102313 &
E. coli QL 11010-2
Milk & Bottled
Water
5°C 48 to 72 Hours
~1.0 x 103
~1.0 x 105
Fish and Seafood Products
Raw Products Tuna Sushi
~1.0 x 102 Enterobacter amnigenus
ATCC 51816 & E. coli QL 16160-16
Milk, Minnesota
& Sprouts
5°C 48 to 72
Hours ~1.0 x 103
~1.0 x 105
Smoked
Products Smoked Salmon
~1.0 x 102 Enterobacter amnigenus
QL 112413-1 & E. coli 3M REC8
Milk & Food
Isolate
5°C 48 to 72 Hours
~1.0 x 103
~1.0 x 105
Vegetable Products
Raw Vegetable
products Bunched Spinach
~1.0 x 102 Klebsiella oxytoca ATCC 13182 &
E. coli 3M REC19
Pharyngeal
tonsil & Not
Available
5°C 48 to 72
Hours ~1.0 x 103
~1.0 x 105
Heat Processed
Vegetable Juices Pasteurized Carrot Juice
~1.0 x 102 Klebsiella oxytoca ATCC 700324 &
E. coli QL 16160-9
Not Avaliable &
Fresh Baby
Spinach
Heat
50ºC for
10 mins
0.63 ~1.0 x 103
~1.0 x 105
92/92
Accuracy Study
Category Type Item
Target
Inoculum
Level
Inoculating
Organism Origin Spiking Seeding
%
Injury
Accuracy Study
Category Type Item
Target
Inoculum
Level
Inoculating
Organism Origin Spiking Seeding
%
Injury
Ready to Eat and Ready to Reheat
Cold Ready
Meals
Ready-made
Sandwiches
~1.0 x 102
Kluyvera intermedia ATCC 33110 &
E. coli 3M REC17
Surface Water &
Not Avaliable
5°C 48 to 72
Hours
~1.0 x 103
~1.0 x 105
Mayonnaise
based deli
salads with raw
ingredients
Raw Vegetable Salad
with Dressing
~1.0 x 102 Kluyvera intermedia
QL 081215 & E. coli QL 16160-12
Water &
Irrigation Rinsate
Water
5°C 48 to 72
Hours ~1.0 x 103
~1.0 x 105
Animal Feed
Finished
Products fro
Livestock
Chicken Feed
~1.0 x 102 Enterobacter cloacae NBRC 13536 &
E. coli QL 16160-5
Not Avaliable &
Flavored
Seasoning
Ambient 2 weeks ~1.0 x 103
~1.0 x 105
Plant Origin
Ingredients Soybean Meal
~1.0 x 102 Enterobacter cloacae NBRC 13535 &
E. coli 3M REC11
Spinal Fluid &
Food Isolate Ambient 2 weeks ~1.0 x 103
~1.0 x 105
Samples from the
Industrial
Production
Environment
Surface Samples
Stainless Steel
Environmental Spones
~1.0 x 102 Enterobacter gergoviae
QL 123009-1 & E. coli 3M REC1
Clinical &
Mozzerella
Cheese
Ambient
16 to 24 Hours ~1.0 x 103
~1.0 x 105
Sealed Concrete
Environmental Sponges
~1.0 x 102 Enterobacter gergoviae
ATCC 33028 & E. coli QL 16160-10
Urine, France &
Raw Pork
Ambient 16 to 24 Hours
~1.0 x 103
~1.0 x 105