ACCREDITATION
N°1-0144
PORTEE DISPONIBLE
SUR WWW.COFRAC.FR
3M
Boulevard de l’Oise
F-95029 CERGY PONTOISE
NF VALIDATION
Validation study according to the EN ISO 16140 standard
Summary report
EN ISO 16140 validation of the
3MTM PetrifilmTM Coliform Count plates (CC)
for thermotolerant colonies enumeration
with regard to the NF V08-060
reference method
Quantitative method
This document includes 39 pages, with 3 appendixes.
Only copies including the totality of this report are authorised.
Competences of the laboratory are certified by COFRAC accreditation for the
analysis marked with symbol.
Version 0
March 27, 2014
ADRIA DEVELOPPEMENT
Creac’h Gwen - F. 29196 QUIMPER Cedex - Tél. (33) 02.98.10.18.18 - Fax (33) 02.98.10.18.08
E-mail : adria.developpement@adria.tm.fr - Site web : http://www.adria.tm.fr
ASSOCIATION LOI DE 1901 - N° SIRET 306 964 271 00036 - N° EXISTENCE 532900006329 - N°TVA FR4530696427100036
3M
1
2
INTRODUCTION _______________________________________________ 4
1.1
Dates of the initial validation and renewal studies _____________________ 4
1.2
Protocol and principle of the alternative method ______________________ 4
1.3
Standard method _______________________________________________ 5
INITIAL VALIDATION (1989) AND RENEWAL STUDIES RESULTS ______ 6
2.1
2.2
2.3
Method Comparison Study ________________________________________ 6
2.1.1
Linearity ________________________________________________________ 6
2.1.2
Relative accuracy ________________________________________________ 10
2.1.3
Detection limit (LOD) and quantification limit (LOQ) _____________________ 17
2.1.4
Relative sensitivity _______________________________________________ 18
2.1.5
Specificity ______________________________________________________ 19
2.1.6
Practicability ____________________________________________________ 20
Inter-laboratory study ___________________________________________ 20
2.2.1
Study organisation _______________________________________________ 20
2.2.2
Verification of experimental parameters ______________________________ 21
2.2.3
Results ________________________________________________________ 22
Conclusion ____________________________________________________ 26
Appendix 1 - Linearity: raw data ________________________________________________________ 28
Appendix 2 - Specificity / Selectivity: raw data _____________________________________________ 31
Appendix 3 - Inter-laboratory study results ________________________________________________ 32
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Quality assurance documents related to this study can be consulted upon
request by 3M.
The technical protocol and the result interpretation were realized according to
the EN ISO 16140 and the AFNOR technical rules.
o Company:
3M
Boulevard de l’Oise
F-95029 CERGY PONTOISE
o Expert Laboratory:
ADRIA Développement
ZA Creac’h Gwen
F-29196 QUIMPER Cedex
o Studied method:
3MTM Petrifilm Coliform Count Plate (CC)
for thermotolerant coliform enumeration
o Validation standard:
NF EN ISO 16140 (October 2003): Food
microbiology – Protocol for the validation of
alternative methods
o Standard method:
NF V08-060: Enumeration of thermotolerant
coliforms by colony-count technique at 44°C
(Routine method)
o Scope:
All human food products
o
AFNOR Certification

Certification organism:
Analysis performed according to the COFRAC accreditation
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1
INTRODUCTION
1.1
Dates of the initial validation and renewal studies
3MTM Petrifilm Coliform Count Plate (Petrifilm CC) performances were
assessed on September 29, 1989 (certificate number 3M - 01/02 - 09/89 C).
The method was renewed in June 1993, September 1998, May 2002, June
2006, April 2010 and March 2014.
1.2
Protocol and principle of the alternative method
The Petrifilm CC plate is a ready-to-use system for the enumeration of the
total coliforms. It is constituted by a cold water soluble dehydrated gel fixed
between a support of polyethylene and a sheet leaf in polypropylene. The
Petrifilm CC plate is based on the VRBA medium formulation.
After an incubation for 24 h ± 2 h at 44° ± 1°C, gas producing coliforms
appear as red colonies surrounded with gas bubbles, whereas non gas
producing coliforms appear as red colonies without gas bubbles.
The general protocol is presented below :
Figure 1 - Flow diagram of the alternative method
Sample

Suspension (1/10) in peptone-salt (PS)
If necessary, adjust the pH of the suspension between 6.6 and 7.2

Decimal dilutions in PS

One Petrifilm inoculation per dilution with 1ml suspension

Incubation for 24 h  2 h at 44°C  1°C

Numerate all the typical colonies (red colonies with or without gas bubbles)
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1.3
Standard method 
The routine method is the NF V08-060 routine method: Microbiology of food
and animal feedings stuffs. Enumeration of thermotolerant coliforms by
colony-count technique at 44°C. The protocol is presented below:
Figure 2 - Flow diagram of the routine method
Suspension and decimal dilutions in peptone-salt

1 ml per Petri dish (1 Petri dish/dilution)

Pour 15 ml VRBA

Solidification

Add a double layer of VRBA

Incubation at 44°C  1°C, 24 h  2 h

Count characteristic colonies (red   0,5 mm sometime surrounded with red colorant)
(take into account the Petri dishes with a maximum of 150 characteristic colonies
and/or non characteristic colonies)

Analysis performed according to the COFRAC accreditation
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2
INITIAL VALIDATION
RESULTS
(1989)
2.1
Method Comparison Study
2.1.1
Linearity
AND
RENEWAL
STUDIES
Linearity is the ability of the method when used with a given matrix to give results that are in
proportion to the amount of analyte present in the sample, that is an increase in analyte
corresponds to a linear or proportional increase in results.
Food matrices
Five 5 “matrix / strain” pairs were tested. Five contamination levels were
analysed in duplicate. The tested samples and the inoculated strains are
presented in the table below:
Matrix
Strain
Ground beef
Enterobacter cloacae 58
Pasteurised milk
Enterobacter sakazakii 95
Egg product
Klebsiella pneumoniae 89
Raw fish fillet
Escherichia coli Ad 228
Green peas
Escherichia coli 19
Results
The raw data (in French) are given in Appendix 1.
The bi-dimensional graphs are shown figure 3.
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Figure 3 - Linearity: bi-dimensional graphs
Pasteurised milk
9
9
8
8
log(Alternative method)
log(Alternative method)
Ground beef
7
6
5
4
3
7
6
5
4
3
2
2
1
1
0
0
0
1
2
3
4
5
6
7
8
9
0
1
2
log(Reference method)
4
5
6
7
8
9
7
8
9
log(Reference method)
Raw fish fillet
Egg product
9
9
8
8
log(Alternative mehtod)
log(Alternative method)
3
7
6
5
4
3
7
6
5
4
3
2
2
1
1
0
0
0
1
2
3
4
5
6
7
8
9
log(Reference method)
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2
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4
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Green peas
log(Alternative method)
9
8
7
6
5
4
3
2
1
0
0
1
2
3
4
5
6
7
8
9
log(Reference method)
Statistical interpretations
Statistical interpretation results are shown below:
Matrix
R
Selected
Regression
Rob.F
Critical
value
P%
Correlation
coefficient
Regression equation*
Ground beef
1.50
GMFR
1.05
5.41
45
0.998
log Alt = 0.930 log Ref. + 0.463
Milk
0.67
GMFR
8.57
5.41
2
0.994
log Alt = 1.081 log Ref. - 0.202
Raw fish
0.80
GMFR
2.63
5.41
16
0.999
log Alt = 0.987 log Ref. + 0.069
Egg product
2.60
OLS1
4.26
5.41
8
0.992
log Alt = 0.922 log Ref. + 0.267
Green peas
0.60
GMFR
1.70
5.41
28
0.990
log Alt = 0.950 log Ref. + 0.025
* x-axis and y-axis choice depends on the selected regression.
Statistical Interprétation :
P > 5 % : not significant
1 % < P < 5 % : significant
P < 1 % : highly significant
The regression lines are shown Figure 4:
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Figure 4 - Linearity: regression lines
GMFR Regression
Ground beef
GMFR Regression
Milk
6,00
6,00
y = 0,9303x + 0,4633
5,00
4,00
Alternative
Alternative
5,00
3,00
2,00
y = 1,0809x - 0,2024
4,00
3,00
2,00
1,00
1,00
0,00
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00
0,00
1,00
2,00
Reference
6,00
5,00
6,00
6,00
y = 0,9865x + 0,0689
5,00
Alternative
Alternative
5,00
OLS 1 Regression
Egg product
6,00
4,00
3,00
3,00
2,00
1,00
1,00
1,00
2,00
3,00
4,00
5,00
0,00
0,00
6,00
Reference
y = 0,9224x + 0,2665
4,00
2,00
0,00
0,00
4,00
Reference
GMFR Regression
Raw fish
5,00
3,00
1,00
2,00
3,00
4,00
Reference
GMFR Regression
Green peas
6,00
Alternative
5,00
y = 0,9501x + 0,0248
4,00
3,00
2,00
1,00
0,00
0,00
1,00
2,00
3,00
4,00
5,00
6,00
Reference
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Conclusion
The determined correlation coefficients are all upper or equal to 0,99
whatever the tested matrix.
The linearity test is not significant for the following matrices: ground beef, raw
fish fillet, egg product and green peas. The correlation coefficient observed
for pasteurised milk, associated to a P value under 5%, shows a high value
which decreases the non linearity test robustness. The linearity cannot be
refuted for pasteurized milks.
The Petrifilm CC plate shows satisfying linearity.
2.1.2
Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference
value. The bias is the difference between the expectation of the test results and an accepted
reference value.
The data obtained in 1997 were completed by analyses realized in 2006 for
all categories.
Number and nature of the samples
Categories and types of analysed food are presented in table 1.
Table 1 – Number and nature of the samples
Categories
Meat products
Milk products
Types
Raw meats, raw and cooked
delicatessen, ready-to-eat foods
Raw milk, cheeses, creams, milk
powders
Egg products and
Egg products, pastries, cooked eggs
pastries
Frozen vegetables, salads, cooked
Vegetables
vegetables, ready-to-eat food
Raw fish and shellfish, ready-to-eat
Fish products
foods
All products
Number of analysed
samples
Number of results
used in statistical
tests
22
16
15
12
12
10
26
11
15
11
90
60
Cross contaminations with egg products were done for three samples.
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Results
Samples were analysed in duplicate for both methods.
Table 2 - Petrifilm CC plate / NF V08-060
Food category
Contamination scale (log CFU/g)
Meat products
1.00 to 2.99
Milk products
1.48 to 7.30
Egg products and pastries
1.48 to 3.74
Vegetables
1.30 to 4.08
Fish products
1.00 to 3.74
Bi-dimensional graphs for each category and for all samples are presented
figure 5.
Figure 5 – Relative accuracy: bi-dimensional graphs
Milk products
9,00
9,00
8,00
8,00
7,00
7,00
log(Alternative method)
log(Alternative method)
Meat products
6,00
5,00
4,00
3,00
6,00
5,00
4,00
3,00
2,00
2,00
1,00
1,00
0,00
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
log(Reference method)
log(Reference method)
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9,00
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Fish products
8,00
8,00
7,00
7,00
log(Alternative method)
log(Alternative method)
Vegetables
6,00
5,00
4,00
3,00
2,00
1,00
0,00
0,00
6,00
5,00
4,00
3,00
2,00
1,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
0,00
0,00
8,00
1,00
log(Reference method)
4,00
5,00
6,00
7,00
8,00
6,00
7,00
8,00
All products
8,00
8,00
7,00
7,00
log(Alternative method)
log(Alternative method)
3,00
log(Reference method)
Egg products and pastries
6,00
5,00
4,00
3,00
2,00
1,00
0,00
0,00
2,00
6,00
5,00
4,00
3,00
2,00
1,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
log(Reference method)
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Comments on samples showing enumeration differences between the
two methods
o Vegetables category: sample n° 242
The results obtained by both methods are the following:
Duplicate 1
Duplicate 2
NF V08-60 (log CFU/g)
2.70
2.72
Petrifim CC plate (log CFU/g)
1.30
1.30
API 20E galleries from 4 colonies isolated from VRBA and Petrifilm CC plate,
were performed.
Colonies
1
2
3
4
NF V08-060
Serratia liquefaciens
Oxydase + *
Klebsiella pneumoniae
Klebsiella pneumoniae
Petrifim CC plate
Enterobacter sakazakii
Enterobacter sakazakii
Enterobacter sakazakii
Enterobacter sakazakii
* Indeed, it is not Enterobacteria or coliform.
o Fish products category: sample n° 243
The results obtained with both methods are presented in the table below. The
results obtained with the routine method (V08-060) using VRBA from an
other manufacturer are also given:
Replicate 1
Replicate 2
NF V08-60
(VRBA A)
log CFU/g
3.78
4.00
Petrifim CC plate
log CFU/g
2.85
2.78
NF V08-60
(VRBA B)
log CFU/g
2.00
3.92
The API 20E galleries on 4 colonies, isolated from VRBA and Petrifim CC
plate, were performed: all the colonies were identified to Klebsiella
pneumoniae species.
The minimal, optimal and maximal growth temperatures for two Klebsiella
pneumoniae strains and for two Serratia liquefaciens strains are presented in
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the table forward1. These parameters are presented for three fecal coliform
species: Escherichia coli, Enterobacter cloacae, Enterobacter kobei.
Klebsiella
pneumoniae
(2 strains)
Serratia liquefaciens
(2 strains)
Enterobacter cloacae
Enterobacter kobei
Escherichia coli
(2 strains)
Tmin
2.77
[- 2.44 ; 7.96]
1.07
[- 0.89 ; 3.04]
-2.16
[-3.61 ; -0.76]
-1.22
[-3.37 ; 0.92]
Topt
36.28
[32.41 ; 40.16]
37.20
[35.76 ; 38.64]
32.91
[29.51 ; 34.30]
31.91
[29.51 ; 34.30]
Tmax
44.00
[37.41 ; 52.58]
45.00
[41.64 ; 48.36]
37.30
[37.15 ; 37.44]
38.08
[36.73 ; 39.44]
2.05
[0.69 ; 3.40]
2.16
[- 2.92 ; 7.24]
5.52
[3.24 ; 7.80]
5.34
[3.63 ; 7.06]
38.17
[37.28 ; 39.06]
36.58
[33.99 ; 39.16]
38.89
[37.83 ; 41.95]
40.85
[39.09 ; 42.61]
43.07
[42.40 ; 43.74]
46.44
[38.84 ; 54.04]
47.00
[44.69 ; 49.31]
45.87
[44.95 ; 46.80]
Tmin: minimal growth temperature
Topt: optimal t growth emperature
Tmax: maximal growth temperature
Klebsiella pneumoniae and Serratia liquefaciens cardinal temperatures are
clearly different from fecal coliforms ones. Depending on the VRBA media
formulation, these strains may have some difficulties to grow.
One colony on four tested, isolated on the VRBA from sample n° 242, is
oxidase positive and thus oxidase negative phenotype is characteristic of
Enterobacteria or coliforms.
1
(ACTIA 01.5 « Coliformes : diversité des souches, diversité des comportements en fonction de la
température et signification hygiénique » - Report n°3 )
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Statistical interpretations
Regression lines (graph and equation representations) for each food
category and for all matrices are presented in Figure 6.
Table 3 - Petrifilm CC plate / NF V08-060
Categories
n
R
P%
Selected
regression
a
t(a)
b
t(b)
Critical T
Ordinate
at 0
Slope
at 1
Meat products
16
0.73
GMFR
- 0.096
0.382
1.028
0.278
2.144
71
82
Milk products
12
1.00
GMFR
- 0.230
0.951
1.029
0.528
2.228
36
61
Egg products and
10
0.71
GMFR
- 0.023
0.088
0.970
0.336
2.306
94
77
Vegetables (all results)
11
3.50
OLS1
-0.272
0.531
1.043
0.243
2.262
61
81
Fish products (all points)
11
0.70
GMFR
-0.313
1.010
0.844
1.341
2.262
34
21
All products
60
0.80
GMFR
1.048
0.994
0.332
2.001
30
74
pastries
-0.061
Statistical Interpretation :
P>5%
:
not significant
P<1%:
highly significant
1 % < P < 5 % : significant
Bias D
Alternative method
repeatability limit
Reference method
repeatability limit
Meat products
- 0.020
0.405
0.543
Milk products
- 0.168
0.300
0.294
Egg products and pastries
- 0.168
0.352
0.499
Vegetables (all results)
-0.035
0.411
0.117
Fish products (all points)
0.055
0.206
0.294
- 0.040
0.360
0.440
Category
All products
Conclusion
Bias between both methods show low values whatever the categories
and vary -0.168 to 0.055 Log CFU/g.
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Figure 6 - Relative accuracy: regression equation
GMFR Regression
Meat products
GMFR Regression
Milk products
6,00
y = 1,029x - 0,2303
y = 1,0276x - 0,0964
5,00
Alternative
Alternative
5,00
6,00
4,00
3,00
2,00
1,00
0,00
0,00
4,00
3,00
2,00
1,00
1,00
2,00
3,00
4,00
5,00
0,00
0,00
6,00
1,00
Reference
5,00
Alternative
Alternative
y = 0,9701x - 0,0233
3,00
2,00
1,00
6,00
5,00
6,00
y = 0,8441x + 0,3128
4,00
3,00
2,00
1,00
1,00
2,00
3,00
4,00
5,00
0,00
0,00
6,00
Reference
1,00
2,00
3,00
4,00
Reference
OLS 1 Regression
Vegetables
GMFR Regression
ALL PRODUCTS
8,00
6,00
y = 1,0427x - 0,2722
7,00
Alternative
Alternative
5,00
6,00
4,00
5,00
4,00
GMFR Regression
Fish products
6,00
0,00
0,00
3,00
Reference
GMFR Regression
Egg products and Pastries
5,00
2,00
4,00
3,00
2,00
y = 0,9944x - 0,0386
6,00
5,00
4,00
3,00
2,00
1,00
1,00
0,00
0,00
1,00
2,00
3,00
4,00
5,00
0,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
6,00
Reference
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2.1.3
Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null), but
not quantified as an exact value. Below this value, it cannot be sure that the true value is not
null.
The detection limit is defined as being higher than the critical level because it involves a
power, the probability 1-, which has to be well over 50 %, for example 95 %.
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual
number of organisms) which can be measured and quantified with defined precision and
accuracy under the experimental conditions by the method under validation.
Protocol
The detection limit of the alternative method was done with pure culture of
Escherichia coli. Three different levels of inoculation were tested, with six
replicates per level, i.e. a total of 18 analyses by the alternative method.
Quantification limit was calculated for six independent determinations done
with sterile dilution solution.
Results
Data are intrinsic to the method used and are presented in the following
tables:
Table 4 - Petrifilm CC plate
Level
Positive samples nb
Standard deviation
Bias
0.5
4/6
0.753
1
1
3/6
0.816
0.5
5
6/6
1.033
3
Formulas
Obtained values
LC
S0 + X0
2.5
LOD
S0 + X0
3.5
LOQ
S0 + X0
8.5
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2.1.4
Relative sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different
amounts of analyte measured by the reference method within a given matrix, at a specified
average value, or over the whole measurement range ; that is, it is the minimal quantity
variation (increase of the analyte concentration x) which gives a significant variation of the
measured signal (response y)).
Data are intrinsic to the method used and are obtained from the results of the
linearity study.
Accuracy patterns obtained for different matrices are presented figure 7.
Figure 7 - Accuracy patterns for the different matrices used
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2.1.5
Specificity
The specificity is defined as the degree to which a method is affected (or not) by the other
components present in a multi-component sample. That is the ability of a method to measure
exactly a given analyte, or its amount, within the sample without interference from non-target
components such as a matrix effect, or background noise.
1997 study
Among the 24 thermotolerant coliform strains tested (defined as growing on
VRBL at 44.5°C), 24 strains grew.
Among the 36 non-coliform strains or non-thermotolerant coliform strains
tested, 6 grew on 3M Petrifilm Coliform Count plates: Enterobacter
agglomerans, Citrobacter freundii, Hafnia alvei (2 strains), Klebsiella oxytoca,
Salmonella enteritidis. The Salmonella enteritidis strain grew both on 3M
Petrifilm Coliform Count plates and on VRBL at 44.5°C ± 1°C.
2006 study
7 thermotolerant coliform strains were tested in duplicate with each of the two
methods. All gave characteristic colonies on both methods.
6 strains that grew previously on 3M Petrifilm Coliform Count plates, but not
on VRBL agar at 44.5°C, were retested on VRBL at 44.5°C. The 6 strains
grew on both methods (44°C).
Exclusivity and inclusivity are equivalent on both tested methods.
The raw data (in French) are given in Appendix 2.
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2.1.6
Practicability
The saving of time is mainly obtained by the fact that:
- the Petrifilm CC plate is ready-to-use and allows to avoid the medium
preparation.
- it is not necessary to pour a double layer of medium.
The time delays to obtain results are equivalent between both methods, i.e.
24 h  2 h. The space needed for incubation is much smaller for the Petrifilm
CC plate than for NF V08-060. The Petrifilm CC plate minimizes the volume
of wastes and the manipulation between two different analysis steps.
2.2
Inter-laboratory study
2.2.1
Study organisation
14 laboratories participated to this study. Pasteurised semi-skimmed milk
was inoculated by Escherichia coli 94, isolated from dairy product.
Inoculation levels targeted were:
-
0 CFU/ml,
10 – 100 CFU/ml,
100 – 1 000 CFU/ml,
1 000 – 10 000 CFU/ml.
Each laboratory received eight flasks of 25 ml sample, i.e. two flasks per
inoculation level. Furthermore, one non-inoculated sample was added to the
package for total viable count microflora (NF ISO 4833 method).
Coded samples (code is only known by the expert laboratory) were placed in
isothermal boxes which contained cooling blocks, and express-shipped to the
different laboratories.
A temperature control flask containing temperature probe was added to the
package in order to register temperature profile during transport and at
reception. Samples were shipped in 24 h to laboratories of the collaborative
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study. Sample temperature should be lower or equal to 8°C during transport,
and between 0°C - 8°C at arrival.
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods.
A stability study of the strain inoculated was run in order to verify there is no
evolution during the transport.
2.2.2
Verification of experimental parameters
Strain stability during transport
In order to evaluate the Escherichia coli 94 strain variability during transport,
bacterial count of all inoculated flasks was checked at different time, i.e.
inoculation time, after 24 h and 48 h of conservation at 2°C. Results are
reported in table 5.
Table 5 - Escherichia coli 94 count with the alternative method
and reference method (in log CFU/ml)
Day 0
Day 1
Day 2
Level 1
Reference
Atlernative
method
method
71 / 76
75 / 78
65 / 45
64 / 60
68 / 96
58 / 56
Level 2 2
Reference
Atlernative
method
method
580 / 670
540 / 450
560 / 620
360 / 390
830 / 910
570 / 470
Level 3
Reference
Atlernative
method
method
5 900 / 6 700 3 700 / 4 000
3 900 / 5 400 4 000 / 5 500
6 500 / 1 900 4 600 / 4 200
No evolution of the strain was observed after 48 h of storage at 4°C in the
isothermal box.
Sample temperature on receipt
Measured temperatures on receipt are listed in the table 6.
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Table 6 – Sample temperature on receipt
Laboratories
Temperature measured
by the temperature probe (°C)
A
2.2 (Day 2)
B
2.0
C
5.3
D
1.7
E
2.7
F
3.3
G
2.8
H
4.9
I
3.5
J
1.5
K
1.8
L
3.9
N
2.5
O
5.0
Conclusion
Any temperature upper than 8°C was noted during transport.
2.2.3
Results
Mesophilic aerobic microflora
The mesophilic aerobic microflora was done on the matrix with ISO 4833
method. The results varied from 70 to 3 600 CFU/ml.
Enumeration of Escherichia coli
The results obtained by the expert laboratory for Escherichia coli count for
the both method are given table 7.
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Table 7 - Expert laboratory results (in log CFU/g)
Targeted rate
(log CFU/g)
Reference method
Duplicate 1
0
Alternative method
Duplicate 2
Duplicate 1
Duplicate 2
0
0
0
0
1 to 2
1.67
1.62
1.79
1.81
2 to 3
2.80
2.74
2.68
2.72
3 to 4
3.85
3.76
3.54
3.54
Contamination levels targeted were reached.
Summary of results obtained by both methods
The laboratory A received the package at Day 1, but realised the analyses
only at Day 2 and the laboratory I did not realise two successive dilutions.
These results were not taken into account for statistical interpretation.
The results from 12 laboratories were retained for statistical interpretation.
The raw data (in French) are given in Appendix 3.
A results synthesis is presented in table 8.
Table 8 - Synthesis of results obtained by ISO 4832 method
and Petrifilm CC plate (CFU/ml)
Level 0
Labs Reference Alternative
method
method
A
0
0
0
0
B
0
0
0
0
C
0
0
0
0
D
0
0
0
0
E
0
0
0
0
F
0
0
0
0
G
0
0
0
0
H
0
0
0
0
J
0
0
0
0
K
0
0
0
0
L
0
0
0
0
N
0
0
0
0
O
0
0
0
0
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Level 1
Reference Alternative
method
method
57
73
36
45
67
68
74
84
35
39
57
57
70
73
57
77
51
88
85
68
55
45
64
66
67
69
78
70
59
49
86
78
80
72
63
78
62
63
58
61
25
31
66
72
65
35
84
95
94
93
73
92
23/39
Level 2
Reference Alternative
method
method
680 660 360 450
800 670 550 760
470 490 480 380
490 630 560 500
960 680 580 680
530 470 540 510
660 780 620 490
780 870 560 600
650 780 560 570
670 750 580 660
390 510 540 500
760 1000 600 650
680 990 590 730
Level 3
Reference Alternative
method
method
3100 8500 3600 3300
6300 6100 5000 4500
5200 3900 3900 2900
6300 7800 4800 4000
7900 5900 7000 6000
6700 6500 5500 4500
6800 9600 5800 5600
8200 8700 5000 5400
5800 10000 6100 3600
4500 5800 4500 4900
4700 5400 3300 5400
10000 8000 7700 7000
9500 6500 4800 6600
March 27, 2014
3M
Comparison of the trueness and precision characteristics of
the reference method and the alternative method
The statistical interpretation was realised according to the draft of the
amendment 1 of the EN ISO 16140 (September 2009).
The statistical values are summarised hereafter:
Reference method
Alternative method
Median
Repeatability
standard
deviation
Reproducibility
standard
deviation
1
1.8109
0.0558
2
2.8542
3
3.8400
Level
Repeatability Reproducibility
ratio
ratio
Median
Repeatability
standard
deviation
Reproducibility
standard
deviation
0.1659
1.8572
0.0575
0.0766
1.031
0.462
0.0773
0.1075
2.7576
0.0587
0.0635
0.759
0.590
0.0977
0.1174
3.6864
0.0907
0.1109
0.928
0.945
 Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level, Dij and t are calculated according to the following equations:


Dij  Y ij , Alt  Y ij , Ref
t
median i ( Dij )
 / (2 p)  Diff
If the t value is superior to 2, the alternative method is significantly biased
with regard to the reference method.
The values are given in table 9.
Table 9 – t (d) values obtained by level
Level
Bias
t (d)
Conclusion
1
0,0612
1,14
Not significant bias
2
-0,0780
2,96
Significant bias
3
-0,1248
4,45
Significant bias
The statistical tests concluded to a non significant bias for Level 1 and a
significant bias for Levels 2 and 3 with a low bias values ( - 0.1 log CFU/g).
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 Comparison of the repeatability standard deviations
If the ratio Srj, Alt / Srj, Ref. of the repeatability standard deviations of the
alternative method and the reference method is larger than 2, the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method. If this ratio is smaller than 0.5, the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method.
The ratio values are given in table 10.
Table 10
Level
1
2
3
Level
Repeatability standard deviation
Alternative method /
Reference method
Alternative method
reference method ratio
0,0558
0,0575
1,031
0,0773
0,0587
0,759
0,0977
0,0907
0,928
Repeatability limit
Reference method
Alternative method
0,1561
0,2163
0,1610
0,1643
0,2737
0,2539
1
2
3
The ratios of the repeatability standard deviation are comprised between
0.8 and 1.0.
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method.
 Comparison of the reproducibility standard deviations
If the ratio Srj, Alt / Srj, Ref. of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2, the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method. If this ratio is smaller than 0.5, the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method.
The ratio values are given in table 11.
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Table 11
Reproducibility standard deviation
Alternative method /
Reference method
Alternative method
reference method ratio
0.1659
0.0766
0.462
0.1075
0.0635
0.590
0.1174
0.1109
0.945
Level
1
2
3
Reproducibility limit
Level
1
2
3
Reference method
Alternative method
0.4646
0.2145
0.3011
0.3287
0.1777
0.3105
The ratios of the reproducibility standard deviations are comprised between
0.5 and 0.9.
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
2.3
Conclusion
Conclusions for the method comparison study:
The Petrifilm CC plate shows satisfying linearity.
The Petrifilm CC plate shows satisfying relative accuracy for all products.
The time delays to obtain results are equivalent between both methods, i.e.
24 h  2 h. The Petrifilm CC plate minimizes the incubation space, the
volume of wastes and the manipulation between two different analysis steps.
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Conclusions for the inter-laboratory study:
The bias between the reference method and the alternative method
show low satisfying values:
- Level 1 (100 - 1 000 CFU/g), bias = 0.061 log CFU/g
- Level 2 (1 000 - 10 000 UFC/g), bias = - 0.078 log CFU/g
- Level 3 (10 000 - 100 000 UFC/g), bias = - 0.125 log CFU/g
The repeatability and the reproducibility limits are similar between both
methods.
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Appendix 1 - Linearity: raw data
Test 3MTM PetrifilmTM 24H TC
N° éch.
Produit
1693
1694
1695
Lait
1696
1697
1443
1444
1445
1446
1447
Poisson cru
surgelé
Dilution
10
100
10
100
10
100
100
1000
1000
10000
10
100
10
100
10
100
100
1000
1000
10000
Ne : estimation des petits nombres
ADRIA Développement
(Summary report - Version 0)
Série 1
18
3
105
2
184
17
76
8
162
13
16
0
60
2
127
13
59
8
76
7
NF V08-060
Série 2 ufc/g série1 ufc/g série2 log série1 log série2
17
0
70
9
198
14
116
8
197
19
15
2
70
7
133
15
67
7
56
7
190
160
2,28
2,20
970
720
2,99
2,86
1800
1900
3,26
3,28
7600
11000
3,88
4,04
160000
200000
5,20
5,30
150
160
2,18
2,20
560
700
2,75
2,85
1300
1300
3,11
3,11
6100
6700
3,79
3,83
75000
57000
4,88
4,76
Série1
Série2
13
0
96
12
183
10
80
2
90
20
11
0
59
5
128
11
66
6
56
7
17
1
71
13
198
29
139
12
56
21
15
1
65
5
131
13
72
3
66
4
130
Ne
980
160
log ufc/g
série1
2,11
760
2,99
2,88
1000
N’
7500
2900
N’
14000
3,00
3,46
3,88
4,15
100000
70000
5,00
4,85
110
150
2,04
2,18
580
640
2,76
2,81
1300
1300
3,11
3,11
6500
6800
3,81
3,83
57000
64000
4,76
4,81
ufc/série1 ufc/g série2
log ufc/g
série2
2,20
N’: moyenne arithmétique
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March 27, 2014
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N°
éch.
Test 3M Petrifilm CC 24H TC
Produit
1544
1545
1546
Steack haché
1547
1548
1723
1724
1725
1726
1727
Petits pois
surgelés
Dilution
10
100
10
100
10
100
100
1000
1000
10000
10
100
10
100
10
100
10
100
1000
10000
Ne : estimation des petits nombres
ADRIA Développement
(Summary report - Version 0)
Série 1
NF V08-060
Série 2 ufc/g série1 ufc/g série2 log série1 log série2
20
1
83
5
>150
16
75
1
61
25
1
87
7
>150
20
77
14
75
2
0
9
0
12
2
4
2
4
0
1
0
10
1
25
0
6
3
6
2
190
240
2,28
2,38
800
860
2,90
2,93
1600
N’
6900
2000
N’
8300
3,20
3,30
3,84
3,92
55000
68000
4,74
4,83
20
Ne
90
Ne
120
Ne
400
Ne
4000
Ne
10
Ne
100
Ne
230
1,30
1,00
1,95
2,00
2,08
2,36
600
Ne
6000
Ne
2,60
2,78
3,60
3,78
Série1
Série2
6
0
55
5
112
7
49
5
43
2
5
1
5
0
26
0
9
1
6
3
12
0
39
7
105
13
60
2
34
6
1
0
10
3
18
1
4
0
12
1
ufc/série1 ufc/g série2
log ufc/g
série1
1,78
log ufc/g
série2
2,08
2,74
2,62
60
Ne
550
120
Ne
420
1100
1100
3,04
3,04
4900
5600
3,69
3,75
41000
36000
4,61
4,56
50
Ne
50
Ne
240
10
e
100
Ne
170
1,70
1,00
1,70
2,00
2,38
2,23
900
Ne
6000
Ne
400
Ne
12000
Ne
2,95
2,60
3,78
4,08
N’ : moyenne arithmétique
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March 27, 2014
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N°
éch.
Test 3M Petrifilm CC 24H TC
Produit
Dilution
Coule d'œuf
10
100
10
100
10
100
100
1000
1000
10000
1729
1730
1731
1732
1733
Ne : estimation des petits nombres
ADRIA Développement
(Summary report - Version 0)
Série 1
4
2
37
5
79
9
58
8
39
4
NF V08-060
Série 2 ufc/g série1 ufc/g série2 log série1 log série2
12
0
50
4
77
11
83
7
65
4
40
e
380
120
e
490
1,60
2,08
2,58
2,69
800
800
2,90
2,90
6000
8200
3,78
3,91
39000
63000
4,59
4,80
Série1
Série2
5
0
48
6
75
8
79
4
57
4
4
0
31
6
76
13
81
6
64
3
ufc/série1 ufc/g série2
log ufc/g
série1
1,70
log ufc/g
série2
1,60
2,69
2,53
50
Ne
490
40
Ne
340
760
810
2,88
2,91
7500
7900
3,88
3,90
55000
61000
4,74
4,79
N’ : moyenne arithmétique
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March 27, 2014
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Appendix 2 - Specificity / Selectivity: raw data
Souches positives
Souche
Origine
PCA
Test 3MTM
PetrifilmTM
V08-060
Escherichia coli 19
Carottes râpées
20
17
19
19
16
9
66
47
30
65
60
28
149
136
132
149
130
153
81
0
0
89
0
0
150
119
0
157
130
0
112
96
115
89
72
103
38
46
52
76
47
59
Test 3M
Petrifilm CC
V08-060
11
10
10
17
38
37
38
18
61
72
72
86
53
63
38
76
5
10
9
6
45
48
40
54
Escherichia coli 20
Eau de puits
Escherichia coli 14
Enterobacter agglomerans 74
Lait cru
Fromage
Enterobacter sakazakii L22
Enterobacter sakazakii 90
Enterobacter cloacae Fb2
Lait UHT
Eclair à la vanille
Alimentaire
Souche
Enterobacter agglomerans 11
Citrobacter freundii 59
Hafnia alvei 168
Hafnia alvei Mi1051295
Klebsiella oxytoca 42
Origine
Fromage à pâte cuite
Produit alimentaire
VSM
Charcuterie
Produit alimentaire
Salmonella enteritidis cip 8297
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Appendix 3 - Inter-laboratory study results
Méthode de référence V08-060
Laboratoire
A
n° éch.
Dilution
A1
A6
Flore totale:
A2
1,3.103/ml
A7
A3
A8
A4
A5
B
B1
B6
Flore totale:
B2
1,1.102/ml
B7
B3
B8
B4
B5
ADRIA Développement
(Summary report - Version 0)
ufc/boite a
1
0
10
0
1
0
10
0
1
57
10
6
1
71
10
9
10
57
100
18
10
65
100
7
100
34
1000
0
100
84
1000
9
1
0
10
0
1
0
10
0
1
65
10
9
1
64
10
11
10
80
100
8
10
66
100
8
100
58
1000
11
100
60
1000
7
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
57
1,76
73
1,86
680
660
3100
8500
<1
2,83
2,82
3,49
3,93
0,00
<1
0,00
67
1,83
68
1,83
800
670
6300
6100
32/39
2,90
2,83
3,80
3,79
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
48
10
3
1
49
10
4
10
36
100
4
10
45
100
4
100
40
1000
0
100
33
1000
3
1
0
10
0
1
0
10
0
1
74
10
7
1
83
10
9
10
58
100
2
10
75
100
8
100
53
1000
2
100
43
1000
6
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
36
1,56
45
1,65
360
2,56
450
2,65
3600
3,56
3300
3,52
<1
0,00
<1
0,00
74
1,87
84
1,92
550
2,74
760
2,88
5000
3,70
4500
3,65
March 27, 2014
3M
Méthode de référence V08-060
Laboratoire
C
n° éch.
Dilution
C1
C6
Flore totale:
C2
7,6.102/ml
C7
C3
C8
C4
C5
D
D1
D6
Flore totale:
D2
1,6.103/ml
D7
D3
D8
D4
D5
ADRIA Développement
(Summary report - Version 0)
ufc/boite a
1
0
10
0
1
0
10
0
1
38
10
1
1
41
10
2
10
43
100
9
10
50
100
4
100
47
1000
10
100
41
1000
2
1
0
10
0
1
0
10
0
1
70
10
7
1
72
10
8
10
48
100
6
10
62
100
7
100
60
1000
9
100
80
1000
6
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
35
1,54
39
1,59
470
490
5200
3900
<1
2,67
2,69
3,72
3,59
0,00
<1
0,00
70
1,85
73
1,86
490
630
6300
7800
33/39
2,69
2,80
3,80
3,89
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
60
10
3
1
56
10
7
10
43
100
10
10
37
100
5
100
40
1000
3
100
31
1000
1
1
0
10
0
1
0
10
0
1
61
10
2
1
81
10
4
10
59
100
3
10
51
100
4
100
51
1000
2
100
43
1000
1
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
57
1,76
57
1,76
480
2,68
380
2,58
3900
3,59
2900
3,46
<1
0,00
<1
0,00
57
1,76
77
1,89
560
2,75
500
2,70
4800
3,68
4000
3,60
March 27, 2014
3M
Méthode de référence V08-060
Laboratoire
E
n° éch.
Dilution
E1
E6
Flore totale:
E2
1,9.103/ml
E7
E3
E8
E4
E5
F
F1
F6
Flore totale:
F2
1,9.103/ml
F7
F3
F8
F4
F5
ADRIA Développement
(Summary report - Version 0)
ufc/boite a
1
0
10
0
1
0
10
0
1
49
10
7
1
83
10
14
10
94
100
12
10
66
100
9
100
79
1000
8
100
61
1000
4
1
0
10
0
1
0
10
0
1
55
10
5
1
46
10
4
10
53
100
5
10
47
100
5
100
64
1000
10
100
68
1000
4
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
51
1,71
88
1,94
960
680
7900
5900
<1
2,98
2,83
3,90
3,77
0,00
<1
0,00
55
1,74
45
1,65
530
470
6700
6500
34/39
2,72
2,67
3,83
3,81
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
90
10
3
1
70
10
5
10
60
100
4
10
70
100
5
100
67
1000
10
100
60
1000
6
1
0
10
0
1
0
10
0
1
65
10
5
1
69
10
4
10
49
100
10
10
54
100
2
100
51
1000
10
100
48
1000
1
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
85
1,93
68
1,83
580
2,76
680
2,83
7000
3,85
6000
3,78
<1
0,00
<1
0,00
64
1,81
66
1,82
540
2,73
510
2,71
5500
3,74
4500
3,65
March 27, 2014
3M
Méthode de référence V08-060
Laboratoire
G
n° éch.
Dilution
G1
G6
Flore totale:
G2
70/ml
G7
G3
G8
G4
G5
H
H1
H6
Flore totale:
H2
3,6.103/ml
H7
H3
H8
H4
H5
ADRIA Développement
(Summary report - Version 0)
ufc/boite a
1
0
10
0
1
0
10
0
1
66
10
8
1
70
10
6
10
65
100
7
10
79
100
7
100
68
1000
7
100
100
1000
6
1
0
10
0
1
0
10
0
1
59
10
6
1
47
10
7
10
75
100
11
10
89
100
7
100
83
1000
7
100
87
1000
9
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
67
1,83
69
1,84
660
780
6800
9600
<1
2,82
2,89
3,83
3,98
0,00
<1
0,00
59
1,77
49
1,69
780
870
8200
8700
35/39
2,89
2,94
3,91
3,94
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
79
10
7
1
73
10
4
10
63
100
5
10
50
100
4
100
60
1000
4
100
56
1000
6
1
0
10
0
1
0
10
0
1
87
10
8
1
81
10
5
10
55
100
6
10
57
100
9
100
51
1000
4
100
56
1000
3
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
78
1,89
70
1,85
620
2,79
490
2,69
5800
3,76
5600
3,75
<1
0,00
<1
0,00
86
1,93
78
1,89
560
2,75
600
2,78
5000
3,70
5400
3,73
March 27, 2014
3M
Méthode de référence V08-060
Laboratoire
J
n° éch.
Dilution
J1
J6
Flore totale:
J2
1,9.103/ml
J7
J3
J8
J4
J5
K
K1
K6
Flore totale:
K2
1,5.103/ml
K7
K3
K8
K4
K5
ADRIA Développement
(Summary report - Version 0)
ufc/boite a
1
0
10
0
1
0
10
0
1
79
10
9
1
73
10
6
10
66
100
5
10
79
100
7
100
54
1000
10
100
107
1000
7
1
0
10
0
1
0
10
0
1
61
10
7
1
63
10
6
10
68
100
6
10
75
100
7
100
45
1000
4
100
58
1000
6
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
<1
<1
<1
80
1,90
72
1,86
650
780
5800
10000
<1
2,81
2,89
3,76
4,00
<1
<1
<1
62
1,79
63
1,80
670
750
4500
5800
36/39
2,83
2,88
3,65
3,76
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
66
10
3
1
79
10
7
10
55
100
7
10
55
100
8
100
63
1000
4
100
37
1000
3
1
0
10
0
1
0
10
0
1
58
10
6
1
61
10
6
10
59
100
5
10
67
100
6
100
45
1000
5
100
49
1000
5
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
63
1,80
78
1,89
560
2,75
570
2,76
6100
3,79
3600
3,56
<1
0,00
<1
0,00
58
1,76
61
1,79
580
2,76
660
2,82
4500
3,65
4900
3,69
March 27, 2014
3M
Méthode de référence V08-060
Laboratoire
L
n° éch.
Dilution
L1
L6
Flore totale:
L2
2,2.103/ml
L7
L3
L8
L4
L5
N
N1
N6
Flore totale:
N2
2,7.103/ml
N7
N3
N8
N4
N5
ADRIA Développement
(Summary report - Version 0)
ufc/boite a
1
0
10
0
1
0
10
0
1
24
10
3
1
30
10
4
10
40
100
3
10
53
100
3
100
49
1000
3
100
54
1000
5
1
0
10
0
1
0
10
0
1
65
10
6
1
30
10
9
10
73
100
10
10
104
100
7
100
99
1000
12
100
75
1000
13
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
25
1,40
31
1,49
390
510
4700
5400
<1
2,59
2,71
3,67
3,73
0,00
<1
0,00
65
1,81
35
1,54
760
1000
10000
8000
37/39
2,88
3,00
4,00
3,90
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
67
10
6
1
71
10
8
10
53
100
6
10
52
100
3
100
34
1000
2
100
54
1000
5
1
0
10
0
1
0
10
0
1
83
10
9
1
95
10
9
10
59
100
7
10
62
100
9
100
77
1000
8
100
72
1000
5
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
66
1,82
72
1,86
540
2,73
500
2,70
3300
3,52
5400
3,73
<1
0,00
<1
0,00
84
1,92
95
1,98
600
2,78
650
2,81
7700
3,89
7000
3,85
March 27, 2014
3M
Méthode de référence V08-060
Laboratoire
O
n° éch.
Dilution
O1
O6
Flore totale:
O2
1,5.103/ml
O7
O3
O8
O4
O5
ADRIA Développement
(Summary report - Version 0)
ufc/boite a
1
0
10
0
1
0
10
0
1
94
10
9
1
87
10
15
10
69
100
6
10
99
100
10
100
94
1000
10
100
68
1000
4
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
94
1,97
93
1,97
680
990
9500
6500
38/39
2,83
3,00
3,98
3,81
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
74
10
6
1
90
10
11
10
59
100
6
10
72
100
8
100
48
1000
5
100
65
1000
8
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
73
1,86
92
1,96
590
2,77
730
2,86
4800
3,68
6600
3,82
March 27, 2014
3M
Méthode de référence V08-060
Laboratoire
ADRIA
n° éch.
Dilution
450
451
Flore totale:
452
1,7.103/ml
453
454
455
456
457

ufc/boite a
1
0
10
0
1
0
10
0
1
67
10
5
1
42
10
7
10
57
100
4
10
61
100
7
100
40
1000
3
100
51
1000
8
Test 3MTM PetrifilmTM
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
65
1,81
45
1,65
560
620
3900
5400
2,75
2,79
3,59
3,73
Dilution
ufc/boite
1
0
10
0
1
0
10
0
1
64
10
6
1
59
10
7
10
37
100
2
10
40
100
3
100
42
1000
2
100
58
1000
3
ufc/ml
log ufc/ml
<1
0,00
<1
0,00
64
1,81
60
1,78
360
2,56
390
2,59
4000
3,60
5500
3,74
Analysis performed according to the COFRAC accreditation
ADRIA Développement
(Summary report - Version 0)
39/39
March 27, 2014