rapport de synthèse 3m 01-13 07-14 (en) - nf validation...2007/01/13 · the bi-dimensional graphs...
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ACCREDITATION
N°1-0144
PORTEE DISPONIBLE
SUR WWW.COFRAC.FR
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
Boulevard de l’Oise
F-95029 CERGY PONTOISE CEDEX
NF VALIDATION
Validation of alternative analytical methods
Application in food microbiology
Summary report
EN ISO 16140 validation of
3MTM Rapid Yeast and Mold PetrifilmTM plate for
yeasts and molds enumeration in food,
environmental samples, pet food and animal feed
Quantitative method
This report includes 70 pages, with 4 appendixes.
Only copies including the totality of this report are authorized.
Competences of the laboratory are certified by COFRAC accreditation for the analyses
marked with symbol.
Version 0
September 30, 2014
3M
ADRIA Développement 2/70 September 30, 2014
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1 INTRODUCTION _______________________________________________ 4
2 METHODS PROTOCOLS ________________________________________ 4
2.1 Reference methods ______________________________________________ 4
2.2 Alternative method ______________________________________________ 4
3 VALIDATION STUDY RESULTS __________________________________ 5
3.1 Method comparison study ________________________________________ 5
3.1.1 Linearity study ___________________________________________________ 5
3.1.2 Relative accuracy ________________________________________________ 23
3.1.3 Detection limit (LOD) and quantification limit (LOQ) _____________________ 42
3.1.4 Relative sensitivity _______________________________________________ 44
3.1.5 Specificity – Selectivity ____________________________________________ 47
3.2 Practicability __________________________________________________ 49
3.3 Inter-laboratory study ___________________________________________ 51
3.3.1 Study organisation _______________________________________________ 51
3.3.2 Verification of experimental parameters ______________________________ 52
3.3.3 Quality Controls and conclusion ____________________________________ 53
3.3.4 Incubation time of the Petrifilm tests _________________________________ 54
3.3.5 Results analysis _________________________________________________ 55
3.3.6 Statistical interpretations and calculations _____________________________ 55
4 CONCLUSION ________________________________________________ 64
Appendix 1 – Diagram of the reference method test procedure ________________________________ 65
Appendix 2 – Flow diagram of the alternative method _______________________________________ 66
Appendix 3 – Artificial contaminations ___________________________________________________ 67
Appendix 4 – Specificity / selectivity _____________________________________________________ 68
3M
ADRIA Développement 3/70 September 30, 2014
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Before comment
Quality assurance documents related to this study can be consulted upon
request from 3M.
The technical protocol and the result interpretation were realized according to
the EN ISO 16140 and the AFNOR technical rules.
Company: 3M
Boulevard de l’Oise
F-95029 CERGY PONTOISE CEDEX
Expert Laboratory: ADRIA Développement
ZA Creac’h Gwen
F-29196 QUIMPER Cedex
Studied method: 3MTM Rapid Yeast and Mold PetrifilmTM plate
Reference method : - ISO 21527 - 1: Microbiology of food and animal
feeding stuffs -- Horizontal method for the
enumeration of yeasts and moulds -- Part 1:
Colony count technique in products with water
activity greater than 0.95
- ISO 21527- 2: Microbiology of food and animal
feeding stuffs -- Horizontal method for the
enumeration of yeasts and moulds -- Part 2:
Colony count technique in products with water
activity less than or equal to 0.95
Scope: Food products, environmental samples,
pet food and animal feed
Certification organism: AFNOR Certification
Analyses performed according to the COFRAC accreditation
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11 IINNTTRROODDUUCCTTIIOONN
The validation study of the 3MTM Rapid Yeast and Mold PetrifilmTM plate
method was performed in 2014 according to the EN ISO 16140 protocol and
the AFNOR technical rules.
22 MMEETTHHOODDSS PPRROOTTOOCCOOLLSS
2.1 Reference methods
The reference method is the ISO 21527 method: Horizontal method for the
enumeration of yeasts and molds, and the related parts depending on the
water activity of the analyzed sample (See Appendix 1):
- Part 1: Colony-count technique in products with water activity greater than
0.95.
- Part 2: Colony count technique in products with water activity less than or
equal to 0.95 and > 0.60.
2.2 Alternative method
The protocol of the alternative method is described in Appendix 2.
The 3M Rapid Yeast and Mold Petrifilm plate is a ready-made medium
system for the enumeration of yeasts and molds commonly found in foods.
The Petrifilm Yeast and Mold plates contain nutrient supplemented with
antibiotics, a cold-water soluble gelling agent and a dye enhance the
visualisation of growth on the plate.
Yeast colonies appear blue-green or off white and form small defined
colonies. Mold colonies tend to be larger and more diffuse; they are usually
blue in colour, but they may also assume their natural pigmentation (i.e.
black, yellow, green, etc.).
Analysis performed according to the COFRAC accreditation
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The protocol is the following:
- Inoculation with 1 ml of mother suspension or decimal dilutions. For
beverages, the undiluted products should not be plated;
- Incubation time: any time from 60 h to 72 hours.
- Incubation temperature: 25°C ± 1°C or 28°C ± 1°C.
The two incubation temperatures were tested during the study.
The incubation of the Petrifilm plate tests could be done until 120 h, in order
to offer sufficient practicability to the users. According to the AFNOR
technical rules, this was tested in the accuracy part.
33 VVAALLIIDDAATTIIOONN SSTTUUDDYY RREESSUULLTTSS
3.1 Method comparison study
3.1.1 Linearity study
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.
3.1.1.1 Food matrices and protocols
Eight matrix/strain pairs were studied. Five levels of contamination were
tested to cover a minimum, a central, a maximum and two intermediary levels
(from 50 to 106 CFU/g). 3 matrix/strain pairs were analyzed with the part 1 of
the ISO 21527 method, the 4 others with the part 2:
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Table 1
Matrix Strain Contamination levels
(CFU/g)
ISO 21527
method
part
Liver pâté Candida pseudotropicalis Adria Y3
50 – 100
500 – 1000
5.0 103 - 104
5.0 104 – 105
5.0 105 – 106
1
Liver pâté Candida norvegica Ad 1827 1
Milk powder Mucor circinelloides Ad1302 2 2
Whole egg powder Aspergillus flavus Adria M53 2
Fresh fruit juice Pichia anomala Ad1037 1
Apricot syrup Penicillium chrysogenum Ad1114 2
Pellet for pet Fusarium solani Ad 2059 2
Process water Torulaspora delbrueckii Ad 1038 1
3.1.1.2 Calculations and interpretations
The bi-dimensional graphs are given in Figure 1.
The different incubation conditions and available statistical interpretations are
summarized in Table 2.
1 For the liver pâté, a linearity study was repeated with a second Candida sp. strain (Candida norvegica): the
first one tested (Candida pseudotropicalis) didn’t give interpretable results. This was probably due to the
behavior of that specific single Candida sp strain. Indeed, various Candida spp strains were tested in the
inclusivity study: they all show expected growth and colonies. 2 As already known, Mucor strain can show an important spread on the plates. Indeed, it was not possible to
use the data provided with the higher dilutions, and unfortunately, at the lowest dilution, less than 4 colonies
were observed on the Petrifilm test. 3 An additional matrix/strain pair, i.e. whole egg powder/ Aspergillus flavus Adria M5, was tested with an Aw >
0.60, because the milk powder had an Aw value of 0.40.
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Table 2 - Incubation temperatures and time,
available statistical interpretations
Matrix Incubation temperature: 25°C Incubation temperature: 28°C
60 h 72 h 60 h 72 h
Liver pâté
(Candida pseudotropicalis Adria Y3)
No (not enough data)
Yes No (no growth)
No (no growth)
Liver pâté
(Candida norvegica Ad 1827) Yes Yes Yes Yes
Milk powder Yes *
No (impossible to enumerate (spreading))
No (impossible to enumerate (spreading))
No (impossible to enumerate (spreading))
Whole egg powder Yes Yes Yes Yes
Fresh fruit juice Yes Yes Yes Yes
Apricot syrup Yes Yes Yes Yes
Pellet for pet Yes Yes Yes Yes
Process water Yes Yes Yes Yes
* Available data but with enumerated colonies < 4 per plate (should not have been taken into account)
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Figure 1 – Linearity: bi-dimensional graphs
Incubation temperature: 25°C; Incubation time: 60 h
0,00
1,00
2,00
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4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Liver pâté / Candida norvegica-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Apricot jam-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log
(Altern
ative m
eth
od)
log(Reference method)
Milk powder 25°C-60H
Apricot syrup - 25°C - 60H
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Incubation temperature: 25°C; Incubation time: 60 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Egg powder -25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Fruit Juice -25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Feed stuff-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Process water-25°C-60H
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Incubation temperature: 25°C; Incubation time: 72 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Liver pâté-25°C-72HCandida pseudotropicalis
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Liver pâté / Candida norvegica-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00
log (A
ltern
ative m
eth
od)
log(Reference method)
Egg powder -25°C-72H
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Incubation temperature: 25°C; Incubation time: 72 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Fruit Juice -25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log(A
ltern
ative m
eth
od)
log(Reference method)
Apricot jam-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Process water-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log( (Reference method)
Feed stuff-25°C-72H
Apricot syrup - 25°C - 72H
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Incubation temperature: 28°C; Incubation time: 60 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Liver pâté / Candida norvegica-28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Egg powder -28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Fruit Juice -28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Apricot jam-28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Feed stuff-28°C-60HApricot syrup - 28°C - 60H
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Incubation temperature: 28°C; Incubation time: 60 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log (A
ltern
ative m
eth
od)
log (Rerference method)
Process water-28°C-60H
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Incubation temperature: 28°C; Incubation time: 72 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(Altern
ative m
eth
od)
log(Reference method)
Liver pâté / Candida norvegica-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (
Alt
era
nti
ve m
eth
od
)
log (Reference method)
Egg powder -28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Fruit Juice -28°C-72H
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Incubation temperature: 28°C; Incubation time: 72 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log
(A
lte
rna
tiv
e m
eth
od
)
log (Reference method)
Apricot jam-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (A
ltern
ative m
eth
od)
log (Reference method)
Feed stuff-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00
log (
Alt
ern
ati
ve m
eth
od
)
log (Reference method)
Process water-28°C-72H
Apricot syrup - 28°C - 72H
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3.1.1.3 Statistical results
The statistical interpretation results obtained per tested matrix are presented
in Table 3. The regression lines are provided in Figure 2.
3.1.1.4 Conclusion
The linearity test is not significant (P > 5%) in all cases, except for:
- liver pâté (Candida norvegica): 28°C, 60 h (P % = 2),
- process water: 25°C, 72 h (P % = 0),
- process water: 28°C, 72 h (P % = 2),
- egg powder: 28°C, 60 h (P % = 2),
- egg powder: 28°C, 72 h (P % = 3).
The regression coefficients are all higher than 0.99, confirming that the
linearity study provides satisfying results.
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Table 3 – Statistical interpretations
Incubation temperature
Incubation time Matrix R Selected
regression Rob F
Critical value
P% Correlation coefficient
Regression equation
25°C
60 h
Liver pâté 4 One level is missing due to atypical behavior of the strain
Liver pâté 5 0.49 OLS2 1.072 5.51 44 0.999 Log Ref. = 1.034 log Alt. - 0.048
Milk powder 2.57 OLS1 1.211 5.41 40 0.992 Log Alt. = 0.979 log Ref. - 0.138
Egg powder 3.19 OLS1 0.000 5.41 100 0.998 Log Alt. = 1.019 log Ref. - 0.192
Fresh fruit juice 0.28 OLS2 0.589 5.41 65 0.997 Log Ref. = 0.998 log Alt. - 0.188
Apricot syrup 0.18 OLS2 0.000 5.41 100 0.997 Log Ref. = 1.045 log Alt. - 0.124
Pellet for pet 1.50 GMFR 0.000 5.41 100 0.999 Log Alt. = 0.974 log Ref. - 0.012
Process water 1.13 GMFR 1.5333 5.41 32 1.000 Log Alt. = 1.001 log Ref. - 0.100
72 h
Liver pâté 4 1.22 GMFR 5.297 4.53 4 0.999 Log Alt. = 0.942 log Ref. + 0.015
Liver pâté 5 0.49 OLS2 1.072 5.41 44 0.999 Log Ref. = 1.034 log Alt. - 0.048
Milk powder One level is missing for interpretation due to strain spreading
Egg powder 3.19 OLS1 0.000 5.41 100 0.998 Log Alt. = 1.020 log Ref. - 0.192
Fresh fruit juice 0.28 OLS2 0.650 5.41 62 0.997 Log Ref. = 0.996 log Alt. - 0.18
Apricot syrup 0.18 OLS2 0.000 5.41 100 0.996 Log Ref. = 1.041 log Alt. - 0.113
Pellet for pet 0.85 GMFR 0.000 5.41 100 1.000 Log Alt. = 0.935 log Ref. + 0.206
Process water 0.52 GMFR 19.387 5.41 0 0.999 Log Alt. = 1.012 log Ref. - 0.099
* Less than 4 colonies per plate (should not have been taken into account)
4 Inoculated strain: Candida pseudotropicalis
5 Inoculated strain: Candida norvegica
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Incubation temperature
Incubation time Matrix R Selected
regression Rob F
Critical value
P% Correlation coefficient
Regression equation
28°C
60 h
Liver pâté 6 No growth
Liver pâté 7 1.23 GMFR 8.631 5.41 2 0.998 Log Alt. = 0.996 log Ref. - 0.066
Milk powder One level is missing for interpretation due to strain spreading
Egg powder 0.70 GMFR 8.033 5.41 2 1.000 Log Alt. = 0.933 log Ref. + 0.224
Fresh fruit juice 0.80 GMFR 0.524 5.41 68 0.999 Log Ref. = 0.945 log Alt. - 0.396
Apricot syrup 0.18 OLS2 0.000 5.41 100 0.996 Log Ref. = 1.032 log Alt. - 0.049
Pellet for pet 0.40 OLS2 0.133 5.41 94 0.999 Log Ref. = 1.041 log Alt. - 0.177
Process water 1.13 GMFR 0.333 5.41 80 1.000 Log Alt. = 0.986 log Ref. + 0.036
72 h
Liver pâté 4 No growth
Liver pâté 5 1.65 GMFR 2.403 5.41 18 0.998 log Alt. = 0.998 log Ref. - 0.062
Milk powder One level is missing for interpretation due to strain spreading
Egg powder 0.69 GMFR 7.705 5.41 3 1.000 Log Alt. = 0.932 log Ref. + 0.239
Fresh fruit juice 0.80 GMFR 0.521 5.41 68 0.999 Log Alt. = 0.945 log Ref. + 0.396
Apricot syrup 0.18 OLS2 0.000 5.41 100 0.996 Log Ref. = 1.034 log Alt. - 0.055
Pellet for pet 0.64 GMFR 1.172 5.41 41 1.000 Log Alt. = 0.955 log Ref. + 0.247
Process water 0.56 GMFR 9.258 5.41 2 1.000 Log Alt. = 0.991 log Ref. + 0.026
* Less than 4 colonies per plate (should not have been taken into account)
6 Inoculated strain: Candida pseudotropicalis
7 Inoculated strain: Candida norvegica
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Figure 2 – Linearity: regression lines
Incubation temperature: 25°C; Incubation time: 60 h
y = 0,9792x - 0,1382
0,00
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7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Milk powder 25°C 60H
y = 1,0342x - 0,0489
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Liver pâté / Candida norvegica25°C-60H
y = 1,0187x - 0,1917
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Egg powder-25°C-60H
y = 0,9986x - 0,1883
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Fruit Juice-25°C-60H
y = 1,0454x - 0,1241
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Apricot jam-25°C-60H
y = 0,9739x - 0,0115
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ativ
e
Reference
Feed stuff-25°C-60H
y = 1,0098x - 0,0997
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Process water-25°C-60H
Apricot syrup - 25°C - 60H
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Incubation temperature: 25°C; Incubation time: 72 h
y = 0,9422x + 0,0149
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Liver pâté-25°C-72HCandida pseudotropicalis
y = 1,0342x - 0,0489
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Liver pâté / Candida norvegica25°C-72H
y = 1,02x - 0,1923
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Egg powder 25°C-72H
y = 0,9959x - 0,18
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Fruit Juice-25°C-72H
y = 1,0409x - 0,1126
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Apricot jam-25°C-72H
y = 0,9354x + 0,2058
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ativ
e
Reference
Feed stuff-25°C-72H
y = 1,012x - 0,0992
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Process water-25°C-72H
Apricot syrup - 25°C - 72H
3M
ADRIA Développement 21/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 28°C; Incubation time: 60 h
y = 0,9955x - 0,0658
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Référence
Liver pâté / Candida norvegica28°c-60H
y = 0,9331x + 0,2243
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Egg powder-28°C-60H
y = 0,9451x + 0,396
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Fruit juice 28°C 60H
y = 1,0323x - 0,0486
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Apricot jam-28°C-60H
y = 1,0406x - 0,1767
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Feed stuff-28°C-60H
y = 0,986x + 0,0363
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Process water-28°C-60H
Apricot syrup - 28°C - 60H
3M
ADRIA Développement 22/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 28°C; Incubation time: 72 h
y = 0,9982x - 0,0617
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Référence
Liver pâté / Candida norvegica28°c-72H
y = 0,9315x + 0,2385
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Egg powder-28°C-72H
y = 0,9451x + 0,396
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Altern
ative
Reference
Fruit juice 28°C 72H
y = 1,0338x - 0,0546
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Réfé
ren
ce
Alternative
Apricot jam-28°C-72H
y = 0,9551x + 0,2469
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Feed Stuff - 28°C - 72H
y = 0,9914x + 0,0255
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
Alt
ern
ati
ve
Reference
Process water-28°C-72H
Apricot syrup - 28°C - 72H
3M
ADRIA Développement 23/70 September 30, 2014
3M RYM Summary Report (Version 0)
3.1.2 Relative accuracy
The relative accuracy is the closeness of agreement between a test result and the accepted reference
value.
Relative 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, it is the ability of the method to measure
exactly a given analyte, or its amount, within the sample without interference from non-target
components such as matrix effect or background noise.
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 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).
3.1.2.1 Number and nature of samples
Six categories were tested, with three food types minimum, 9 types were
analyzed with the part 1 of the ISO 21527 method, the 9 others with the
part 2.
3M
ADRIA Développement 24/70 September 30, 2014
3M RYM Summary Report (Version 0)
Table 4 - Distribution per tested category and type
Category Type Item (examples) ISO 21527
method part
Number of analysed
samples
Exploitable results at 25°C Exploitable results at 28°C
60H 72H 120H 60H 72H 120H
Meat and fish products
Cooked Terrines, ham, deli turkey 1 8 7 7 7 7 7 7 Cured, marinated, smocked Bacons, roll herrings, smoked fishes 1 8 4 5 5 5 5 5 Dehydrated Ambient culinary products 2 6 3 3 4 2 3 4
Total 22 14 15 16 14 15 16
Dairy and egg products
Pasteurized and fermented Creams, yoghurts, butters, grated cheeses 1 17 13 13 13 11 13 13
Pasteurized Dairy desserts, ice creams, drinks, liquid eggs, fresh pasta
1 4 2 2 2 2 2 2
Dehydrated Milk powders, egg powders 2 4 3(2) 3(2) 4(2) 3(2) 4(2) 3(2)
Total 25 18 (17) 18 (17) 19
(17) 18
(17) 19
(17) 18
(17)
Fruits, vegetables and
cereals
Juices Fruit juices 1 6 4 3 3 3 3 3 Dehydrated Spices, aromatic herbs, cereals 2 12 8(4) 8(4) 10(5) 10(6) 9(6) 10(7) Syrups, concentrates Jams, fruits concentrates 2 14 6 7 7 7 7 7
Total 32 18
(14) 18
(14) 20
(15) 20 (16) 19 (16) 20 (17)
Chocolate and bakery products
Patisserie & bakery products
Confectionaries, pancakes, breads, raw puff pastries
1 15 6 6 6 6 6 6
Dry & sugared low moisture Cakes, pralines, marzipans 2 4 2 2 2 2 2 2 Dry & sugared low moisture Biscuits, chocolates, candy syrups 2 8 5 6 4 5 5 4
Total 27 13 14 12 13 13 12
Feed stuffs
Dehydrated Cereals, flours 2 4 4 4 3 4 4 4 Dry Pellets, treats, low moisture croquettes 2 5 3 3 4(3) 4(3) 4(3) 4(3) Wet High moisture croquettes, sausages 1 5 4 4 4 4 2 4
Total 14 11 11 11 (10) 12 (11) 12 (11) 12 (11)
Environmental samples
Equipments and surfaces Swabs, sponges 1 11 3 3 3 3 3 3 Production environment Dusts 2 5 4 4 4 4 3 3 Waters used in the manufacturing process
Recycled washing waters, process waters 1 7 4 4 4 4 4 4
Total 23 11 11 11 11 10 10
TOTAL 143 85 (80) 87 (82) 89 (83) 88 (82) 88 (82) 88 (82)
( ): number of samples analyzed with Aw > 0.6
3M
ADRIA Développement 25/70 September 30, 2014
3M RYM Summary Report (Version 0)
3.1.2.2 Artificial contaminations
18 samples were artificially contaminated. Depending on the tested
conditions, 13 samples or less gave exploitable results. Mold spores were
inoculated and 4 with yeast cells. Note that several treatments were tested to
injure the yeast strains, but no effect was observed. The percentage of
naturally contaminated samples used in the interpretation is higher than 80%
whatever the conditions.
The inoculated strains, the inoculation level are provided in the Appendix 3.
3.1.2.3 Results
The samples were analyzed in duplicate by the reference and the alternative
methods. The contamination range is presented below:
Table 5
Categories
Contamination level
25°C 28°C
60 h 72 h 120 h 60 h 72 h 120 h
Meat and
fish products
1.60 to 7.66 1.70 to 7.66 1.70 to 7.51 1.48 to 6.91 1.60 to 7.66 1.60 to 7.66
Dairy and
egg products
1.60 to 7.67 1.70 to 7.67 1.70 to 7.67 1.78 to 7.67 1.78 to 7.67 1.78 to 7.67
Fruits, vegetables
and eggs
1.60 to 7.83 1.70 to 7.83 1.70 to 7.83 1.78 to 7.83 1.78 to 7.83 1.78 to 7.83
Chocolate and
bakery products
1.60 to 7.71 1.60 to 7.71 1.78 to 7.71 1.60 to 7.71 1.60 to 7.71 1.60 to 7.71
Feed stuff 1.78 to 4.81 1.68 to 4.81 1.78 to 4.83 1.60 to 4.83 1.78 to 4.83 1.78 to 4.83
Environmental
samples
2.23 to 6.32 2.23 to 6.32 2.23 to 6.34 1.85 to 6.32 1.85 to 6.32 2.20 to 6.32
All products 1.60 to 7.83 1.60 to 7.83 1.60 to 7.83 1.48 to 7.72 1.60 to 7.83 1.60 to 7.83
All products Aw > 0.6 1.60 to 7.83 1.60 to 7.83 1.60 to 7.83 1.48 to 7.83 1.60 to 7.83 1.60 to 7.83
The bi-dimensional graphs are given figure 3.
3M
ADRIA Développement 26/70 September 30, 2014
3M RYM Summary Report (Version 0)
3.1.2.4 Statistical interpretation
The results of the statistical interpretation are given in Table 4. Regression
lines (graph and equation representations) for each food category and for all
matrices are presented Figure 3.
Figure 3 – Relative accuracy: bi-dimensional graphs
Incubation temperature: 25°C; Incubation time: 60 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Meat and fish products-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Dairy and egg products-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Fruits and vegetables-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Chocolate and bakery products-25°C-60H
3M
ADRIA Développement 27/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 25°C; Incubation time: 60 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Feed stuffs-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Environmental samples-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-25°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-25°C-60H AwAw > 0.6
3M
ADRIA Développement 28/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 25°C; Incubation time: 72 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Meat and fish products-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Dairy and egg products-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Fruits and vegetables-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Chocolate and bakery products-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Feed stuffs-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Environmental samples-25°C-72H
3M
ADRIA Développement 29/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 25°C; Incubation time: 72 h
Incubation temperature: 25°C; Incubation time: 120 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-25°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-25°C-72HAw > 0.6
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-25°C-120H
3M
ADRIA Développement 30/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 28°C; Incubation time: 60 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Meat and fish products-28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Dairy and egg products-28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Fruits and vegetables-28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Chocolate and bakery products-28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Feed stuffs -28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Environmental samples-28°C-60H
3M
ADRIA Développement 31/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 28°C; Incubation time: 60 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-28°C-60H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-28°C-60H-Aw>0,6
3M
ADRIA Développement 32/70 September 30, 2014
3M RYM Summary Report (Version 0)
Incubation temperature: 28°C; Incubation time: 72 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Meat and fish products-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Dairy and egg products-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Fruits and vegetables-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Chocolate and bakery products-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
Feed stuffs -28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00
log
(A
ltern
ati
ve m
eth
od
)
log (Reference method))
Environmental samples-28°C-72H
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Incubation temperature: 28°C; Incubation time: 72 h
Incubation temperature: 28°C; Incubation time: 120 h
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-28°C-72H
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-28°C-72HAw > 0.6
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00
log
(A
ltern
ative m
eth
od)
log (Reference method))
All products-28°C-120H
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Table 6 – Statistical interpretation
Incubation temperature
Incubation time
Food category n R Regression a t(a) b t(b) critical
T
P%
Bias
Repeatability limit
Slope Intercept Reference
method Alternative
method
25°C
60 h
Meat and fish products 14 1,21 GMFR -0.065 0.463 0.990 0.311 2.179 76 65 -0.088 0.206 0.254
Dairy and egg products 18 0,76 GMFR -0.384 3.278 1.044 1.984 2.120 6 0 -0.116 0.221 0.172
Fruits vegetables and eggs 18 0,79 GMFR -0.658 2.061 1.132 1.821 2.120 9 6 -0.158 0.255 0.205
Chocolate and bakery products 13 0,98 GMFR 0.187 0.630 0.944 0.862 2.201 41 54 -0.037 0.174 0.174
Feed stuff 11 1,01 GMFR -0.170 0.367 1.043 0.310 2.262 76 72 -0.066 0.217 0.224
Environmental samples 11 0.42 OLS2 0.475 1.388 0.933 0.782 2.262 44 18 -0.226 0.212 0.090
All products 85 0.89 GMFR -0.220 1.903 1.025 0.959 1.989 34 6 -0.093 0.213 0.194
All products Aw>0.6 80 0.95 GMFR -0.207 1.684 1.023 0.838 1.991 40 10 -0.088 0.213 0.206
72 h
Meat and fish products 15 0,86 GMFR -0.144 1.128 1.015 0.507 2.160 62 28 -0.056 0.220 0.186
Dairy and egg products 18 0,81 GMFR -0.289 3.030 1.032 1.737 2.120 10 1 -0.103 0.221 0.184
Fruits vegetables 18 0,64 GMFR -0.312 1.236 1.087 1.461 2.120 16 23 -0.067 0.252 0.165
Chocolate and bakery products 14 1,19 GMFR 0.055 0.159 0.968 0.421 2.179 68 88 -0.042 0.191 0.232
Feed stuff 11 0,91 GMFR 0.220 0.627 0.945 0.526 2.262 61 55 -0.066 0.217 0.201
Environmental samples 11 0.62 GMFR -0.181 0.338 0.986 0.107 2.262 92 74 -0.235 0.212 0.135
All products 87 0.80 GMFR -0.132 1.226 1.014 0.585 1.988 56 22 -0.068 0.213 0.174
All products Aw>0.6 82 0.80 GMFR -0.127 1.113 1.013 0.552 1.990 60 27 -0.069 0.210 0.172
120 h All products 89 0.95 GMFR -0.197 2.239 1.033 1.681 1.988 10 3 -0.065 0.213 0.207
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Incubation temperature
Incubation time
Food category n R Regression a t(a) b t(b) critical
T
P% Bias
Repeatability limit
Slope Intercept Reference
method Alternative
method
28°C
60 h
Meat and fish products 14 1.14 GMFR -0.359 1.804 1.024 0.512 2.179 62 10 -0.252 0.206 0.239
Dairy and egg products 18 1.42 GMFR -0.356 2.442 1.038 1.377 2.120 19 3 -0.113 0.221 0.321
Fruits vegetables 20 0.84 GMFR -0.330 1.090 1.027 0.370 2.101 72 29 -0.206 0.344 0.294
Chocolate and bakery products 13 0.77 GMFR -0.132 0.230 0.952 0.385 2.201 71 82 -0.055 0.174 0.137
Feed stuff 12 0.59 GMFR -0.705 0.501 1.109 0.272 2.228 79 63 -0.064 0.200 0.121
Environmental samples 11 1.92 GMFR -0.206 0.332 0.928 0.498 2.262 63 75 -0.566 0.212 0.415
All products 88 1.23 GMFR -0.360 2.023 1.016 0.390 1.988 70 5 -0.160 0.210 0.264
All products Aw>0.6 82 1.23 GMFR -0.262 1.637 1.004 0.125 1.990 90 11 -0.149 0.210 0.264
72 h
Meat and fish products 15 1.04 GMFR -0.268 1.831 1.021 0.616 2.160 55 9 -0.128 0.213 0.227
Dairy and egg products 19 1.27 GMFR -0.463 2.954 1.056 1.831 2.110 8 1 -0.127 0.182 0.237
Fruits vegetables 19 0.81 GMFR -0.241 0.967 1.038 0.623 2.110 54 35 -0.154 0.395 0.327
Chocolate and bakery products 13 0.82 GMFR -0.720 1.259 1.070 0.566 2.201 58 23 -0.076 0.207 0.174
Feed stuff 12 0.76 GMFR 0.051 0.047 0.926 0.243 2.228 81 96 -0.053 0.200 0.154
Environmental samples 10 1.82 GMFR -0.247 0.391 0.971 0.197 2.306 85 71 -0.386 0.226 0.420
All products 88 1.30 GMFR -0.340 2.211 1.028 0.799 1.988 43 3 -0.128 0.215 0.284
All products Aw>0.6 82 1.30 GMFR -0.263 1.896 1.018 0.575 1.990 57 6 -0.118 0.215 0.284
120 h All products 88 1.04 GMFR -0.249 1.856 1.021 0.685 1.988 49 7 -0.105 0.218 0.232
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Figure 4 – Relative accuracy: regression lines
Incubation temperature: 25°C; Incubation time: 60 h
y = 0,9901x - 0,0649
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Meat and fish products - 25°C, 60 hGMFR Regression
y = 1,0443x - 0,3837
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Dairy and egg products - 25°C, 60 hGMFR Regression
y = 1,1316x - 0,6584
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Fruits and vegetables - 25°C, 60 hGMFR Regression
y = 0,944x + 0,1871
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Chocolate and bakery products - 25°C, 60 hGMFR Regression
y = 1,0426x - 0,1704
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Feed stuff - 25°C, 60 hGMFR Regression
y = 0,9332x + 0,4745
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Refe
ren
ce
Alternative
Environmental samples, 25°C, 60 hOLS2 Regression
y = 1,0247x - 0,2203
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 25°C, 60 hGMFR Regression
y = 1,0225x - 0,2066
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 25°C, 60 h - AwGMFR Regression
Aw > 0.6
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Incubation temperature: 25°C; Incubation time: 72 h
y = 1,0151x - 0,1444
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Meat and fish products - 25°C, 72 hGMFR Regression
y = 1,0316x - 0,2889
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Dairy and egg produts - 25°C, 72 hGMFR Regression
y = 1,0865x - 0,3117
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Fruits and vegetables - 25°C, 72 hGMFR Regression
y = 0,9683x + 0,0545
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Chocolate and bakery products - 25°C, 72 hGMFR Regression
y = 0,9453x + 0,2199
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Feed stuff - 25°C, 72 hGMFR Regression
y = 0,9861x - 0,1809
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Environmental samples - 25°C, 72 hGMFR Regression
y = 1,0143x - 0,1322
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 25°C, 72 hGMFR Regression
y = 1,0132x - 0,127
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 25°C, 72 h - AwGMFR Regression
Aw > 0.6
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Incubation temperature: 25°C; Incubation time: 120 h
y = 1,0332x - 0,1967
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 25°C, 120 hGMFR Regression
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Incubation temperature: 28°C; Incubation time: 60 h
y = 1,0236x - 0,3586
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Meat and fish products - 28°C, 60 hGMFR Regression
y = 1,0383x - 0,3558
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Dairy and egg products - 28°C, 60 h GMFR Regression
y = 1,0274x - 0,3295
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Fruits and vegetables - 28°C, 60 hGMFR Regression
y = 0,9517x - 0,1318
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Chocolate and bakery products - 28°C, 60 hGMFR Regression
y = 1,1091x - 0,7048
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Feed stuff - 28°C, 60 hGMFR Regression
y = 0,9276x - 0,2057
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Environmental samples - 28°C, 60 hGMFR Regression
y = 1,0175x - 0,3582
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 28°C, 60 hGMFR Regression
y = 1,0044x - 0,2622
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 28°C, 60 h - AwGMFR Regression
Aw > 0.6
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Incubation temperature: 28°C; Incubation time: 72 h
Incubation temperature: 28°C; Incubation time: 120 h
y = 1,0208x - 0,268
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Meat and fish products - 28°C, 72 hGMFR Regression
y = 1,0559x - 0,4627
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Dairy and egg products - 28°C, 72 hGMFR Regression
y = 1,0376x - 0,2406
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Fruits and vegetables - 28°C, 72 hGMFR Regression
y = 1,0689x - 0,7198
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Chocolate and bakery products - 28°C, 72 hGMFR Regression
y = 0,926x + 0,0506
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Feed stuff - 28°C, 72 hGMFR Regression
y = 0,971x - 0,2471
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
Environmental samples - 28°C, 72 hGMFR Regression
y = 1,0277x - 0,3399
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 28°C, 72 hGMFR Regression
y = 1,0171x - 0,2632
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 28°C, 72 h - AwGMFR Regression
Aw > 0.6
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3.1.2.5 Discussion
The statistical tests are not significant concluding to the equivalence between
the compared methods, except in the following cases:
Incubation temperature: 25°C
- Dairy and egg products:
* 60 h, intercept P % = 0, with log Alt. = 1.04 log Ref. - 0.38
* 72 h, intercept P % = 1, with log Alt. = 1.03 log Ref. - 0.29
- All products:
* 120 h, intercept P % = 3, with log Alt. = 1.03 log Ref. - 0.20
Incubation temperature: 28°C
- Dairy and egg products:
* 60 h, intercept P % = 3, with log Alt. = 1.04 log Ref. - 0.36
* 72 h, intercept P % = 1, with log Alt. = 1.06 log Ref. - 0.46
- All products:
* 72 h, intercept P % = 3, with log Alt. = 1.02 log Ref. - 0.25
But looking at the graphs and the regression equations, satisfying results are
observed in all the cases, whatever time and temperature incubation.
The target analyte is here an important micro-organism group, and some of
them may have clearly different behaviors. Therefore, their recovery may be
sometime better with the Petrifilm Test growth conditions, or in the opposite
y = 1,0201x - 0,2479
0,00
2,00
4,00
6,00
8,00
0,00 2,00 4,00 6,00 8,00
Alt
ern
ati
ve
Reference
All products - 28°C, 120 hGMFR Regression
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with the ISO method growth conditions. Indeed, few outliers are observed
with difference in the enumeration data higher than 0.5 log CFU/g between
the compared methods:
- At 25°C, 13 outliers after 60 h incubation time (8 with a negative bias and
5 with a positive bias);
- At 28°C, 19 outliers are observed (18 with a negative bias and 1 with a
positive bias).
3.1.3 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
3.1.3.1 Protocol
The detection and quantification limits were determined with a pure culture of
Saccharomyces cerevisiae Ad 999 and Aspergillus niger ATCC 16404
strains.
Five different levels of inoculation were tested, with six replicates per level,
i.e. a total of 30 analyses by the alternative method.
Quantification limit was calculated for six independent blank samples
determinations.
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3.1.3.2 Results
These data are intrinsic to the alternative method and are presented in the
following tables:
Table 7 - Yeasts
Incubation temperature : 25°C
Inoculation level
Number of positive samples
Standard deviation
(S0)
Bias X0
(Xoi median)
Calculation formula
Calculated values (cfu/ml)
LC(1ml) 1.65 So + Xo 1.4
T0 0 / /
LOD(1ml) 3.3 So + Xo 2.3
T1 0/6 0.548 0.5
LOQ(1ml) 10So + Xo 6.0
T2 3/6 0.548 0.5
T3 5/6 1.033 1.0
T4 6/6 1.643 4.0
T5 6/6 3.983 8.0
Incubation temperature : 28°C
Inoculation level
Number of positive samples
Standard deviation
(S0)
Bias X0
(Xoi median)
Calculation formula
Calculated values (cfu/ml)
LC(1ml) 1.65 So + Xo 2.2
T0 0 / /
LOD(1ml) 3.3 So + Xo 3.5
T1 1/6 0.408 0.0
LOQ(1ml) 10So + Xo 8.5
T2 4/6 0.753 1.0
T3 3/6 0.548 0.5
T4 6/6 3.347 4.0
T5 6/6 2.639 8.0
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Table 8 - Molds
Incubation temperature : 25°C
Inoculation level
Number of positive samples
Standard deviation
(S0)
Bias X0
(Xoi median)
Calculation formula
Calculated values (cfu/ml)
LC(1ml) 1.65 So + Xo 1.4
T0 0 / /
LOD(1ml) 3.3 So + Xo 2.3
T1 0/6 0.000 0.0
LOQ(1ml) 10So + Xo 6.0
T2 3/6 0.548 0.5
T3 4/6 0.753 1.0
T4 3/6 0.548 0.5
T5 6/6 0.837 2.0
Incubation temperature : 28°C
Inoculation level
Number of positive samples
Standard deviation
(S0)
Bias X0
(Xoi median)
Calculation formula
Calculated values (cfu/ml)
LC(1ml) 1.65 So + Xo 1.4
T0 0 / /
LOD(1ml) 3.3 So + Xo 2.3
T1 0/6 0.000 0.0
LOQ(1ml) 10So + Xo 6.0
T2 2/6 0.837 0.0
T3 2/6 0.516 0.0
T4 3/6 0.548 0.5
T5 5/6 0.816 1.5
3.1.4 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 and are obtained from the results of the
linearity study.
Sensitivity patterns obtained for tested (matrix/strain) pairs are presented in
figure 5.
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Figure 5 – Sensitivity patterns for the tested (matrix/strain) pairs
0,0%
5,0%
10,0%
15,0%
20,0%
25,0%
30,0%
35,0%
40,0%
0 1 2 3 4 5 6 7
CV
(<x(y
)>)
Reference method log cfu/g (x)
Precision profiles obtained for the different food matrices25°C-60H
Milk powder
Egg powder
Apricot jam
Feed stuff
Process water
Liver pâté/ Candida norvegica
Yoghourt
0,0%
5,0%
10,0%
15,0%
20,0%
25,0%
0 1 2 3 4 5 6 7
CV
(<x(y
)>)
Reference method log cfu/g (x)
Precision profiles obtained for the different food matrices25°C-72H
Liver pâté/Candia pseudotropicalis
Egg powder
Apricot jam
Feed stuff
Process water
Liver pâté/Candia norvegica
Yoghourt
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0,0%
5,0%
10,0%
15,0%
20,0%
25,0%
30,0%
0 1 2 3 4 5 6 7
CV
(<x(y
)>)
Reference method log cfu/g (x)
Precision profiles obtained for the different food matrices28°C-60H
Egg powder
Apricot syrup
Feed stuff
Process water
Liver pâté/ Candida norvegica
Yoghourt
0,0%
5,0%
10,0%
15,0%
20,0%
25,0%
30,0%
35,0%
40,0%
45,0%
0 1 2 3 4 5 6 7
CV
(<x(y
)>)
Reference method log cfu/g (x)
Precision profiles obtained for the different food matrices28°C-72H
Egg powder
Apricot syrup
Feed stuff
Process water
Liver pâté/ Candida norvegica
Yoghourt
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3.1.5 Specificity – Selectivity
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.
The selectivity is defined as a measure of the degree of non-interference in the presence of
non-target analytes. A method is selective if it can be used to detect the analyte under
examination, and that a guarantee can be provided that the detected signal can only be a
product by that specific analyte.
3.1.5.1 Protocol
The target strains were grown in Sabouraud broth at 25°C and spreaded
onto the different media in duplicate. The non target strains were grown in
appropriate media and appropriate temperatures; they were spreaded in
duplicate on the different media.
3.1.5.2 Results
The raw data are given in the Appendix 4.
The target analyte is a wide range of microorganisms. Indeed, some
differences may be observed in the recovery ability, depending on the growth
conditions.
Inclusivity
43 positive strains were tested. Note that various Candida spp strains were
tested to confirm the singularity of the Candida pseudotropicalis strain tested
in the linearity PART. The provided data confirms that hypothesis.
- Incubation temperature: 25°C
For 37 strains, same results were observed on DG18, DRBC, YGC and
3MTM Rapid Yeast and Mold PetrifilmTM, whatever incubation time. Some
differences of behavior are mentioned below.
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Table 9 - Growth after different incubation times at 25°C
Strains 60 h 72 h
Aspergillus fumigatus Adria 1053 - +
Aspergillus versicolor Adria 1057 - +
Byssochlamys nivea Ad 1316 - +*
Paecilomyces variotii Adria M6 - +
Rhototorula sloffiae Adria 1031 - -
Wallemia sebi Adria 1627 - -
* small colonies
4 strains recovered after 72 h incubation time; Rhototorula sloffiae and
Wallemia sebi didn’t grow whatever the incubation time. Note that
Wallemia sebi Adria 1627 grews only on DG18 media.
2 other strains didn’t grow on DRBC medium (Mucor Plumbeus Adria M10
and Saccharomyces rouxii Ad 2058).
- Incubation temperature: 28°C
37 strains showed a same recovery whatever the tested conditions.
6 strains didn’t recover or showed small colonies on 3MTM Rapid Yeast and
Mold PetrifilmTM plate; they are listed below:
Table 10 - Growth after different incubation times at 28°C
Strains 60 h 72 h
Aspergillus versicolor Adria 1057 - +
Byssochlamys nivea Ad 1316 +* +
Pichia delfensis CLIB 291 - -
Rhototorula sloffiae Adria 1031 +* +*
Penicillium expansum UMIP 135082 +/- +/-
Wallemia sebi Adria 1627 - -
* small colonies
Exclusivity
20 strains were tested; no growth was observed for all the tested strains,
whatever the incubation time and temperature.
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3.2 Practicability
The alternative method practicability was evaluated according to the AFNOR
criteria relative to method comparison study.
Packaging of test
components
The components needed for analysis are the 3MTM PetrifilmTM Rapid
Yeast and Mold Count Plate.
Volume of reagents One pouch contains 25 3MTM PetrifilmTM Plates.
Storage conditions and
shelf-life
Unopened 3MTM PetrifilmTM Plates are stored refrigerated or frozen
(- 20 to 8°C). Prior to use, the pouches are allowed to come to room
temperature before opening (20 - 25°C). The shelf-life is given on the
pouch.
Utilisation modalities after
initial use
Opened pouches are stored in a cool dry place (20 - 25+C) for no
longuer than 4 weeks.
Specific equipment No specific equipment is required.
Reagents ready-to-use or for
constitution The 3MTM PetrifilmTM Plates are ready-to-use.
Training period for operator
with no experience of the
method
Half a day is required for technician with microbiology knowledge
Handling time and flexibility
as a function of number of
samples to be analysed (in
minutes) for 10 samples
Steps
Reference method
ISO 21527-1
or ISO 21527-2
Alternative method
RYM method
Sampling 30 30
Stomaching 15 15
Dilution, inoculation with
ready-to-use plates* 30* 20
Reading 15 15
Total / sample 9 8
*When using not ready-to-use plates, please add the time required to prepare and sterilize the culture medium,
and to prepare and dry the plates
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Time to result Steps Reference method Alternative
method
RYM method ISO 21527-1 ISO 21527-2
Sampling Day 0 Day 0 Day 0
Inoculation Day 0 Day 0 Day 0
Enumeration Day 5 Day 7 60 h as a minimum
and up to 120 hours
Technician background Technician qualified in microbiology
Common step with the
reference method Sampling step
Traceability of the results No specific traceability procedure is required
Maintenance No specific maintenance is required.
The use of the 3MTM PetrifilmTM Rapid Yeast and Mold Count Plates reduces
the handling time.
Time to results is significantly decreased with the alternative method in
comparison to the ISO 21527 - Parts 1 and 2: 60 hours are needed for
enumeration instead of five or seven days for the ISO standards.
In comparison to the ISO 21527-1 and 2 standards, the 3MTM PetrifilmTM
Rapid Yeast and Mold Count Plate decreases the volume of materials and,
indeed the volume required at the incubation step and the volume of wastes.
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3.3 Inter-laboratory study
3.3.1 Study organisation
Samples were sent to 16 laboratories.
Dairy based dessert samples were inoculated with Saccharomyces
cerevisiae Ad 999, isolated from cheese.
Inoculation levels targeted were:
- Level 0: <10 CFU/g,
- Level 1: 100 – 1 000 CFU/g,
- Level 2: 1 000 – 10 000 CFU/g,
- Level 3: 10 000 – 100 000 CFU/g.
Each laboratory received 8 samples of 10 g, i.e. 2 samples per inoculation
level. Furthermore, one non-inoculated sample was added to the package for
total viable count microflora enumeration by the ISO 4833 method.
Blind 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 register was added to the
package in order to register temperature profile during transport and package
delivery.
Samples were shipped in 48h to laboratories of the collaborative 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 method and the reference method
(ISO 21527-1).
In order to evaluate the Saccharomyces cerevisiae Ad 999 strain variability
during transport, enumerations were performed at different time, i.e.
inoculation time, after 24 h and 48 h of storage at 2°C – 8°C.
The collaborative study instructions were sent on April 29, 2014.
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3.3.2 Verification of experimental parameters
3.3.2.1 Strain stability during transport
In order to evaluate the Saccharomyces cerevisiae Ad 999 strain stability
during transport, bacterial count of samples were checked at different times,
i.e. inoculation time, after 24 h and 48 h of storage at 4°C.
Six samples (3 contamination levels x 2 samples) were enumerated. The
results are reported in table 11.
Table 11 – Saccharomyces cerevisiae count with ISO 21527 method –
Part 1 (in CFU/g)
Day of
analysis
Inoculation
level
Saccharomyces cerevisiae enumeration
(cfu/g)
Reference method Alternative method
Replicate1 Replicate2 25°C 28°C
Replicate 1 Replicate 2 Replicate 1 Replicate 2
D0
Level 1 680 580 460 440 430 430
Level 2 4800 6700 4400 4400 3500 3500
Level 3 61000 53000 49000 48000 49000 52000
D1
Level 1 420 500 410 300 390 550
Level 2 5500 4100 4500 4000 4700 4300
Level 3 45000 66000 51000 40000 60000 45000
D2
Level 1 480 520 560 560 590 540
Level 2 6400 4700 5700 4800 4500 4000
Level 3 41000 55000 43000 54000 56000 47000
No evolution of the inoculated strain was observed between Day 0 and
Day 2.
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3.3.2.2 Logistic conditions
Temperature conditions are given below:
Table 12 - Sample temperatures at receipt
Laboratories Temperature measured
by the probe (°C)
Temperature measured
at receipt (°C)
Receipt date
and time
A 2.5 3.5 20/05/2014 14h45
B 4.0 5.5 20/05/2014 11h30
C 4.5 6.7 20/05/2014 12h00
D 6.5 7.8 20/05/2014 11h55
E 5.5 6.8 20/05/2014 12h00
F 2.0 3.4 20/05/2014 11h00
G 6.0 7.5 21/05/2014 13h00
H 3.5 6.3 20/05/2014 16h15
I 3.0 4.6 20/05/2014 11h30
J 2.5 8.0 21/05/2014 08h15
K 2.5 4.0 20/05/2014 /
L 4.0 3.3 20/05/2014 09h00
M 3.0 4.2 20/05/2014 10h30
N 6.7 / 20/05/2014 06h31
O 3.0 4.3 20/05/2014 13h00
P 11.5 12.0 21/05/2014 08h15
All the labs received their package in good conditions, except Lab P which
received it at 12°C.
3.3.3 Quality Controls and conclusion
Two labs didn’t store the temperature probe with the samples between
reception and analysis, but they confirmed that the samples were stored at
the right temperature (2 – 8°C).
For one lab (L), incubators were out of use the day before the study and the
DRBC plates were incubated at 21 – 24°C and the Petrifilm tests at 30°C
instead of 28°C. This lab was not retained for interpretation.
Lab P received his package above the temperature limit.
Finally, 14 labs were retained: A, B, C, D, E, F, G, H, I, J, K, M, N and O.
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3.3.4 Incubation time of the Petrifilm tests
The possibility was offered to the labs to incubate the RYM tests between
48 h to 60 h; the real incubation applied is reported in Table 13
Table 13 – Incubation time of the RYM tests
Laboratories Incubation time (hours)
A 55
B 57
C 56
D 56
E 54
F 48
G 54
H 54
I 63
J 54
K 54
L 60
M 59
N 54
O 53
P 57
Q (ADRIA) 57
One lab (I) incubated the RYM test for 63 h instead of 60 h; their results were
in agreement with those of the other labs. They were kept for interpretation.
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3.3.5 Results analysis
Depending on the Lab results, the aerobic mesophilic flora enumeration
levels varied from < 10 to > 3 000 000 CFU/g.
3.3.6 Statistical interpretations and calculations
The calculations were realized according to the amendment number 1 of the
ISO 16140 standard (2011):
Accuracy: closeness of agreement between a measurement result and the
accepted reference value. Note: Accuracy refers to a combination of
trueness and precision
Trueness: closeness of agreement between the expectation of a
measurement result and the accepted reference value. Note: the measure
of trueness is usually expressed in terms of bias
Precision: closeness of agreement between independent measurement
results obtained under stipulated conditions. Note: quantitative measures
of precision depend critically on the stipulated conditions. Repeatability
conditions and reproducibility conditions are particular sets of extreme
stipulated conditions
Repeatability: precision under repeatability conditions
Repeatability conditions: measurement conditions where independent
measurement results are obtained with the same method on identical
measurement items in the same laboratory by the same operator using the
same equipment within short interval of time.
Repeatability standard deviation: standard deviation of measurement
results obtained under repeatability conditions
Repeatability limit (r): value less than or equal to which the absolute
difference between two measurement results obtained under repeatability
conditions is expected to be with a probability of 95%
Reproducibility: precision under reproducibility conditions
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Reproducibility conditions: measurement conditions where measurement
results are obtained with the same method on identical measurement
items in different laboratories with different operators using different
equipment
Reproducibility standard deviation: standard deviation of measurement
results obtained under reproducibility conditions
Reproducibility limit (R): value less than or equal to which the absolute
difference between two measurement results obtained under
reproducibility conditions is expected to be with a probability of 95%
All the available results are summarized in Tables 14 and 15 for 25°C
incubation temperature and Tables 16 and 17 for 28°C incubation
temperature.
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Table 14 – Results synthesis (CFU/g) – Incubation temperature: 25°C
Labs Level 0 Level 1 Level 2 Level 3
Reference method Alternative method Reference method Alternative method Reference method Alternative method Reference method Alternative method
A <10 <10 <10 <10 700 640 740 270 5600 5400 5500 5700 64000 57000 55000 46000
B <10 <10 <10 <10 520 470 380 510 6200 5200 5700 5300 68000 63000 71000 50000
C <10 <10 <10 <10 410 430 520 530 5500 5700 4500 3000 40000 67000 53000 45000
D <10 <10 <10 <10 590 340 580 460 5800 4600 4300 5800 65000 61000 85000 42000
E <10 <10 <30 <10 540 540 660 530 4900 5200 5600 6400 50000 62000 54000 66000
F <10 <10 <10 <10 560 610 510 560 6000 5600 5300 5100 60000 61000 73000 58000
G <10 <10 <10 <10 590 490 620 550 5600 12000 5700 5800 98000 60000 59000 46000
H <10 <10 <10 <10 700 650 700 600 6000 3000 4300 6400 64000 68000 60000 60000
I <10 <10 <10 <10 530 770 580 580 5800 3500 5500 6600 63000 55000 60000 75000
J <10 <10 <10 <10 550 450 350 590 7400 6800 5300 4100 84000 68000 44000 42000
K <10 <30 <10 <10 490 460 520 480 4900 5200 4000 5900 47000 55000 41000 40000
M <10 <10 <10 <10 600 760 630 720 4900 5100 6400 6500 68000 59000 58000 59000
N <10 <10 <10 <10 510 750 510 620 2500 4900 7300 3800 57000 45000 35000 40000
O <10 <10 <10 <10 630 530 620 460 4300 5900 5500 5000 60000 52000 54000 48000
Table 15 – Results summary (log CFU/g) - Incubation temperature: 25°C
Lab Level 0 Level 1 Level 2 Level 3
Reference method Alternative method Reference method Alternative method Reference method Alternative method Reference method Alternative method
A <1.00 <1.60 <1.00 <1.00 2.845 2.806 2.869 2.431 3.748 3.732 3.740 3.756 4.806 4.756 4.740 4.663
B <1.00 <1.00 <1.00 <1.00 2.716 2.672 2.580 2.708 3.792 3.716 3.756 3.724 4.833 4.799 4.851 4.699
C <1.00 <1.00 <1.00 <1.00 2.613 2.633 2.716 2.724 3.740 3.756 3.653 3.477 4.602 4.826 4.724 4.653
D <1.00 <1.00 <1.00 <1.00 2.771 2.531 2.763 2.663 3.763 3.663 3.633 3.763 4.813 4.785 4.929 4.623
E <1.00 <1.00 <1.60 <1.00 2.732 2.732 2.820 2.724 3.690 3.716 3.748 3.806 4.699 4.792 4.732 4.820
F <1.00 <1.00 <1.00 <1.00 2.748 2.785 2.708 2.748 3.778 3.748 3.724 3.708 4.778 4.785 4.863 4.763
G <1.00 <1.00 <1.00 <1.00 2.771 2.690 2.792 2.740 3.748 4.079 3.756 3.763 4.991 4.778 4.771 4.663
H <1.00 <1.00 <1.00 <1.00 2.845 2.813 2.845 2.778 3.778 3.477 3.633 3.806 4.806 4.833 4.778 4.778
I <1.00 <1.00 <1.00 <1.00 2.724 2.886 2.763 2.763 3.763 3.544 3.740 3.820 4.799 4.740 4.778 4.875
J <1.00 <1.00 <1.00 <1.00 2.740 2.653 2.544 2.771 3.869 3.833 3.724 3.613 4.924 4.833 4.643 4.623
K <1.00 <1.60 <1.00 <1.00 2.690 2.663 2.716 2.681 3.690 3.716 3.602 3.771 4.672 4.740 4.613 4.602
M <1.00 <1.00 <1.00 <1.00 2.778 2.881 2.799 2.857 3.690 3.708 3.806 3.813 4.833 4.771 4.763 4.771
N <1.00 <1.00 <1.00 <1.00 2.708 2.875 2.708 2.792 3.398 3.690 3.863 3.580 4.756 4.653 4.544 4.602
O <1.00 <1.00 <1.00 <1.00 2.799 2.724 2.792 2.663 3.633 3.771 3.740 3.699 4.778 4.716 4.732 4.681
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Table 16 – Results synthesis (CFU/g) – Incubation temperature: 28°C
Lab. Level 0 Level 1 Level 2 Level 3
Reference method Alternative method Reference method Alternative method Reference method Alternative method Reference method Alternative method
A <10 <10 <10 <10 700 640 420 600 5600 5400 7400 6100 64000 57000 72000 47000
B <10 <10 <10 <10 520 470 430 510 6200 5200 5600 6400 68000 63000 59000 49000
C <10 <10 <10 <10 410 430 560 500 5500 5700 5400 3500 40000 67000 45000 51000
D <10 <10 <10 <10 590 340 560 460 5800 4600 4700 5100 65000 61000 100000 38000
E <10 <10 <10 <10 540 540 390 560 4900 5200 5500 4600 50000 62000 55000 58000
F <10 <10 <10 <10 560 610 470 620 6000 5600 6100 5600 60000 61000 61000 46000
G <10 <10 <10 <10 590 490 670 560 5600 12000 6600 6100 98000 60000 59000 43000
H <10 <10 <10 <10 700 650 530 500 6000 3000 5400 6200 64000 68000 58000 69000
I <10 <10 <10 <10 530 770 640 460 5800 3500 3900 6100 63000 55000 53000 55000
J <10 <10 <10 <10 550 450 490 440 7400 6800 4500 3500 84000 68000 53000 51000
K <10 <30 <10 <30 490 460 460 470 4900 5200 3500 4600 47000 55000 48000 40000
M <10 <10 <10 <10 600 760 640 530 4900 5100 6800 5000 68000 59000 70000 55000
N <10 <10 <10 <10 510 750 580 520 2500 4900 6200 4000 57000 45000 43000 46000
O <10 <10 <10 <10 630 530 520 360 4300 5900 5000 4300 60000 52000 61000 51000
Table 17 – Results summary (log CFU/g) - Incubation temperature: 28°C
Lab Level 0 Level 1 Level 2 Level 3
Reference method Alternative method Reference method Alternative method Reference method Alternative method Reference method Alternative method
A <1.00 <1.60 <1.00 <1.00 2.845 2.806 2.623 2.778 3.748 3.732 3.869 3.785 4.806 4.756 4.857 4.672
B <1.00 <1.00 <1.00 <1.00 2.716 2.672 2.633 2.708 3.792 3.716 3.748 3.806 4.833 4.799 4.771 4.690
C <1.00 <1.00 <1.00 <1.00 2.613 2.633 2.748 2.699 3.740 3.756 3.732 3.544 4.602 4.826 4.653 4.708
D <1.00 <1.00 <1.00 <1.00 2.771 2.531 2.748 2.663 3.763 3.663 3.672 3.708 4.813 4.785 5.000 4.580
E <1.00 <1.00 <1.00 <1.00 2.732 2.732 2.591 2.748 3.690 3.716 3.740 3.663 4.699 4.792 4.740 4.763
F <1.00 <1.00 <1.00 <1.00 2.748 2.785 2.672 2.792 3.778 3.748 3.785 3.748 4.778 4.785 4.785 4.663
G <1.00 <1.00 <1.00 <1.00 2.771 2.690 2.826 2.748 3.748 4.079 3.820 3.785 4.991 4.778 4.771 4.633
H <1.00 <1.00 <1.00 <1.00 2.845 2.813 2.724 2.699 3.778 3.477 3.732 3.792 4.806 4.833 4.763 4.839
I <1.00 <1.00 <1.00 <1.00 2.724 2.886 2.806 2.663 3.763 3.544 3.591 3.785 4.799 4.740 4.724 4.740
J <1.00 <1.00 <1.00 <1.00 2.740 2.653 2.690 2.643 3.869 3.833 3.653 3.544 4.924 4.833 4.724 4.708
K <1.00 <1.60 <1.00 <1.60 2.690 2.663 2.663 2.672 3.690 3.716 3.544 3.663 4.672 4.740 4.681 4.602
M <1.00 <1.00 <1.00 <1.00 2.778 2.881 2.806 2.724 3.690 3.708 3.833 3.699 4.833 4.771 4.845 4.740
N <1.00 <1.00 <1.00 <1.00 2.708 2.875 2.763 2.716 3.398 3.690 3.792 3.602 4.756 4.653 4.633 4.663
O <1.00 <1.00 <1.00 <1.00 2.799 2.724 2.716 2.556 3.633 3.771 3.699 3.633 4.778 4.716 4.785 4.708
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3.3.6.1 Scrutiny of the measurement results for consistency
In order to identify other measurement results or laboratories that could be
inconsistent, two graphical consistency techniques were realized: the robust
Mandel’s h-ank-statistics.
Mandel indicators h and k at 5 % significance highlight some possible
inconsistent data:
Table 18
Incubation temperature: 25°C
Mandel’s values Number of values above the threshold
Reference method Alternative method
h > 1 % / Lab C Level 2
h > 5 % Lab G Level 2
Lab N Level 2
Lab C Level 2
k> 1 % Lab C Level 2 Lab G Level 2
Lab A Level 1 Lab D Level 3
k > 5 % Lab C Level 2
Lab D Level 1
Lab H Levels 2 and 3
Lab H Level 2
Lab I Level 2
Lab N Level 2
Lab A Level 1
Lab D Level 3
Lab J Level 1
Lab N Level 2
Incubation temperature: 28°C
Mandel’s values Number of values above the threshold
Reference method Alternative method
h > 1 % / /
h > 5 % Lab G Level 1
Lab N Level 1
/
k> 1 % Lab C Level 3 Lab G Level 2
Lab D Level 3
k > 5 % Lab C Level 3
Lab D Level 1
Lab G Levels 2 and 3
Lab H Level 2
Lav I Level 2
Lab N Level 2
Lab D Level 3
No data were excluded.
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3.3.6.2 Comparison of the trueness and precision characteristics of the reference
method and alternative methods
The statistical values are summarized hereafter:
Table 19 – Statistical values
Incubation temperature: 25°C
Level
Reference method Alternative method Ratios
repeatability Ratios
reproducibility Median Repeatability
s.d. (Sr) Reproducibility
s.d. (SR) Median
Repeatability s.d.(Sr)
Reproducibility s.d. (SR)
1 2.747 0.076 0.086 2.728 0.084 0.090 1.101 1.043
2 3.708 0.083 0.099 3.721 0.086 0.086 1.043 0.869
3 4.781 0.060 0.074 4.742 0.077 0.104 1.268 1.393
Incubation temperature: 28°C
Level Reference method Alternative method
Ratios repeatability
Ratios reproducibility Median
Repeatability s.d. (Sr)
Reproducibility s.d. (SR)
Median Repeatability
s.d. (Sr) Reproducibility
s.d. (SR)
1 2.747 0.076 0.086 2.709 0.085 0.085 1.119 0.986
2 3.708 0.084 0.100 3.699 0.096 0.113 1.150 1.132
3 4.781 0.060 0.074 4.731 0.082 0.082 1.358 1.102
Bias of the alternative method
In order to estimate the bias of the alternative method with respect to the
reference method for each level, Dij and t are calculated as described below:
Ref,Alt, ijYijYDij
Diffp
Dijit
)2(/
)(median
If t is larger than 2, the alternative method is significantly biased with respect
to the reference method.
The values obtained for t are given in table 20.
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Table 20 – Values obtained for t(d)
Incubation temperature: 25°C
Level Bias D t (d) Interpretation
1 - 0.026 1.33 Non significant bias
2 - 0.003 0.08 Non significant bias
3 - 0.041 1.22 Non significant bias
According to the statistical tests, the bias is non significant for all the tested
inoculation levels. They vary from – 0.041 to - 0.003 log CFU/g.
Incubation temperature: 28°C
Level Bias D t (d) Interpretation
1 - 0.043 1.51 Non significant bias
2 0.001 0.03 Non significant bias
3 - 0.036 2.25 Significant bias
According to the statistical tests, the bias is non significant for Level 1 and
Level 2, and significant for Level 3 while the value is very low
(- 0.03 log CFU/g). They vary from – 0.043 to 0.001 log CFU/g, which
correspond to very low and satisfying values.
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 21.
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Table 21
Incubation temperature: 25°C
Contamination
level
Reference method Alternative method Ratio
Sr Alt. / Sr Ref. Sr Ref. r Ref. Sr Alt. r Alt.
1 0.076 0.213 0.084 0.234 1.101
2 0.083 0.232 0.086 0.242 1.043
3 0.060 0.169 0.104 0.215 1.268
Incubation temperature: 28°C
Contamination
level
Reference method Alternative method Ratio
Sr Alt. / Sr Ref. Sr Ref. r Ref. Sr Alt. r Alt.
1 0.076 0.213 0.085 0.238 1.119
2 0.084 0.235 0.113 0.270 1.150
3 0.060 0.169 0.082 0.230 1.358
The ratios of the repeatability standard deviations are comprised below 2 for
all the tested contamination levels whatever the incubation temperature; the
precision under repeatability conditions of the alternative method is
equivalent to that of the reference method, whatever the tested
incubation temperature.
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 22.
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Table 22
Incubation temperature: 25°C
Contamination
level
Reference method Alternative method Ratio
SR Alt/SR Ref. SR Ref. R Ref. SR Alt. R Alt.
1 0.086 0.242 0.090 0.252 1.043
2 0.099 0.278 0.086 0.242 0.869
3 0.074 0.209 0.104 0.291 1.393
Incubation temperature: 28°C
Contamination
level
Reference method Alternative method Ratio
SR Alt/SR Ref. SR Ref. R Ref. SR Alt. R Alt.
1 0.086 0.242 0.085 0.238 0.986
2 0.100 0.279 0.113 0.317 1.132
3 0.074 0.209 0.082 0.230 1.102
The ratios of the reproducibility standard deviations are comprised below 2
for the contamination levels; the precision under reproducibility
conditions of the alternative method is equivalent to that of the
reference method, whatever the tested incubation temperature.
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44 CCOONNCCLLUUSSIIOONN
The method comparison study conclusions are:
The target analyte gathers yeast and molds, i.e. an important
group of microorganisms. Therefore, some differences are
sometimes observed between the data obtained after an
incubation of the Petrifilm test at 25°C or 28°C, as well as the data
provided by the ISO method. However, the statistical tests
conclude to the performances assessment of the Petrifilm test
whatever the incubation conditions:
In the linearity study, the correlation coefficients are higher than
0.99 with slopes close to 1 and ordinates close to 0, assessing
the linearity performances of the alternative method.
Whatever the tested conditions in the accuracy study, the
alternative method is reliable when compared to ISO 21527
methods.
The inter-laboratory study conclusions are:
The bias values between the reference and the alternative
methods vary between – 0.041 to - 0.003 log CFU/g at 25°C and
between – 0.043 to 0.001 log CFU/g at 28°C.
The repeatability and reproducibility of the alternative method are
clearly similar to the repeatability and the reproducibility of the
reference method, whatever the incubation temperature tested.
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Appendix 1 – Diagram of the reference method test procedure
ISO 21527 method “Microbiology of food and animal feeding stuffs -
Horizontal method for the enumeration of yeasts and molds”.
Part 1: Colony-count technique in products with water activity greater than 0.95
10 g sample + 90 ml peptone water (0.1 %)
Stomach
Plate 2 x 0.1 ml aliquots of each dilution onto
2 Petri dishes containing DRBC media
(or 2 x 1 ml onto 2 x 3 Petri Dishes
for low contaminated samples)
Incubate 5 days at 25°C 1°C
(If fast growing molds are a problem, count colonies/propagules/germs
after 2 days, and again after 5 days)
Enumeration
Part 2: Colony-count technique in products with water activity less than or
equal to 0.95 and > 0.60
10 g sample + 90 ml peptone water (0.1 %)
Stomach
Plate 2 x 0.1 ml aliquots of each dilution onto
2 Petri dishes containing DG18 media
(or 2 x 1 ml onto 2 x 3 Petri Dishes
for low contaminated samples)
Incubate 5 to 7 days at 25°C 1°C
(If fast growing molds are a problem, count colonies/propagules/germs
after 2 days, and again after 5 and 7 days)
Enumeration
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Appendix 2 – Flow diagram of the alternative method
10 g + 90 ml peptone water 0.1 %
or liquid product*
1 ml of each dilution
per Petrifilm plate
Incubation at 25°C ± 1°C or 28°C ± 1°C
read the plate any time between 60 h and 72 hours
Numeration of yeasts and molds
For practicability, the possibility is offered to incubate the RYM plates until 120 hours.
* For beverages, apply 1/10 dilution
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Appendix 3 – Artificial contaminations
N°sample Product (French name) Product (English name) Strain inoculated Origin Injury
protocol Inoculation level (CFU/g)
370 Poudre de blanc d'œuf Egg white powder Penicillium chlorophylum M11 Dairy product Spores 1000
371 Poudre d'œuf entier Whole egg powder Penicillium chlorophylum M11 Dairy product Spores 10000
373 Lait écrémé en poudre Skimmed milk powder Byssochlamis nivea Dairy product Spores 500
492 Nuoc mam Nuoc mam (culinary product) Candida saké Ad1826 Environmental sample
10000
493 Viandox Culinary product Eupenicillium meridianum DSM2209 Environmental sample Spores 100
494 Fond de veau Culinary product Candida norvegica Ad1827 Environmental sample
1000
495 Cœur de bouillon Culinary product Phialophora richarsiae UMIP 2645.08 Environmental sample Spores 5000
496 Sirop d'érable Syrup Penicillium chlorophylum M11 Bakery Spores 1000
497 Biscuits cuillère Biscuits Penicillium chlorophylum M11 Bakery Spores 10000
631 Aliment complet pour chat (bœuf, carotte)
Pet food (cat) Cladosporium cladosporoides Ad1405
Spores 500
632 Aliment complet pour chat (veau, poivron))
Pet food (cat) Penicillium citeonigrum Ad1052 Environmental sample (fruit) Spores 5000
633 Complément alimentaire pour chien
Pet food (dog) Cladosporium cladosporoides Ad1405
Spores 1000
634 Complément alimentaire pour chien
Pet food (dog) Penicillium citeonigrum Ad1052 Environmental sample (fruit) Spores 10000
635 Complément alimentaire pour chien
Pet food (dog) Penicillium citeonigrum Ad1052 Environmental sample (fruit) Spores 50000
638 Eau de process Stephan (fromage de chèvre)
Process water Hanseniaspora uvarum Ad1033 Environmental sample (fruit)
1000
639 Eau de rinçage fabrication béchamel
Rinsing water Hanseniaspora uvarum Ad1033 Environmental sample (fruit)
5000
640 Eau de process pompe homogénéisateur
Process water Phialophora parasitica LMSA 1-01-237 Vegetables Spores 100
641 Eau de rinçage robot coupe
Rinsing water Phialophora parasitica LMSA 1-01-237 Vegetables Spores 500
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3M Rapid Yeast and Molds Petrifim
Appendix 4 – Specificity / selectivity
INCLUSIVITY
Strain n° Origin
Sabouraud 3M Rapid Yeast and Molds Petrifilm - 25°C 3M Rapid Yeast and Molds Petrifilm - 28°C ISO 21527-1
(DRBC) ISO 21527-2
(DG18) 60h 72h 60h 72h
rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2
Aspergillus flavus Adria M5 Pastry / Backery 57(-5) 73(-5) 58(-5) 38(-5) 61(-5) 39(-5) 48(-5) 45(-5) 49(-5) 47(-5) 33(-5) 25(-5) 49(-5) 65(-5)
Aspergillus fumigatus Adria 1053 Environment 11(-6) 12(-6) 0(-6) 0(-6) 7(-6) 8(-6) 12(-6) 14(-6) 13(-6) 14(-6) 9(-6) 19(-6) 11(-6) 12(-6)
Aspergillus versicolor Adria 1057 Environment 80(-5) 47(-5) 1(-5) 0(-5) 61(-5) 41(-5) 0(-5) 0(-5) 33(-5) 45(-5) 151(-5) 128(-5) 114(-5) 72(-5)
Byssochlamys nivea Ad1316 Dairy 55(-2) / 0(-2) 0(-2) 32(-2) 30(-2) 17(-2) 26(-2) 48(-2) 47(-2) 34(-3) 33(-3) 24(-3) 17(-3)
Cladosporium cladosporioïdes Ad1405 Bakery 90(-6) 82(-6) 101(-6) 93(-6) 101(-6) 93(-6) 98(-6) 88(-6) 98(-6) 88(-6) 107(-6) 81(-6) 92(-6) 100(-6)
Cryptococcus liquefaciens Adria 1041 Environment 110(-4) 120(-4) 99(-4) 110(-4) 99(-4) 110(-4) 129(-4) 90(-4) 129(-4) 91(-4) 107(-4) 99(-4) 120(-4) 115(-4)
Cryptococcus magnus Adria 1040 Environment 19(-5) 27(-5) 34(-5) 29(-5) 35(-5) 30(-5) 33(-5) 29(-5) 33(-5) 29(-5) 33(-5) 20(-5) 25(-5) 23(-5)
Debaryomyces castellii CLIB 424 Environment 63(-5) 82(-5) 67(-5) 71(-5) 67(-5) 71(-5) 69(-5) 65(-5) 69(-5) 65(-5) 81(-5) 76(-5) 64(-5) 66(-5)
Debaryomyces hansenii var. hansenii CLIB 197 Brewery 140(-5) 138(-5) 91(-5) 99(-5) 91(-5) 99(-5) 95(-5) 109(-5) 95(-5) 109(-5) 116(-5) 140(-5) 143(-5) 109(-5)
Hanseniaspora uvarum CLIB 303 Oenology 37(-6) 41(-6) 24(-6) 27(-6) 24(-6) 27(-6) 28(-6) 24(-6) 28(-6) 24(-6) 27(-6) 30(-6) 30(-6) 30(-6)
Kluyveromyces lactis var. lactis CLIB 196 Dairy 61(-5) 52(-5) 17(-5) 12(-5) 17(-5) 12(-5) 10(-5) 28(-5) 10(-5) 28(-5) 65(-5) 49(-5) 69(-5) 60(-5)
Kluyveromyces marxianus CLIB 720 Dairy 16(-6) 25(-6) 21(-6) 26(-6) 21(-6) 26(-6) 21(-6) 16(-6) 21(-6) 16(-6) 25(-6) 21(-6) 30(-6) 22(-6)
Metschenikowia pulcherrina CLIB 403 Fruit 75(-5) 84(-5) 72(-5) 77(-5) 72(-5) 77(-5) 71(-5) 67(-5) 71(-5) 67(-5) 85(-5) 85(-5) 74(-5) 74(-5)
Mucor plumbeus Adria M10 Food
ill(-7) ill(-8)
ill(-7) ill(-8)
ill(-6) 4(-7) ill(-6) 4(-7) ill(-7) ill(-7) ill(-6) 1(-7) ill(-6) 1(-7) ill(-6) 1(-7) ill(-6) 1(-7) 0(-4) 0(-4) 26(-6) 37(-6)
Mucor racemosus Adria M9 Food
16(-6) 3(-7)
14(-6) 1(-7)
ill(-6) 3(-7) ill(-6) 3(-7) ill(-6) 3(-7) ill(-6) ill(-7) ill(-6) 3(-7) ill(-6) 3(-7) ill(-6) 3(-7) ill(-6) 3(-7) 39(-6) 2(-7)
37(-6) 3(-7)
56(-6) 7(-7)
49(-6) 4(-7)
Paecilomyces variotii Adria M6 Food 63(-5) 82(-5) 2(-5) 1(-5) 48(-5) 40(-5) 93(-5) 69(-5) 96(-5) 71(-5) 106(-5) 121(-5) 99(-5) 107(-5)
Penicillium chlorylophilum Adria M11 Pastry / Backery 20(-4) 29(-4) 16(-4) 22(-4) 18(-4) 22(-4) 21(-4) 25(-4) 22(-4) 25(-4) 12(-4) 17(-4) 25(-4) 19(-4)
Penicillium citreonigrum Adria 1052 Environment 17(-6) 18(-6) 19(-6) 19(-6) 19(-6) 19(-6) 15(-6) 18(-6) 15(-6) 18(-6) 24(-6) 20(-6) 13(-6) 26(-6)
Penicillium roqueforti Adria M1 Dairy 145(-6) 153(-6) 91(-6) 94(-6) 101(-6) 100(-6) 122(-6) 123(-6) 124(-6) 128(-6) 171(-6) 145(-6) 151(-6) 185(-6)
Phoma glomerata Adria M4 Food 117(-5) 112(-5) 122(-5) 127(-5) 125(-5) 128(-5) 147(-5) 132(-5) 147(-5) 147(-5) 137(-5) 155(-5) 76(-5) 70(-5)
Pichia delftensis CLIB 291 Cider brewery 76(-5) 73(-5) 44(-5) 59(-5) 46(-5) 73(-5) 0(-5) 0(-5) 0(-5) 0(-5) 70(-5) 64(-5) 84(-5) 57(-5)
Rhodotorula graminis Adria 1032 Environment 80(-5) 126(-5) 81(-5) 87(-5) 85(-5) 89(-5) 87(-5) 88(-5) 88(-5) 92(-5) 89(-5) 93(-5) 78(-5) 93(-5)
Rhodotorula slooffiae Adria 1031 Environment 34(-5) 40(-5) 0(-5) 0(-5) 0(-5) 0(-5) 8(-5) 14(-5) 8(-5) 15(-5) 28(-5) 35(-5) 51(-5) 32(-5)
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3M Rapid Yeast and Molds Petrifim
INCLUSIVITY
Strain n° Origin
Sabouraud 3M Rapid Yeast and Molds Petrifilm - 25°C 3M Rapid Yeast and Molds Petrifilm - 28°C ISO 21527-1
(DRBC) ISO 21527-2
(DG18) 60h 72h 60h 72h
rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2
Saccharomyces bayanus CLIB 181 Beer brewery 53(-5) 48(-5) 46(-5) 38(-5) 48(-5) 41(-5) 42(-5) 40(-5) 43(-5) 43(-5) 60(-5) 85(-5) 65(-5) 67(-5)
Endomyces copis vini CLIB 7 Oenology 49(-3) 43(-3) 25(-3) 37(-3) 29(-3) 43(-3) 41(-3) 41(-3) 41(-3) 44(-3) 50(-3) 67(-3) 48(-3) 52(-3)
Trichoderma virride Adria M2 Food 7(-7) 6(-7) 4(-7) 5(-7) 5(-7) 5(-7) 6(-7) 6(-7) 6(-7) ill(-7) 13(-7) 13(-7) 7(-7) 10(-7)
Yarrowia lipolytica CLIB 183 Food 97(-5) 122(-5) 101(-5) 103(-5) 101(-5) 103(-5) 115(-5) 112(-5) 115(-5) 112(-5) 89(-5) 72(-5) 87(-5) 102(-5)
Zygosaccharomyces rouxii Adria 1046 Fruit 46(-5) 26(-5) 164(-4) 11(-5) 168(-4) 19(-5)
176(-4) 12(-5)
170(-4) 21(-5)
165(-4) 15(-5) 185(-4) 14(-5) 165(-4) 16(-5) 186(-4) 15(-5) 152(-4) 14(-5)
158(-4) 13(-5)
25(-5) 16(-5)
Penicillium expansum UMIP 1350.82 / 39(-4) 38(-4) 22(-4) 25(-4) 29(-4) 33(-4) 2(-4) 1(-4) 5(-4) 2(-4) 51(-4) 39(-4) 27(-4) 26(-4)
Saccharomyces rouxii Ad2058 Salad 104(-4) 119(-4) 86(-4) 79(-4) 94(-4) 87(-4) 111(-4) 104(-4) 112(-4) 104(-4) 2(-4) 1(-4) 29(-4) 25(-4)
Wallemia sebi Ad1627 Pastry 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 25(-5) 11(-5)
Candida pseudotropicalis Adria Y3 Deli salad 80(-5) 89(-5) 39(-5) 25(-5) 111(-5) 81(-5) 0(-5) 0(-5) 0(-5) 0(-5) 17(-6) 29(-6) 102(-5) 100(-5)
Geotrichum candidum ATCC 204307 Dairy 63(-5) 59(-5) 48(-5) 51(-5) 48(-5) 51(-5) 64(-5) 49(-5) 64(-5) 49(-5) 59(-5) 64(-5) 75(-5) 61(-5)
Candida fermenticarens Ad1152
71(-4) 84(-4) 22 very pale(-4) 19 very pale(-4) 52pale(-4) 78 pale(-4) 57 very pale(-4) 61 very pale(-4) 75(-4) 75(-4) 150(-4) 184(-4) 214(-4) 150(-4)
Candida guillermondii Adria 381
39(-6) 26(-6) 31(-6) 35(-6) 31(-6) 35(-6) 34(-6) 39(-6) 34(-6) 39(-6) 41(-6) 31(-6) 43(-6) 36(-6)
Candida inconspicua Ad1825
15(-5) 15(-5) 20(-5) 12(-5) 22(-5) 12(-5) 13(-5) 12(-5) 14(-5) 12(-5) 35(-5) 18(-5) 21(-5) 33(-5)
Candida kefir Ad1056
103(-5) 120(-5) 140(-5) 114(-5) 142(-5) 115(-5) 105(-5) 128(-5) 105(-5) 128(-5) 117(-5) 118(-5) 143(-5) 137(-5)
Candida norvegica Ad1827 Environment 65(-5) 62(-5) 57(-5) 58(-5) 59(-5) 64(-5) 56(-5) 66(-5) 56(-5) 67(-5) 83(-5) 81(-5) 66(-5) 64(-5)
Candida parapsilosis Ad1055
115(-5) 98(-5) 95 pale(-5) 97 pale(-5) 108(-5) 109(-5) 78(-5) 93(-5) 78(-5) 93(-5) 24(-6) 21(-6) 124(-5) 117(-5)
Candida pulcherrina Adria 210
33(-6) 22(-6) 20(-6) 25(-6) 20(-6) 25(-6) 21(-6) 30(-6) 21(-6) 30(-6) 37(-6) 36(-6) 31(-6) 36(-6)
Candida sake Ad1826 Environment 90(-5) 111(-5) 62 very pale(-5) 76 very pale(-5) 90 pale(-5) 104 pale(-5) 0(-5) 0(-5) 5 very pale(-5) 1 very pale(-5) 23(-6) 21(-6) 12(-6) 16(-6)
Candida tropicalis Ad1157
101(-5) 76(-5) 114(-5) 155(-5) 114(-5) 155(-5) 123(-5) 127(-5) 123(-5) 127(-5) 19(-6) 25(-6) 20(-6) 14(-6)
Candida zeylanoides Ad1154
58(-5) 55(-5) 68 pale(-5) 57 pale(-5) 70(-5) 62(-5) 56 very pale(-5) 60 very pale(-5) 60 very pale(-5) 67 very pale(-5) 76(-5) 74(-5) 80(-5) 71(-5)
Osmophil strains
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3M Rapid Yeast and Molds Petrifim
EXCLUSIVITY
Strain n°
PCA 3M Rapid Yeast and Molds Petrifilm - 25°C 3M Rapid Yeast and Molds Petrifilm - 28°C
ISO 21527-1 (DRBC) ISO 21527-2 (DG18) 60h 72h 60h 72h
rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2
Bacillus subtilis 836 45(-5) 50(-5) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4)
Bacillus weihenstephanensis 778 56(-6) 61(-6) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4)
Brochotrix thermosphacta EN 15/29 7(-4) 2(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4)
Bruttiaux agrestis 117 110(-6) 103(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6)
Carnobacterium piscicola 369 61(-7) 58(-7) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6)
Corynebacterium spp 361 62(-5) 59(-5) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3)
Enterococcus faecalis 288 101(-8) 118(-8) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5)
Lactobacillus plantarum 70 109(-7) 117(-7) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6)
Leclercia adecarboxylata 707 44(-5) 34(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5)
Leuconostoc oenos 73 57(-7) 56(-7) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5)
Listeria innocua 644 100(-7) 95(-7) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5)
Micrococcus luteus 438 101(-5) 90(-5) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3)
Moraxella
51.11 52(-6) 42(-6) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3)
Plesiomonas shigelloïdes 673 30(-7) 29(-7) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5)
Pseudomonas fluorescens 16 95(-6) 92(-6) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5) 0(-5)
Pseudomonas putida 4 48(-7) 57(-7) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6)
Rhanella aquatilis 67 115(-7) 120(-7) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6)
Shewanella putrefaciens EN 15/34 130(-6) 119(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6) 0(-6)
Staphylococcus aureus 904 24(-7) 30(-7) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4) 0(-4)
Staphylococcus epidermidis 931 55(-5) 52(-5) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3) 0(-3)
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