rapport de synthèse 3m 01-13 07-14 (en) - nf validation...2007/01/13 · the bi-dimensional graphs...

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 : [email protected] - 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

mailto:[email protected]

http://www.adria.tm.fr/

3M

ADRIA Développement 2/70 September 30, 2014

3M RYM Summary Report (Version 0)

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



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

3M



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

3M



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:

3M



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.

3M



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))



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)

3M



Figure 1 – Linearity: bi-dimensional graphs

Incubation temperature: 25°C; Incubation time: 60 h

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Liver pâté / Candida norvegica-25°C-60H

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Apricot jam-25°C-60H

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Milk powder 25°C-60H

Apricot syrup - 25°C - 60H

3M




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Fruit Juice -25°C-60H

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Feed stuff-25°C-60H

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Process water-25°C-60H

3M




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Liver pâté-25°C-72HCandida pseudotropicalis

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3M




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log( (Reference method)



3M




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Feed stuff-28°C-60HApricot syrup - 28°C - 60H

3M




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log (Rerference method)


3M




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3M




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3M



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.

3M



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


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

3M




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


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


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


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


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

3M



Figure 2 – Linearity: regression lines


y = 0,9792x - 0,1382

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Reference

Milk powder 25°C 60H

y = 1,0342x - 0,0489

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Alternative

Liver pâté / Candida norvegica25°C-60H

y = 1,0187x - 0,1917

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Egg powder-25°C-60H

y = 0,9986x - 0,1883

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Fruit Juice-25°C-60H

y = 1,0454x - 0,1241

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y = 0,9739x - 0,0115

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y = 1,0098x - 0,0997

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3M




y = 0,9422x + 0,0149

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Reference

Liver pâté-25°C-72HCandida pseudotropicalis

y = 1,0342x - 0,0489

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ce

Alternative

Liver pâté / Candida norvegica25°C-72H

y = 1,02x - 0,1923

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Reference

Egg powder 25°C-72H

y = 0,9959x - 0,18

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Alternative

Fruit Juice-25°C-72H

y = 1,0409x - 0,1126

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Alternative


y = 0,9354x + 0,2058

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Reference


y = 1,012x - 0,0992

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Reference



3M




y = 0,9955x - 0,0658

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Référence

Liver pâté / Candida norvegica28°c-60H

y = 0,9331x + 0,2243

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Reference


y = 0,9451x + 0,396

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Reference

Fruit juice 28°C 60H

y = 1,0323x - 0,0486

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ce

Alternative


y = 1,0406x - 0,1767

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Alternative


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



3M




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

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Alt

ern

ati

ve

Reference


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


y = 0,9551x + 0,2469

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

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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



3M



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



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



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.

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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


0,00

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log (Reference method))

Meat and fish products-25°C-60H

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Dairy and egg products-25°C-60H

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Fruits and vegetables-25°C-60H

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Chocolate and bakery products-25°C-60H

3M




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Feed stuffs-25°C-60H

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Environmental samples-25°C-60H

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All products-25°C-60H

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All products-25°C-60H AwAw > 0.6

3M




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Feed stuffs-25°C-72H

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3M





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All products-25°C-72HAw > 0.6

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3M




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Feed stuffs -28°C-60H

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3M




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All products-28°C-60H-Aw>0,6

3M




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(A

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Feed stuffs -28°C-72H

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)



3M





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All products-28°C-72HAw > 0.6

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3M



Table 6 – Statistical interpretation


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



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

3M




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




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




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

3M



Figure 4 – Relative accuracy: regression lines


y = 0,9901x - 0,0649

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2,00

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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

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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

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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

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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

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4,00

6,00

8,00

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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

3M




y = 1,0151x - 0,1444

0,00

2,00

4,00

6,00

8,00

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Alt

ern

ati

ve

Reference


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


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


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


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


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


Aw > 0.6

3M




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


3M




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


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


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


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


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


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


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


Aw > 0.6

3M





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


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


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


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


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


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


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


Aw > 0.6

3M



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


- All products:



- Dairy and egg products:



- All products:


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


3M



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


Inoculation level


Standard deviation

(S0)

Bias X0

(Xoi median)

Calculation formula


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


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


Inoculation level


Standard deviation

(S0)

Bias X0

(Xoi median)

Calculation formula


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


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


Inoculation level


Standard deviation

(S0)

Bias X0

(Xoi median)

Calculation formula


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


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

)>)



Liver pâté/Candia pseudotropicalis

Egg powder

Apricot jam

Feed stuff

Process water

Liver pâté/Candia norvegica

Yoghourt

3M



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

)>)



Egg powder

Apricot syrup

Feed stuff

Process water


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

)>)



Egg powder

Apricot syrup

Feed stuff

Process water


Yoghourt

3M



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.

3M



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.

3M



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

3M



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.

3M



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.

3M



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.

3M



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.

3M



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.

3M



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

3M



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.

3M



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


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


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


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


Mandel’s values Number of values above the threshold


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


Mandel’s values Number of values above the threshold


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


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


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)


Level Bias D t (d) Interpretation

1 - 0.026 1.33 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.


Level Bias D t (d) Interpretation


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


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


Contamination

level


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


Contamination

level


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


Contamination

level


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.

3M



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.

3M



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

3M



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


Pet food (dog) Penicillium citeonigrum Ad1052 Environmental sample (fruit) Spores 10000


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

3M


Summary Report (Version 0)

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)

3M




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


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

3M




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


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)

rapport de synthèse 3m 01-13 07-14 (en) - nf validation...2007/01/13 · the bi-dimensional graphs...

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