Microbiology of the food chain - Method validation - Part 3: Protoc
International Standard ISO 16140-3:2021
Quantitative methods - Technical protocol for verification; ca
Calculation and evaluation tool for verification of side-by-side method c
Evaluation of results
The absolute value of this bias must be ≤ 0.5 log CFU/g . ∣eBias∣≤0.5
Sample Info
Sample ID Matrix Type Contamination Level Replicate
L1-R1 Sausage Low 1
L1-R2 Sausage Low 2
L2-R1 Sausage Medium 1
L2-R2 Sausage Medium 2
L3-R1 Sausage High 1
L3-R2 Sausage High 2
Sample Info
Sample ID Matrix Type Contamination Level Replicate
L1-R1 solid Low 1
L1-R2 solid Low 2
L2-R1 solid Medium 1
L2-R2 solid Medium 2
L3-R1 solid High 1
L3-R2 solid High 2
Sample Info
Sample ID Matrix Type Contamination Level Replicate
L1-R1 semi solid Low 1
L1-R2 semi solid Low 2
L2-R1 semi solid Medium 1
L2-R2 semi solid Medium 2
L3-R1 semi solid High 1
L3-R2 semi solid High 2
Sample Info
Sample ID Matrix Type Contamination Level Replicate
L1-R1 semi solid Low 1
L1-R2 semi solid Low 2
L2-R1 semi solid Medium 1
L2-R2 semi solid Medium 2
L3-R1 semi solid High 1
L3-R2 semi solid High 2
Sample Info
Sample ID Matrix Type Contamination Level Replicate
L1-R1 Liquid Low 1
L1-R2 Liquid Low 2
L2-R1 Liquid Medium 1
L2-R2 Liquid Medium 2
L3-R1 Liquid High 1
L3-R2 Liquid High 2
Sample Info
Sample ID Matrix Type Contamination Level Replicate
L1-R1 Liquid Low 1
L1-R2 Liquid Low 2
L2-R1 Liquid Medium 1
L2-R2 Liquid Medium 2
L3-R1 Liquid High 1
L3-R2 Liquid High 2
dation - Part 3: Protocol for the verification of reference methods and validated alternative
Cell colour coding: Enter data
NOTE: initially, this excel sheet shows the valid
ol for verification; calculation and lookup tool for side-by-side comparison against va
f side-by-side method comparison
g . ∣eBias∣≤0.5
Reference Method Alternative Method
Test Portion (g) cfu/g log10 (cfu/g) cfu/g log10(cfu/g)
10 74 1.8692 70 1.8451
10 80 1.9031 75 1.8751
10 1450 3.1614 1400 3.1461
10 1500 3.1761 1420 3.1523
10 8500 3.9294 8300 3.9191
10 9000 3.9542 8800 3.9445
Reference Method Alternative Method
Test Portion (g) cfu/g log10 (cfu/g) cfu/g log10(cfu/g)
10 74 1.8692 70 1.8451
10 80 1.9031 75 1.8751
10 1450 3.1614 1400 3.1461
10 1500 3.1761 1420 3.1523
10 8500 3.9294 8300 3.9191
10 9000 3.9542 8800 3.9445
Reference Method Alternative Method
Test Portion (g) cfu/g log10 (cfu/g) cfu/g log10(cfu/g)
10 74 1.8692 70 1.8451
10 80 1.9031 75 1.8751
10 1450 3.1614 1400 3.1461
10 1500 3.1761 1420 3.1523
10 8500 3.9294 8300 3.9191
10 9000 3.9542 8800 3.9445
Reference Method Alternative Method
Test Portion (g) cfu/g log10 (cfu/g) cfu/g log10(cfu/g)
10 74 1.8692 70 1.8451
10 80 1.9031 75 1.8751
10 1450 3.1614 1400 3.1461
10 1500 3.1761 1420 3.1523
10 8500 3.9294 8300 3.9191
10 9000 3.9542 8800 3.9445
Reference Method Alternative Method
Test Portion (g) cfu/g log10 (cfu/g) cfu/g log10(cfu/g)
10 74 1.8692 70 1.8451
10 80 1.9031 75 1.8751
10 1450 3.1614 1400 3.1461
10 1500 3.1761 1420 3.1523
10 8500 3.9294 8300 3.9191
10 9000 3.9542 8800 3.9445
Reference Method Alternative Method
Test Portion (g) cfu/g log10 (cfu/g) cfu/g log10(cfu/g)
10 74 1.8692 70 1.8451
10 80 1.9031 75 1.8751
10 1450 3.1614 1400 3.1461
10 1500 3.1761 1420 3.1523
10 8500 3.9294 8300 3.9191
10 9000 3.9542 8800 3.9445
dated alternative methods in a single laboratory
Calculated results
heet shows the valid data (Laboratory sample numbers 2,3,4,5,6,7,8,9,10,12) from the EXAMPLE in Table 10 and
rison against validated method.
Mean of Ref Mean of Alt leBiasl
Mean[log₁₀(Ref)] Mean[log₁₀(Alt)] Mean[log₁₀(Alt)] - Mean[log₁₀(Ref)]
2.9989 2.9804 0.0186
ACCEPTED
Mean of Ref Mean of Alt leBiasl
Mean[log₁₀(Ref)] Mean[log₁₀(Alt)] Mean[log₁₀(Alt)] - Mean[log₁₀(Ref)]
2.9989 2.9804 0.0186
ACCEPTED
Mean of Ref Mean of Alt leBiasl
Mean[log₁₀(Ref)] Mean[log₁₀(Alt)] Mean[log₁₀(Alt)] - Mean[log₁₀(Ref)]
2.9989 2.9804 0.0186
ACCEPTED
Mean of Ref Mean of Alt leBiasl
Mean[log₁₀(Ref)] Mean[log₁₀(Alt)] Mean[log₁₀(Alt)] - Mean[log₁₀(Ref)]
2.9989 2.9804 0.0186
ACCEPTED
Mean of Ref Mean of Alt leBiasl
Mean[log₁₀(Ref)] Mean[log₁₀(Alt)] Mean[log₁₀(Alt)] - Mean[log₁₀(Ref)]
2.9989 2.9804 0.0186
ACCEPTED
Mean of Ref Mean of Alt leBiasl
Mean[log₁₀(Ref)] Mean[log₁₀(Alt)] Mean[log₁₀(Alt)] - Mean[log₁₀(Ref)]
2.9989 2.9804 0.0186
ACCEPTED
he EXAMPLE in Table 10 and Table 11, but these can be replaced by your own data
End of this tab
Microbiology of the food chain - Method validation - Part
International Standard ISO 16140-3:2021
Food Category
Food type
Plate type AC
Spike Level Count result
Food Name
(CFU/g) (CFU/g)
R1 50 43
R2 50 32
R3 50 55
R4 50 51
R5 50 54
R6 50 58
R7 50 60
R8 50 49
R9 50 46
R10 50 53
Mean
SD
%CV
MU
hain - Method validation - Part 3: Protocol for the verification of reference methods and va
16140-3:2021
Cell colour coding: Enter data
Food Category
Food type
Plate type CC
Spike Level
log10 CFU/g Food Name
(CFU/g)
1.6335 R1 50
1.5051 R2 50
1.7404 R3 50
1.7076 R4 50
1.7324 R5 50
1.7634 R6 50
1.7782 R7 50
1.6902 R8 50
1.6628 R9 50
1.7243 R10 50
1.6938
0.0795
4.6962
0.1591
erence methods and validated alternative methods in a single laboratory
Enter data Calculated results
Food Category
Food type
Plate type
Count result
log10 CFU/g Food Name
(CFU/g)
43 1.6335 R1
32 1.5051 R2
55 1.7404 R3
51 1.7076 R4
54 1.7324 R5
58 1.7634 R6
60 1.7782 R7
49 1.6902 R8
46 1.6628 R9
53 1.7243 R10
Mean 1.6938
SD 0.0795
%CV 4.6962
MU 0.1591
EC
Spike Level Count result
log10 CFU/g
(CFU/g) (CFU/g)
50 43 1.6335
50 32 1.5051
50 55 1.7404
50 51 1.7076
50 54 1.7324
50 58 1.7634
50 60 1.7782
50 49 1.6902
50 46 1.6628
50 53 1.7243
Mean 1.6938
SD 0.0795
%CV 4.6962
MU 0.1591
Food Category
Food type
Plate type YM-R
Spike Level Count result
Food Name log10 CFU/g
(CFU/g) (CFU/g)
R1 50 43 1.6335
R2 50 32 1.5051
R3 50 55 1.7404
R4 50 51 1.7076
R5 50 54 1.7324
R6 50 58 1.7634
R7 50 60 1.7782
R8 50 49 1.6902
R9 50 46 1.6628
R10 50 53 1.7243
Mean 1.6938
SD 0.0795
%CV 4.6962
MU 0.1591
End of this tab
Microbiology of the food chain - Method validation - Part
International Standard ISO 16140-3:2021
Food Category
Food type
Analyst A
Total Count (CFU/g)Log10 CFU/g
Food Name
Low Level Low Level
R1
R2
D1 R3
R4
R5
R1
R2
D2 R3
R4
R5
SD #DIV/0!
Variance (SD^2) #DIV/0!
F-test (Day)
Fcrit (4x4) 5.05
D1 SD #DIV/0!
Variance (SD^2) #DIV/0!
F-test (Day)
Fcrit (4x4) 6.39
D2 SD #DIV/0!
Variance (SD^2) #DIV/0!
* F = (Larger Variance/Lower variance)
* Comparing 2 groups (Analyst A & B / Day 1 & 2)
df₁ = degrees of freedom for the group with larger variance
df₂ = degrees of freedom for the group with smaller variance
N = number of replicates
N = 6-1 = 5
hence, Fcrit = F-table (5x5) = 5.05
Acceptance Criteria: F-test value < Fcrit value
ain - Method validation - Part 3: Protocol for the verification of reference methods and valid
140-3:2021
Cell colour coding: Enter data
Analyst B
Total Count (CFU/g)Log10 CFU/g
Low Level Low Level
#DIV/0!
#DIV/0! #DIV/0! SD
#DIV/0! #DIV/0! Variance (SD^2)
F-test (Day)
Fcrit (4x4)
#DIV/0! #DIV/0! SD
#DIV/0! #DIV/0! Variance (SD^2)
Measurement of Uncertainty
Pooled SD
MU
methods and validated alternative methods in a single laboratory
Enter data Calculated results
#DIV/0! #DIV/0!
#DIV/0! #DIV/0!
6.39
#DIV/0! #DIV/0!
#DIV/0! #DIV/0!
ent of Uncertainty
Low level high level
#DIV/0! #DIV/0!
#DIV/0! #DIV/0!
End of this tab
Food Category Food Matrix Sample Type
Solid 2
Trueness RTE Semi Solid 2
Liquid 2
only 1 represent
Repeatability RTE 1
routine test
only 1 represent
Reproducibility RTE 1
routine test
Easy Plate
Replication/
Spike Level Analyst Days AC CC
sample type
Low - - 4 4
2 Med - - 4 4
High - - 4 4
Low - - 4 4
2 Med - - 4 4
High - - 4 4
Low - - 4 4
2 Med - - 4 4
High - - 4 4
10 Low - - 10 10
A 2 10 10
5 Low
B 2 10 10
Total Plate Required 66 66
Easy Plate Petrifilm
EC YM-R AC CC EC YM-R
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
4 4 4 4 4 4
10 10
10 10
10 10
66 66 36 36 36 36
