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ICMSF Lecture on Microbiological

Sampling Plans

Susanne Dahms
IAFP, San Diego, 2002

Client - meeting - _____ - 1


Overview

 Introduction
 Sampling plans: Design and means to study their
performance
• Two-class attributes plans for qualitative data
• Two-class and three-class attributes plans for
grouped quantitative data
• Variables plans for quantitative data
 Microbiological sampling plans and Food Safety
Objectives
 Summary

Client - meeting - _____ - 2


ICMSF Books on Microbiological
Sampling Plans

Microorganisms in Foods 2.
Sampling for Microbiological Analysis: Principles and
Specific Applications.
(2nd ed. 1986, University of Toronto Press, out of
print)

Topics:
The statistical principles underlying attributes
sampling plans and their application to foods.

Client - meeting - _____ - 3


ICMSF Books on Microbiological
Sampling Plans
Microorganisms in Foods 7.
Microbiological Testing in Food Safety Management.
(2002, Kluwer Academic/Plenum Publishers)

Topics:
 Concepts of probability and sampling
 Appropriate sampling plans
 Selection of cases and attributes plans
 Establishing micro criteria for lot acceptance

Client - meeting - _____ - 4


ICMSF Cases

15 cases which reflect severity of the hazard, effect of


handling/preparation on the hazard, and intended population

Type of hazard Conditions Conditions Conditions may


reduce hazard cause no change increase hazard
in hazard
Utility Case 1 Case 2 Case 3

Indicator Case 4 Case 5 Case 6

Moderate Case 7 Case 8 Case 9

Serious Case 10 Case 11 Case 12

Severe Case 13 Case 14 Case 15

Client - meeting - _____ - 5


Types of Microbiological Sampling
Plans

Attributes plans:
Qualitative analytical results (presence/absence) or
quantitative results that have been grouped
(e.g. <10 cfu/g, 10 to 100 cfu/g, >100 cfu/g)

Variables plans:
Non-grouped quantitative analytical results
Require distributional assumptions be made

Client - meeting - _____ - 6


Two-Class Attributes Sampling Plans

Two-class sampling plans designed to decide on


acceptance or rejection of a lot consist of

 n – number of sample units to be chosen


independently and randomly from the lot
 m – a microbiological limit (i.e. in cfu/g);
a sample is defined to be positive, if its microbial
content exceeds this limit
 c – maximum allowable number of sample units
yielding a positive result (presence/absence testing)
or exceeding the microbiological limit m;
for pathogens c is usually set to 0

Client - meeting - _____ - 7


OC Curve for Two-Class Plans

Operation characteristics (OC) or performance for


two-class sampling plans:
Probability of lot acceptance calculated for possible
proportions defective in lot

Acceptance probability
Plot of OC curve to visualize
 sampling plan performance
 dependency on n and c

Proportion defective

Client - meeting - _____ - 8


Probability of Acceptance by Proportion Defective
1.0

n=5, c=0
0.8
Probability of Acceptance

0.6 P(rejection)

0.4

0.2
P(acceptance)

0.0

0.0 0.2 0.4 0.6 0.8


Proportion Defective
Probability of Acceptance by Proportion Defective
1.0
P(acceptance)=95%
n=5, c=0
0.8
Probability of Acceptance

0.6

0.4

0.2

P(rejection)=95%
0.0

0.0 0.2 0.4 0.6 0.8


Proportion Defective
Probability of Acceptance by Proportion Defective
1.0

n=5, c=0
0.8 n=10, c=0
n=20, c=0
Probability of Acceptance

0.6

0.4

0.2

0.0

0.0 0.2 0.4 0.6 0.8


Proportion Defective
Three-Class Attributes Sampling Plans

Three-class sampling plans consist of


 n – number of sample units to be chosen
independently and randomly from the lot
 m – a microbiological limit that separates good
quality from marginally acceptable quality
 M – a microbiological limit above which sampling
results are unacceptable or defective
 c – maximum allowable number of sample units
yielding results between m and M (marginally
acceptable);
the number of sample units allowed to exceed M is
usually set to 0

Client - meeting - _____ - 12


OC Function for Three-Class Plans

Operation characteristics (OC) or performance for


three-class plans:
Probability of lot acceptance depending on two
proportions
 marginally acceptable: between m and M
 defective: above M

Acceptance probability
ta bl e
c ce p
a l lya
in
OC function plotted as a arg
rop. m
P
three-dimensional graph
Proportio
n defectiv
e
Client - meeting - _____ - 13
OC Curve Referring to Mean Log CFU/G

Alternative approach for quantitative data:


 Distributional assumption for sampling results
e.g. log-normal with standard deviation known from
previous experience

Client - meeting - _____ - 14


Frequency Distribution Describing Lot Quality
0.6

mean
0.5 s.d.: standard deviation
(=0.8)
0.4
Probability Density

0.3
s.d. s.d.

0.2

0.1

0.0

0 1 2 3 4 5 6
Log cfu/g
OC Curve Referring to Mean Log CFU/G

Alternative approach for quantitative data:


 Distributional assumption for sampling results
e.g. log-normal with standard deviation known from
previous experience
 Determine proportions acceptable,
(marginally acceptable), and defective
for possible mean log cfu/g

Client - meeting - _____ - 16


Two-class sampling plan:
0.6
m

0.5

0.4
Probability Density

0.3

0.2 Proportion defective

0.1

0.0

0 1 2 3 4 5 6
Log cfu/g
Three-class sampling plan:
0.6
m M

0.5

0.4 Proportion marginally


Probability Density

acceptable
0.3

0.2

0.1
Proportion
defective
0.0

0 1 2 3 4 5 6
Log cfu/g
OC Curve Referring to Mean Log CFU/G

Alternative approach for quantitative data:


 Distributional assumption for sampling results
e.g. log-normal with standard deviation known from
previous experience
 Determine proportions acceptable,
(marginally acceptable), and defective
for possible mean log cfu/g
 Calculate acceptance probabilities and
plot against mean log cfu/g

Client - meeting - _____ - 19


m
Probability Density

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Log cfu/g
m

1.0
Proportion defective, pd

0.8

0.6

0.4

0.2

0.0

Mean Log cfu/g


1.0
0.8
0.6
pd
0.4
0.2
0.0

P(accept)
OC curve
n = 10,
c=2
Probability of acceptance

1.0 pd

0.8

0.6

0.4

0.2

0.0

Mean log cfu/g


Probability of Acceptance by Mean Log cfu/g (s.d.=0.8)
1.0

P(acceptance)=95% n=5, c=0, m=100 cfu/g


0.8
Probability of Acceptance

0.6

0.4

0.2

P(rejection)=95%
0.0

-2 -1 0 1 2 3 4
Mean Log cfu/g
Probability of Acceptance by Mean Log cfu/g (s.d.=0.8)
1.0

n=5, c=0, m=100 cfu/g


0.8 n=10, c=0, m=100 cfu/g
n=20, c=0, m=100 cfu/g
Probability of Acceptance

0.6

0.4

0.2

0.0

-2 -1 0 1 2 3 4
Mean Log cfu/g
Probability of Acceptance by Mean Log cfu/g (s.d.=0.8)
1.0

n=5, c=0, m=100 cfu/g


0.8 n=10, c=0, m=100 cfu/g
n=20, c=0, m=1 cfu/g
Probability of Acceptance

0.6

0.4

0.2

0.0

-2 -1 0 1 2 3 4
Mean Log cfu/g
Probability of Acceptance by Mean Log cfu/g (s.d.=0.8)
1.0

n=5, c=0, m=1 cfu/25g


0.8 n=10, c=0, m=100 cfu/g
n=20, c=0, m=1 cfu/g
Probability of Acceptance

0.6

0.4

0.2

0.0

-2 -1 0 1 2 3 4
Mean Log cfu/g
Performance of Sampling Plans

Sampling plan stringency, steepness of OC curve,


location of critical lot qualities (95% probability of
rejection, 95% probability of acceptance)
depend on
 Plan specifications n and c
 Microbiological limits m and M

Client - meeting - _____ - 27


Probability of Acceptance by Mean Log cfu/g, s.d.=0.8

1.0

Two-Class Plan:
0.8
n=5, c=1,
m=1000 cfu/g
Probability of Acceptance

0.6 Three-Class Plan:


n=5, c=1,
m=1000 cfu/g,
0.4 M=10 000 cfu/g

0.2

0.0

1.0 1.5 2.0 2.5 3.0 3.5 4.0


Mean Log cfu/g
Probability of Acceptance by Mean Log cfu/g, s.d.=1.2

1.0

Two-Class Plan:
0.8
n=5, c=1,
m=1000 cfu/g
Probability of Acceptance

0.6 Three-Class Plan:


n=5, c=1,
m=1000 cfu/g,
0.4 M=10 000 cfu/g

0.2

0.0

1.0 1.5 2.0 2.5 3.0 3.5 4.0


Mean Log cfu/g
Probability of Acceptance by Mean Log cfu/g
3-Class Plan: n=5, c=1, m=1000 cfu/g, M=10000 cfu/g
1.0

s.d.=0.8
0.8 s.d.=0.4
s.d.=0.2
Probability of Acceptance

0.6

0.4

0.2

0.0

1.0 1.5 2.0 2.5 3.0 3.5 4.0


Mean Log cfu/g
Performance of Sampling Plans

Sampling plan stringency, steepness of OC curve,


location of critical lot qualities (95% probability of
rejection, 95% probability of acceptance)
depend on
 Plan specifications n and c
 Microbiological limits m and M
 Standard deviation s.d.
 Difference M-m in relation to s.d.

Client - meeting - _____ - 31


ICMSF Three-Class Plans: Mean CFU/G
Rejected With 95% Probability

Case 4: Case 5: Case 6:


n=5, c=3 n=5, c=2 n=5, c=1
5128 cfu/g 3311 cfu/g 1819 cfu/g

Case 7: Case 8: Case 9:


n=5, c=3 n=5, c=1 n=10, c=1
3311 cfu/g 1819 cfu/g 575 cfu/g

With:
m = 1000 cfu/g, M = 10 000 cfu/g,
and standard deviation s.d. = 0.8

Client - meeting - _____ - 32


ICMSF Three-Class Plans: Mean CFU/G
Accepted With 95% Probability

Case 4: Case 5: Case 6:


n=5, c=3 n=5, c=2 n=5, c=1
138 cfu/g 115 cfu/g 63 cfu/g

Case 7: Case 8: Case 9:


n=5, c=3 n=5, c=1 n=10, c=1
115 cfu/g 63 cfu/g 35 cfu/g

With:
m=1000 cfu/g, M=10 000 cfu/g,
and standard deviation s.d.=0.8

Client - meeting - _____ - 33


ICMSF Two-Class Plans: Mean CFU/G
Rejected With 95% Probability

Case 10: Case 11: Case 12:


n=5, c=0 n=10, c=0 n=20, c=0
1 cfu / 32g 1 cfu / 83g 1 cfu / 185g

Case 13: Case 14: Case 15:


n=15, c=0 n=30, c=0 n=60, c=0
1 cfu / 135g 1 cfu / 278g 1 cfu / 526g

With:
m = 0 cfu / 25g,
and standard deviation s.d. = 0.8

Client - meeting - _____ - 34


ICMSF Two-Class Plans: Mean CFU/G
Accepted With 95% Probability

Case 10: Case 11: Case 12:


n=5, c=0 n=10, c=0 n=20, c=0
1 cfu / 1515g 1 cfu / 2439g 1 cfu / 3846g

Case 13: Case 14: Case 15:


n=15, c=0 n=30, c=0 n=60, c=0
1 cfu / 3125g 1 cfu / 4761g 1 cfu / 7142g

With:
m = 0 cfu / 25g,
and standard deviation s.d. = 0.8

Client - meeting - _____ - 35


Variables Sampling Plans: Design

Variables Sampling Plans evaluate non-grouped


quantitative analytical results (i.e. log cfu/g).
Specifications:
 Number of sample units: n
 Acceptable quality limit: V (in log cfu/g)
 Maximum proportion above V: p0
 Probability to accept a non-conforming lot: α

Client - meeting - _____ - 36


Variables Sampling Plan (s.d.=0.8)
0.6
Mean0 = V - u1-p0 * s.d.
0.5 V: acceptable
quality limit
0.4
Probability Density

0.3

0.2

p0: acceptable
0.1 proportion
defective
0.0

0 1 2 3 4 5 6
Log cfu/g
Variables Sampling Plans: Decision Rule

Calculate the average of n sample results and a


confidence interval to estimate true lot mean.
For acceptable lots the estimate is expected to be
lower than mean0 corresponding to V and p0.

Decision rule:
 Rejection of lot, if average + k * s.d. > V
 Acceptance of lot, if average + k * s.d. <= V
k is depending on n, p0, and α.

Client - meeting - _____ - 38


Sampling Plans and FSOs: Example

Food Safety Objective:

≤100 Listeria monocytogenes per g in cold-smoked


salmon at time of consumption

Cases and sampling plans:


No inactivation, growth assumed not to occur
case 11: n = 10 samples with c = 0 and m = 100 cfu/g
No inactivation, growth assumed to occur
case 12: n = 20 samples with c = 0 and m = 100 cfu/g

ICMSF (1994) Int. J. Food Microbiol. 22:89-96


CODEX ALIMENTARIUS COMMISSION, August 2001, CX/FH 01/6 ANNEX 3.2
Client - meeting - _____ - 39
Performance of Sampling Plans for
Listeria Monocytogenes

Assumption: standard deviation s.d. = 0.8

Case 11: n = 10 samples with c = 0 and m = 100 cfu/g


Mean cfu/g rejected with 95% probability: 30 cfu/g
Mean cfu/g accepted with 95% probability: 1 cfu/g

Case 12: n = 20 samples with c = 0 and m = 100 cfu/g


Mean cfu/g rejected with 95% probability: 13 cfu/g
Mean cfu/g accepted with 95% probability: 0.5 cfu/g

Client - meeting - _____ - 40


Lot quality rejected with 95% probability (s.d.=0.8)
0.6
Case 11: 2-class plan, n=10, c=0, m=100 cfu/g
0.5 m = FSO
Probability Density

0.4

0.3

0.2 26%
0.1

0.0

-1 0 1 2 3 4

Log cfu/g

0.6
Case 12: 2-class plan, n=20, c=0, m=100 cfu/g
0.5 m = FSO
Probability Density

0.4

0.3

0.2 14%
0.1

0.0

-1 0 1 2 3 4

Log cfu/g
Variables Sampling Plans and FSOs

Example: Listeria monocytogenes in cold-smoked


salmon; FSO = 100 cfu/g at time of consumption
Variables Plan:
 Number of sample units: n = 10
 Acceptable quality limit: V = FSO = 100 cfu/g
 Maximum proportion above V: p0 = 5%
 Probability to reject a non-conforming lot: 1-α = 95%

Approach is based on the assumption that


L. m. number in sample can be reliably quantified !

Client - meeting - _____ - 42


Summary (1)

Though microbiological sampling plans are widely used


and adopted (e.g. microbiological criteria), their
implications are not fully understood:
 Hypothesis tested
 Reliability of decision
 Performance: steepness of OC curve / stringency,
quality rejected / accepted with given, say 95%,
probability
 Justification of number of samples, n

Client - meeting - _____ - 43


Summary (2)

Based on a quantitative description of lot quality in terms


of a frequency distribution for sample results
even attributes plans can be used to assess mean
microbiological concentrations in lots of foods.
Performance depends on:
 Sampling plan specifications (attributes plans: n and c)
 Microbial limits set (attributes plans: m and M)
 Validity of assumptions for the frequency distribution
(standard deviation s.d.)
 Reliability and precision of the analytical technique

Client - meeting - _____ - 44


Summary (3)

Firm understanding of sampling plan


performance and its statistical background is
required to use them effectively,
for instance,
to design sampling plans that are in agreement
with given Food Safety Objectives.

www.icmsf.org
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