Microbiological

Microbiological Sampling
Sampling and
and
Testing
Testing in
in Food
Workshop,
v
Food Safety
Safety
v Management
Management
Workshop, Xiamen,
Xiamen, October
October 2012
2012
 Overview
Overview

• basic statistics of attributes sampling schemes

• ICSMF sampling plan guidance
• testing for compliance to a specified microbiological
level:
– implications of variability, sample size, sample number

• the ICSMF sampling plan spreadsheet

Sampling Workshop, Xiamen, October 2012

 Sampling
Sampling plans
plans

• “attributes” plans
– 2 or 3 class
– test against a (probability of a) specific level of the hazard
– used by buyers, regulators

• “variables” plans
– incorporate all measured levels of the hazard to assess
compliance
– must know mean and standard deviation of hazard levels,
i.e. a history of quantitative results

Sampling Workshop, Xiamen, October 2012

 attributes sampling plans

defined by three (or four) quantities:

• ’m’ – the microbiological limit (e.g., <1 per 25g, 100 cfu/g)
desired

• ‘M’ – applied in some cases to recognise occasional deviations, a

level higher than ‘m’ but one which must never be exceeded

• ‘n’= the number of samples from the lot that must be tested

• ‘c’ = the number of samples that may exceed ‘m’

Sampling Workshop, Xiamen, October 2012

 2 and 3-class sampling plans

2 class:
the criterion is absolute, sample units either “pass” or “fail”
described only by m, c, n

3 class:
the criterion includes a third category, i.e., “marginally
acceptable (“M”)
described by M, m, c, n

Sampling Workshop, Xiamen, October 2012

 22 and
and 33 class
class attributes
attributes plans
plans

2-class plan m 3-class plan m M

unacceptable
marginally

Relative proportion of
Relative proportion of

acceptable

sample units in lot

sample units in lot

acceptable acceptable

unacceptabl

log count / g log count / g

Sampling Workshop, Xiamen, October 2012

Advice on Sampling Plan
Plan Selection

Sampling Workshop, Xiamen, October 2012

 ICMSF
ICMSF Sampling
Sampling Plans
Plans –– ‘risk’
‘risk’ basis
basis
increasing probability of unacceptable hazard
increasing severity of consequence

Sampling Workshop, Xiamen, October 2012

 Examples of Sampling Plans

ICMSF: Microorganisms in Foods, 2. Sampling for Microbiological Analysis. Principles and Specific Applications,
2nd Edition, Blackwell Scientific Publications, 1986 (ISBN-0632-015-675).

Sampling Workshop, Xiamen, October 2012

 Microbiological Sampling and
Testing in Food Safety Management
Workshop, Xiamen, October 2012
 Common
Common Misconception
Misconception about
about
Presence/Absence
Presence/Absence Testing
Testing

“If the test is negative, the batch is free from pathogens”

Wrong!

In fact, we can never demonstrate ‘zero tolerance’, only a

certain level of confidence that the contamination is
below a certain level

Sampling Workshop, Xiamen, October 2012

 Sampling
Sampling and
and the
the Probability
Probability of
of
Detection
Detection

<50% defective?

<10% defective?

<1 % defective?

Sampling Workshop, Xiamen, October 2012

• no sampling plan can ever guarantee absolute
safety of the food
• the sampling plan can only provide a certain
level of confidence

Sampling Workshop, Xiamen, October 2012

 Sampling
Sampling and the Probability
Probability of
of
Detection
Detection

• probability of detection depends on

– actual proportion of samples that are unacceptable
– number of samples examined
– it is easily described mathematically (a Poisson
sampling process)

number of samples examined

Pacceptance = (1 - Pdefective)

Sampling Workshop, Xiamen, October 2012

Probability
Probability of
of acceptance
acceptance with
with 55 negative
negative
samples
samples depends
depends on
on true
true ‘defect’
‘defect’ rate
rate

1
Probability of accepting the lot

0.8

0.6 n=5
0.4

0.2

0
0 0.2 0.4 0.6 0.8 1
Proportion defective sample units in a lot

Sampling Workshop, Xiamen, October 2012

Probability
Probability of
of accepting
accepting aa defective
defective lot
lot
(Operating
(Operating Characteristic
Characteristic Curve)
Curve)

Sampling Workshop, Xiamen, October 2012

• a presence/absence sampling plan is effectively
a (single dilution) multiple tube dilution method

• knowing this, we can calculate the detection

limits (“sensitivity”) of any sampling scheme

Sampling Workshop, Xiamen, October 2012

 Designing an Appropriate Sampling
Plan to Meet a Performance Objective

• binomial distributions
– either we detect a microorganism(s) in the sample or we do
not
• probability of detection depends
– concentration in the lot
– size of the sample
– e.g. if the concentration is 1 per 10 g we expect to detect
(“presence”) in most 25 g samples, but if the concentration is
1 per 50 g, we expect to detect in only every second sample,
“on average”

Sampling Workshop, Xiamen, October 2012

 Limitations
Limitations of Testing

• methods may not recover injured cells

• microbiological condition of the food changes over time

• microoorganisms aren’t uniformly distributed

throughout the lot

• even if they were completely evenly distributed the

samples would not contain identical numbers of cells

Sampling Workshop, Xiamen, October 2012

Use and Interpretation of the
ICMSF
ICMSF Sampling Plan Tool
Tool

Sampling Workshop, Xiamen, October 2012

Variability and acceptable levels

Sampling Workshop, Xiamen, October 2012

Variability and acceptable levels

Sampling Workshop, Xiamen, October 2012

Variability and acceptable levels

Sampling Workshop, Xiamen, October 2012

Variability and acceptable levels

Sampling Workshop, Xiamen, October 2012

 Variability and acceptable levels

• we want to design a sampling plan that:

– accounts for variability in hazard concentration
– provides the required level of confidence that levels in most
samples do not exceed the acceptable concentration

• thus, number and size of sample depends on

– maximum acceptable level
– mean level
– variability (e.g., standard deviation) around the mean
– level of confidence required

Sampling Workshop, Xiamen, October 2012

 Designing an Appropriate Sampling
Plan to Meet a Performance Objective

• Steps in the development of a sampling plan…

• For the specified standard:

– define the ‘just unacceptable lot’ from:
• the standard deviation of contamination of samples within a
lot
• the required level of confidence
– determine the needed performance of the analytical
procedure (probability of false positives and false
negatives; e.g. determined from OC curves)
– determine the number of samples/sample size

Sampling Workshop, Xiamen, October 2012

 Designing an Appropriate Sampling
Plan to Meet a Performance Objective

• often, when testing for pathogens, the acceptable

mean concentration is small
• consequently, only a small proportion of samples to
be tested are expected to be positive (have
detectable pathogens)
• this means we are sampling from a Poisson process
• we need to know how many samples to take to be
highly confident that the mean concentration is below
our acceptable limit

Sampling Workshop, Xiamen, October 2012

 Poisson processes

• a Poisson process is a stochastic (random, chance)

process in which events occur continuously and
independently of one another
• random sampling from a population when assessing a
(binomial) attribute is a Poisson process
• the chances of making a particular number of
observations, in a given time, or space, based on some
true average density, or frequency, is defined by the
Poisson distribution
• for example….

Sampling Workshop, Xiamen, October 2012

 Poisson processes

• if the concentration of a pathogen in a food:

– were exactly 1 cell per cm3, and
– the cells were perfectly evenly distributed, and
– our sample size was exactly one cm3

• our samples of 1 cm3 would not always contain 1 cell….

Sampling Workshop, Xiamen, October 2012

Poisson sampling “error”
This is to illustrate the idea of
Poisson (sampling) process. i.e., that
even if contamination is perfectly evenly
distributed, and if the sample size is exactly
equal to the expected concentration (i.e., so that
you would expect one contaminant per sample) it
is possible to capture none, or more than one
contaminant
per sample.

i.e., mean concentration is one cell per sample ……

Sampling Workshop, Xiamen, October 2012
Poisson sampling “error”

i.e. but a sample could, by chance, contain four cells …

Sampling Workshop, Xiamen, October 2012
Poisson sampling “error”

or could contain two cells …

Sampling Workshop, Xiamen, October 2012
Poisson sampling “error”

or could contain no cells …

Sampling Workshop, Xiamen, October 2012
 Poisson distribution

• the Poisson distribution predicts the probability of

observing a certain number of ‘events’ (e.g. positive
samples) from a population with some known, or
expected, average value of ‘events’.

Sampling Workshop, Xiamen, October 2012

 Poisson distribution

• The probability that a certain number of ‘events’

(e.g., number of positive samples) will be
observed, when the true average of those
‘events’ is known, is given as:

P(observing n positives) = (true_ averagen ) ´ e- ( true_ average)

n!

Sampling Workshop, Xiamen, October 2012

Advice
Advice on Sampling Plan Design
Design

http://www.icmsf.iit.edu/main/home.html

Sampling Workshop, Xiamen, October 2012

ICMSF
ICMSF Sampling
Sampling Plan
Plan Spreadsheet
Spreadsheet

Sampling Workshop, Xiamen, October 2012

 elements
elements of
of the
the spreadsheet
spreadsheet

• mean = required average value of distribution (log10CFU) of counts in

the lot
• sigma = standard deviation known, or assumed (and assumed to be the same
between lots) to occur in the counts in the lot
• m* = detection threshold of test method (e.g. 2 log 10CFU; -1.4
log10CFU; for presence/absence is log (inverse of sample size); often
also the microbiological criterion
• n = number of samples tested
• c = number of samples permitted to fail the test (usually 0 for pathogens)
• Paccept = confidence required (or achieved) in the reliability of the sampling
plan
• various ‘buttons’ – these use ‘Solver’ or Goal Seek to calculate the required
plan for variables entered

Sampling Workshop, Xiamen, October 2012

Sampling Workshop, Xiamen, October 2012
Sampling Workshop, Xiamen, October 2012
Sampling Workshop, Xiamen, October 2012
 Food Control, 20 (2009): 967 - 979

Sampling Workshop, Xiamen, October 2012

 Useful
Useful Reading
Reading

van Schothorst, M., Zwietering, M.H., Ross, T., Buchanan, R.L and Cole,
M.B. International Commission on Microbiological Specifications for
Foods. (2009). Relating microbiological criteria to food safety objectives
and performance objectives. Food Control, 20: 967-979.ICMSF

R.C. Whiting, A. Rainosek, R.L. Buchanan, M. Milioti, D. LaBarre, W. Long,

A. Ruple and S. Schaub. (2006). Determining the microbiological criteria
for lot rejection from the performance objective or food safety objective.
International Journal of Food Microbiology, 110: 263–267.

ICMSF (International Commission on Microbiological Specifications for

Foods), 2002. Microorganisms in Foods, Microbiological Testing in
Food Safety Management, Vol. 7. Kluwer Academic/Plenum Pub, NY.
362 pp.

Sampling Workshop, Xiamen, October 2012

 Summary

• attributes sampling plans can be analysed

quantitatively
• need to know standard deviation, attributes test
results to estimate mean concentration
• the calculations can be complex and are best
done by computers…

Sampling Workshop, Xiamen, October 2012

 Use
Use and
and Interpretation
Interpretation of
of the
the
ICMSF
ICMSF Sampling
Sampling Plan
Plan Tool
Tool

thank you for attention, and questions

Sampling Workshop, Xiamen, October 2012