Sampling and microbiological analysis of

foods

See Chapters 10 and 11 of Food Microbiology (Adams, Moss & McClure, 4th edition 2016)

Ch 10 – Methods for the microbiological examination of foods

Ch 11 - Controlling the microbiological quality of foods
 Lecture content
• Overview of microbiological examination of
food (who, what, why, how)
• Microbiological criteria
• Sampling issues
• Two class and three class sampling plan
• Type of microbiological analysis
• Selective/differential media
• Modern methods (overview)
The Microbiological Examination of Food

Why ???
• To ensure food safety
• To ensure quality and fair trade
• To ensure a sustainable food supply
Who does it concern???
• Food producers, food processors, food retail and
service industry; industries engaged in food trade
• The consumers
• Government agencies—regulation, supply
 What is examined
• The end product
• Raw materials (including water!!)
• Ingredients, additives
• Surfaces of processing equipment, pipes.
pumps, valves, hoses, mixing vessels etc
• Packaging materials
• Food production, food processing and food
service environments, surrounding air
 Outline of Examination Process
• Deciding what to examine and what
microorganisms to test
• Selection and collection of samples
• Selection of analytical methods
• Conducting the analysis
• Comparing analytical data with established
specifications and standards
• Making decision to accept or reject product
Basic Operations in Microbiological Analysis
of Foods
• Selection, collection and transport of
samples to laboratory
• Preparation of sample suspension and
dilutions
• Culture, enumeration and detection of
microorganisms
• Isolation, identification and typing of
specific microorganisms
 The Five Criteria Required to Determine
Microbiological Acceptability
• A statement that describes the sample for testing
• A statement that describes the microorganisms to
be tested
• A statement that describes the analytical methods
to be used to measure the microorganisms
• A plan that describes the number, size and method
of sampling the product
• Statement of a numerical limit for each organism to
be examined in a sample and the number of sample
units in the test batch/lot that must comply with
these limits
International Commission on the Microbiological Specification for Food (ICMSF)
 Application of Microbiological Criteria

1. Microbiological standard. The five criteria are written up as

part of a government regulation and are enforceable by law
and prosecution; mainly used to manage public health issues
2. Microbiological specification. The five criteria become part
of a contract between a supplier and purchaser to manage
quality and safety; it is a legal document that can be used in
court to determine liability and compensation should a
dispute over food quality or safety occur.
3. Guidelines. The five criteria used by industry/companies for
internal monitoring of performance in application of Good
Manufacturing Practice (GMP) and Hazard Analysis and
Critical Control Points (HACCP).
 Use of Microbiological Criteria
• To assess the safety and keeping quality or shelf life of a food
• To monitor adherence of producers and processors to good
manufacturing practices (GMP)

Provide industries and governments with

guidelines to manage food production

Enhance consumer confidence

Facilitate (fair) international trade through a

system of uniformity and harmonization
 Who Sets Microbiological Criteria?
• Experts within the International Commission on the
Microbiological Specification for Foods (ICMSF)

• Food & Agriculture Organisation (FAO) and World

Health Organisation (WHO) in Codex Alimentarius

• The World Trade Organization (WTO) recommends

adoption of recommended criteria
 Setting Microbiological Criteria
• The components of microbiological criteria
(sampling plans, analytical methods,
microbiological limits or acceptance levels) are
generally set only after many years of experience
and extensive study with any given product
• Such criteria should be achievable by existing
good manufacturing practices and technologies
• Need to allow for some margin of variation in
process and production
 Sampling for Analysis
• Cannot test all the products. Why?? Need to take
samples.
• Make decision to accept or reject based on testing a
representative number of samples from the
lot/batch
• A lot or batch is that quantity of product produced,
handled or stored under identical or uniform
conditions within a specified time period. Give an
example of a lot/batch!!
• Final decision is statistically based and considers the
balance of risk to consumer and producer.
 Sampling for Analysis

• The greater the number and amount of

samples tested, the greater the
reliability of the result.
• BUT, there is an economic cost—loss of
product, expense of testing
• How do we achieve the best balance??
 Sampling—Many Variables
• What amount?
• How many?
• From what location?
• At what time/point in the process?
Some factors to consider:
Degree of product hazard
Product uniformity, heterogeneity
Data consistency
Practical limitations
Distribution of Microbial Cells

RANDOM REGULAR CLUMPS or

MICRO-COLONIES
What Factors Affect the Distribution of
Microbial Cells in Foods ?

Let’s discuss
 Sampling Plans
• Working component of microbiological
examination
• Now developed for most products by the
International Commission on Microbiological
Specifications for Foods (ICMSF) through the
International Union of Microbiological Societies
(IUMS) -www.iums.org- and adopted by CODEX
Alimentarius of WHO/FAO
• Used in world trade
 Sampling Plans

Two class sampling plan

• Decision: accept or reject (two classes); consists
of:
• n - number of sample units in batch to be tested
• m - microbial count (eg. cfu/g), above which
sample is considered defective
• c - maximum number of sample units allowed to
exceed m; when above this number, the whole
batch is rejected
 Example: Food Standards Code Australia

• Powdered infant formula products. (25 g

samples to be taken from batch)
• Salmonella (two class): n = 10; c = 0; m= 0.
• This is a two class sampling plan where 10 x
25 g samples of each batch must be analysed;
if Salmonella (ie, a single cell or more) is
detected in any of the 10 x 25 g samples,
then the batch is rejected.
Example: Food Standards Code Australia

PDF included in this weeks materials on Moodle!

Example: Food Standards Code Australia
Example: Food Standards Code Australia

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 Sampling Plans
Three class sampling plan
• Decision: accept, reject, marginally acceptable
• n - number of sample units in batch to be tested
• m - microbial count above which sample is
considered marginally acceptable
• M - count above which sample is considered
defective, and whole batch is rejected if any sample
exceeds this count
• c - the maximum number of samples allowed to
have counts between m and M
 Example: Food Standards Code Australia
•Unpasteurized milk; standard plate count
•n = 5, m = 25,000 cfu/ml, M = 250,000 cfu/ml, c=1 (100 ml samples
to be taken)
•Sample volumes of 100 ml are to be taken from each test batch
•If >1 (ie, c) of these samples gives a count exceeding 25,000 cfu/ml
(ie, m) the batch is considered unacceptable ie, rejected.
•Also, if any of the five samples gives a count exceeding
250,000 cfu/ml (ie, M), the batch is rejected.
•There is a zone of marginal acceptability where one sample of the
batch may have a count within 25,000 - 250,000 cfu/ml.
•While such a batch is legally acceptable, the data signal to the
manufacturer that “something” is not working to the best
performance and urgent checking of GMP or analytical
procedures is required.
 Example: Three Class Plan
Studdard E et al.( 2003) Sampling for microbiological analysis. In Foodborne Microorganisms of
Public Health Significance, 6th ed, Hocking, AH et al (eds)
AIFST, Sydney
Page 457 of Course Text Book
 Sampling Plans

Acceptable probability of making incorrect

decisions
• False negative—product accepted when in
reality it contains the hazardous organism;
serious consequences; should be less than 0.1%?
• False positive– product rejected when it does
not contain the hazardous organism; leads to
economic waste; should be less than1-5%?
 How can we decrease probability of an
incorrect decision?

• Increase n ?
• Decrease m and M ?
• Decrease c ?
• Consult tables on probability of acceptance
(derived from operating characteristic curves)

Cannot sample and test all product

Testing is expensive and time consuming
Always some variation of data
 Operating characteristic curve

Relationship between percentage defectives and acceptance probability

 Operating characteristic curve

Dependence of acceptance probability on n (number of samples tested in a batch)

 Factors Affecting the Stringency of
Sampling Plan
• Hazard severity of organism: severe (eg. Salmonella
serovar Typhi, Clostridium botulinum); moderate
(eg. Salmonella serovar Typhimurium); low (eg.
Bacillus cereus, Clostridium perfringens)
• Consumer group: young , elderly,
immunocompromised at greater risk
• Type of food: raw, cooked, canned, mass service,
catered

Sampleaccording
Sample accordingtotorisk
risk
Consider the interplay between stringency and sampling plans
 Microbiological Examination of Foods

• There are many types of analyses to do.

• For each analysis, there are many methods.

The Challenge!!!
What test/ analysis???
What method do you choose??
 Sampling - Practical Points
• Liquid products: use pipette.
• Solid products: excise, cut out, collect a
representative portion using scalpel, scissors,
tongs, spatula; use gloved (sterile) hand.
• Surfaces of products (eg. animal carcass,
surfaces of equipment, utensils etc); use swabs
or contact media.
• Atmosphere (air); use special impactor pumps
to collect and test a fixed volume of air
 Sampling - Practical Points
• Random sampling
• Label clearly, precisely
• Collect, transport aseptically
• Refrigerate (5oC); do not freeze; ice box
• Analyse within 24 hours/less; check temperature on
receipt for analysis
• Register sample in log book
• Must have accurate records of traceability
• Failures in sampling lead to invalid results

If these are not done properly, your results will be in question!!!

 Microbiological Analyses
• Total plate count (TPC), aka, aerobic plate count, standard plate
count (SPC)
• Drop plates
• Most Probable Number methods
• Yeast and mould counts
• Coliforms, faecal coliforms, Escherichia coli counts (indicator
tests)
• Counts or presence/absence tests for specific pathogenic bacteria
(eg. Salmonella)
• Counts for specific spoilage bacterial groups (psychrotrophs,
proteolytics, lipolytics, lactic acid bacteria, acetic acid bacteria
etc)
• Viruses???
Specific issues in microbiological analysis of
foods
• Target organism may be present in very low populations
—need to use enrichment culture for its detection
• Target organism may occur along with many other
species in total microbial community - need selective
culture to amplify its relative population in mixture and
differential reactions to detect its presence among other
species
• Consider matrix effect of food
• Detect sublethally injured cells
• Need differentiation and identification at sub-species
level - strains (sometimes)
 Formulation of Culture Media
• Selective Growth Medium. Add specific components (eg.
antibiotics, dyes, detergents, organic acids) or use certain
culture conditions (eg. low temperature) that selectively
encourage the growth of the target organism and restrict
growth of non-target organisms
• Differential Growth Medium. Add ingredients that allow
target species to express key biochemical reactions (eg. sugar
fermentation, hydrogen sulphide production, egg yolk
hydrolysis) that distinguish/ differentiate it from other species
• Selective Differential Growth Medium. Combination of above
 Selective Differential Broth
Lauryl Tryptose Broth, for coliforms, E. coli
• Tryptone—nutrient
• Lactose—differential agent
• Phosphate buffer, pH 6.8
• NaCl
• Sodium lauryl sulphate (detergent), selective
agent
 Selective Differential Agar
XLD agar----Salmonella
• Yeast extract—nutrient
• Sodium deoxycholate—selective agent
• Xylose , 3.75 g---differential
• Lactose, 7.5 g----differential
• Sucrose, 7.5g----differential
• Phenol red----differential
• Sodium thiosulphate—differential
• Ferric ammonium citrate--differential
Requirements of Microbiological Methods

• Reliability. Gives data with predictable degree of

accuracy and precision when used by a competent
analyst.
• Accuracy. Ability of method to measure the true
value of the target organism (ie. give ecological
truth!!).
• Precision. Measures the reproducibility of a
method within a laboratory and between different
laboratories. Replicate samples are measured and
the standard deviation or coefficient of variation of
data are calculated to give the degree of precision
Requirements of Microbiological Methods

• Specificity/selectivity. The ability of the

method to detect the target organism; a
false positive result occurs when it detects
the target organism when, in reality, it is not
there; a false negative occurs if it fails to
detect the target organism when it is there.
• Sensitivity. Defines the lowest concentration
of the organism that can be measured with
acceptable accuracy and precision; the limit
of detection
 Reference or Standard Method
• One that has been peer reviewed,
published in an internationally reputable
journal, used over a period of time, and
judged by a consensus of scientific data
and opinion to be accurate.
• Standard method is written into national
and international legislation and is the
required method in legal proceedings
 Question???
How would you determine
the most abundant
microbial species in a food
product??
 Examine Sample Under Microscope

• If a product contains > 106 cfu/g (eg. if spoiled),

these cells can be seen under light microscope by
phase contrast methods or by staining.
• Fast method of assessing quality
• Enables general decision on microbial group
present—bacteria (rods/cocci), yeasts, moulds
• Enables observation of some properties—shape,
spores, motility, Gram reaction
 General Plate Count
• Examine sample, after appropriate dilution, by plate count on a
general nutritive, non-selective medium.
• Note diversity of colony types (morphology) on the plate; count
of each type gives estimation of its population and relative
dominance
• Isolate and purify representatives of each colony type, prepare
a stock culture
• Identify isolate by standard cultural physiological and
biochemical tests, compare data with standard descriptions
• Prepare DNA from isolate, sequence sections of ribosomal DNA;
compare sequences with taxonomic data bases
• Use molecular DNA methods or immunoassays for sub species
(strain) differentiation
 Question??

Will the dominant species in a product be the

one that determines the overall quality of the
product??
• YES??
• NO??
• NOT ALWAYS??
WHY??
 Alternative Methods for Microbiological
Analysis of Foods
• Cultural methods take too long for decision; labour
and materials expensive
• Modern food production requires automated
methods that give faster decisions
•Modern methods have objectives of:
 speed of decision
 automation
 computer processing
 decreased analytical costs
 improved efficiency, accuracy and reliability,
 potential to analyse a greater number of samples.
 Requirements of Alternative Methods

• Offer a benefit- speed, cost, efficiency

• Reliable, accurate, precise, reproducible
• Give data equal to or better than existing
standard methods
• Approval and accreditation by regulatory
authorities
• Safe in use
• Have reliable effective back up technical service
 Alternative Methods
• Speed up, automate determination of total microbial
biomass– electrical impedance/ conductance; optical
techniques; ATP-bioluminescence; quantitative PCR
• Speed up, automate species identification; miniaturized
kits (eg. API, BioLog kits); nucleic acid sequencing
• Speed up detection of specific organisms:
immunoassays (ELISA), slide agglutination; nucleic acid
probes, PCR methods.
• Strain differentiation: PCR and sequencing
• Simplified cultural methods: Petrifilm