Utility of Microbiological Testing for Safety and Quality

1.1 Introduction
This chapter is intended to provide an overview of microbiological testing, as well as an introduction
to the related concepts that are discussed in more detail in subsequent chapters or other ICMSF publications.
Microbiological testing is applied to food safety and quality management in a number of
ways. Governments may use pathogen or indicator testing for lot inspection or verification as a means

of lot acceptance, for example at port of entry or for surveillance activity on products in commerce.
Industry may also use end product tests for pathogens or indicators for lot acceptance in customersupplier
relationships. Industry also uses microbiological testing to design products and verify the
adequacy of performance of process controls for food safety and spoilage control in Hazard Analysis
Critical Control Point (HACCP) programs or Good Hygienic Practices/Good Manufacturing Practices
(GHP/GMP) programs. These tests may be run on end product, in-process or environmental samples.
The target microorganism may be a pathogen, an indicator or a utility microorganism. Investigational
sampling is conducted by both government and industry when a microbiological issue is identified to
gain information and to identify potential causes of a problem and potential solutions. This testing
may examine end product, ingredients, in-process and environmental samples that may be collected
at different points in the food system.
Microbiological criteria can be applied at all stages in the food supply chain, from agricultural and
aquaculture producers to wild harvesters, through production and retail. The quality and safety of
foods at retail may be mandated by governments to protect consumers and meet their expectations,
but to achieve this, microbiological limits may need to be applied at earlier points in the supply chain.
These criteria are often determined and imposed by businesses rather than governments and may be
different than those applicable at the retail level.
When using microbiological tests to evaluate safety or quality of food it is important to select and
apply these with knowledge of their limitations, their benefits and the purposes for which they are
intended. In many instances, other assessments are faster and more effective than microbiological
testing for food safety assurance. It is well recognized that application of prerequisite programs (e.g.,
Good Agricultural Practices (GAP), GHP, GMP etc.) and a HACCP program is the most effective food
safety management strategy (Codex Alimentarius 1997a, ICMSF 1988, 2002a). Control of undesirable
microorganisms in foods is best addressed at appropriate steps in the food chain through application of
these approaches. However, a variety of different approaches to microbiological testing, which may or
may not include pathogen testing, frequently plays an important role in verifying the effectiveness of
food safety management programs when used in a thoughtful, well-planned manner.
Chapter 1
Utility of Microbiological Testing for Safety and Quality
4 1 Utility of Microbiological Testing for Safety and Quality
Identification of criteria relevant for assurance of microbiological food safety and quality, and
their specification within the risk-based food safety management strategies is the main subject of
this text. The book aims to provide guidance on appropriate microbiological testing for food safety
and quality, including relevant microorganisms, limits and steps in the production and distribution
of foods at which testing can be usefully applied. Chapters 2–6 provide more detailed discussions
of specific uses of microbiological testing, while Chaps. 8–26 provide guidance of relevant microbiological
testing and criteria for specific groups of commodities. Chapter 7 describes the structure
of Chaps. 8–26, and explains the approach that led to the suggested microbiological tests and
criteria.
This chapter provides a brief introduction to microbiological testing in the management of microbial
food safety and quality, as well as providing an introduction to the overall text.
1.1.1 Testing as Part of a Food Safety Management Program
The role of food safety in international trade of foods is governed by the World Trade Organization
Sanitary and Phytosanitary (SPS) Agreement (WTO 1994). To determine whether a food should be
considered safe the term appropriate level of protection (ALOP) has been used, defined as “the level
of protection deemed appropriate…to protect human, animal or plant life…” This definition has
caused great difficulties for a number of reasons in part because the idea of what is “appropriate”
differs from country to country, i.e., “acceptable” risk is culturally defined. Hence, there is increased
interest in developing tools to more effectively link the requirements of food safety programs with
their expected public health impact.
The risk analysis framework described by ICMSF (2002a) and the Codex Alimentarius
Commission (2008b) provides a structured approach to the management of the safety of food and
introduced the concept of Food Safety Objective (FSO) as a tool to meet a public health goal such as
an ALOP. FSOs and Performance Objectives (PO) can be used to communicate requisite food safety
levels, e.g., to industry. FSOs and POs are distinct levels of foodborne hazards that cannot be
exceeded at the point of consumption and earlier in the food chain, respectively, and can be met using
good practices (GAPs and GHPs) and HACCP programs. While primarily applied for food safety
assurance, the principles of these programs can also be applied for proactive assurance of food
quality.
The principles of using good practices and HACCP, in order to produce safe foods, do not change
with the introduction of these concepts. GHP, GAP and HACCP are the tools for achieving an FSO
or PO. Assessing processing and preservation parameters is the preferred option to check that an
FSO or a PO is met, but sometimes sampling and testing against microbiological criteria can be
used.
Since the FSO is the maximum frequency or concentration of a hazard at the point of consumption,
this level is frequently very low. Because of this, obtaining a true measure of this level is impossible
in most cases. Compliance with POs set at earlier steps in the food chain can sometimes be
checked by microbiological testing. However, in most cases, validation of control measures, verification
of the results of Critical Control Point (CCP) monitoring, and auditing GHP and HACCP
systems are needed to provide reliable evidence that POs and thus the FSO are met.
To benefit from the flexibility that an outcome based risk management system offers, it is important
to be able to demonstrate that the selected control measures actually are capable of achieving the
intended level of control on a consistent basis. The successful implementation of HACCP depends
on its validation, including the clear identification of hazards, control measures available, critical
control points, critical limits and corrective actions. The outcomes of monitoring and verification
activities within a HACCP system assist in defining when re-validation may be necessary.
1.1 Introduction 5
1.1.2 Principles of Microbiological Testing and Definitions
The International Commission on Microbiological Specifications for Foods (ICMSF) has written
extensively on the principles of controlling microbial hazards in foods (see Introduction). These same
principles apply to the control of microorganisms associated with spoilage as well as general indicators
of GHP/GMP.
Microbiological tests are frequently performed to reach a decision or make a judgment. If the
purpose for collecting a sample cannot be defined, then the analysis should probably not be done. The
rationale for testing should be established prior to use and in the context of food safety management
falls into four general categories:
1. To determine safety
2. To determine adherence to Good Hygienic Practices (GHPs)
3. To determine the utility of a food or ingredient for a particular purpose
4. To predict product stability
Microbiological testing may also be used to gather background information (e.g., baseline data) that
does not involve setting limits. Additionally, microbiological testing may also be done for trace back
in the context of an epidemiologic investigation. This has important implications for liability, trade
and potential identification of root cause. Because this book focuses on use of data to assess process
control and product acceptance, the reader is referred to other references for epidemiological investigation
testing (e.g., CLSI 2007) and use of epidemiologic data to measure the impact of food safety
control programs ICMSF (2006).
Decision-making based on microbiological data requires that limits be established to differentiate
acceptable from unacceptable product or processes. These limits are meaningless without definition
of the sampling plan and analytical procedures employed to generate the data, as well as decisions to
be made and actions to be implemented as a consequence of the results. Microbiological limits that
include methods and sampling plans are defined as microbiological criteria. Microbiological criteria
should specify the number of sample units to be collected, the analytical method and the number of
analytical units that should conform to the limits. Criteria may be established for quality as well as
safety concerns (Codex Alimentarius 1997a) and are used in setting standards, guidelines and purchase
specifications, which are defined as follows:
Microbiological standards: Standards are contained in international, national and regional laws and
regulations. Exceeding a standard for a pathogen, such as Salmonella or Listeria, may lead to a
product recall and potentially punitive action.
Microbiological specification: Purchase specifications are agreements between the vendor and buyer
of a product as a basis for sale. These criteria can be considered mandatory and failure of the
vendor to meet specifications can be used as a basis for product rejection.
Microbiological guidelines: Guidelines are internal, advisory criteria established by a processor, a
trade association or sometimes governments. Failure to meet guidelines serves as an alert to the
processor, indicating that remedial action should be taken. A wide variety of criteria fit into this
category, such as results on pre-operational swabs from equipment, in-process samples from product
or equipment and environmental samples tested for pathogens.
1.1.3 Utility Microorganisms, Indicators or Pathogens
Some microbiological tests provide information regarding general contamination, incipient spoilage
or reduced shelf life, i.e., the utility of the product. The use of a utility test should be supported by
6 1 Utility of Microbiological Testing for Safety and Quality
relevant evidence, e.g., that total aerobic count, rather than enumeration of specific spoilage
microorganisms,
is a measure of incipient spoilage. Such tests may be useful indicators of product
quality. They may involve direct microscopic counts, yeast and mold counts, aerobic plate counts or
specialized tests, such as for cold tolerant microorganisms or for species causing a particular type of
spoilage (e.g., psychrotrophic pseudomonads in aerobically stored meats, lactobacilli in mayonnaise,
or thermophilic spore formers in sugar).
Microorganisms that are not normally harmful but may indicate the presence of pathogenic
microorganisms may be used as indirect indicators of a health hazard. For example, for dried egg
products Enterobacteriaceae or coliforms can be used as indicators of the potential presence of
salmonellae. In dried egg products, any practically applicable sampling plan cannot detect the low
level of salmonellae that may be present but that may represent an unacceptable risk to public
health. The quantitative information provided by indicator tests can be highly useful for trend analysis
and verification of process control. As such, the relative importance of conducting indicator
analysis may be higher than that for end product testing in a well designed program that emphasizes
useful testing for microbiological safety and quality management. Similarly, indicator microorganisms
may be useful in other situations, e.g., when assessing efficiency of cleaning and disinfection
or in investigational sampling. Tests for relevant microorganisms can also indicate whether certain
foods have been under processed, e.g., high numbers of mesophilic spore forming bacteria in lowacid,
shelf-stable canned foods indicate probable under processing when it is certain the container
is not leaking.
It is important to recognize that relationships between pathogen and indicators are not universal
and are influenced by the product and process and, therefore care must be taken when selecting
indicator microorganisms. For instance, coliform counts have been widely used as universal indicators
of hygiene, but in many products (e.g., meat or poultry, vegetables, etc.), psychrotrophic
Enterobacteriaceae will inevitably be present and the apparently high coliform counts do not necessarily
indicate hygienic failure or consumer risk. Similarly, microorganisms naturally present in the
product may also interfere with the analysis and interpretation of results, e.g., aeromonads on seafood
can mimic coliforms in methods.
1.1.4 Risk Based Sampling Using ICMSF Cases
ICMSF sampling plans are described, and their performance evaluated, in Chap. 7, Applications
and Use of Criteria and Other Tests. Sampling plan stringency varies according to the number of
samples tested (n), the upper limit on the acceptable concentration (m), the maximum tolerable
number of results (c) that exceed m and, for three-class plans, the upper limit of the marginally
acceptable level (M). Plans become more stringent as n increases and c, m and M decrease. ICMSF
(1974, 1986, 2002a) presented a comprehensive framework for use of acceptance sampling plans
based on degree of health risk or concern associated with a food and the change in hazard level,
and consequent risk to health, that is expected to occurred between sampling and consumption.
The latter is described as conditions of use. Five levels of hazard related to the microorganism
assessed are differentiated including utility microorganisms, indicator microorganisms and three
levels of hazard for pathogens, depending on the severity of the disease they cause. Three conditions
of use are differentiated:
1. Those that lead to a reduction in the level of the hazard between the time of production and time
of consumption.
2. Those that do not affect the level of the hazard.
3. Those that increase the level of hazard, and thus the risk, between the time of production and time
of consumption.
1.2 GHP and HACCP 7
These combinations lead to 15 different cases, each with its own corresponding sampling plan,
with higher numbered cases corresponding to more stringent plans. See Sect. 7.4 for additional explanation
of cases and how they are used in this book.
Utility tests are not related to health hazard, but to economic and esthetic considerations, therefore
the level of concern is categorized as low. Utility tests are included in cases 1–3 and satisfied by relatively
lenient sampling plans. Because of the uncertain relationship between indicators and specific
pathogens, the level of concern is classified as moderate and it is inappropriate to apply sampling
plans with high stringency for indicator microorganisms.
Three-class plans are typically less stringent than two-class plans, and are appropriate where
health risk is relatively low (cases 1–9). Two-class plans with c = 0 are usually used for situations
where the health risk is significant and more stringent control is needed (cases 10–15).
1.2 GHP and HACCP
As noted above, the production of safe food requires the application of GHP, GAP and similar prerequisite
programs, as well as the principles of HACCP, where they can be applied. These approaches
enable development and implementation of a total food safety management system that will control
most reliably the significant hazards in the food that is being produced. Some hazards are better
addressed through GAP or GHP measures (e.g., controlling the initial levels of a hazard through good
hygiene) while others are clearly best addressed through HACCP by a defined CCP that has been validated
to control the hazard of concern (e.g., reducing the level of a hazard or preventing growth).
It is recognized that in many situations preventative measures such as GHP and HACCP are much more
effective food safety management tools than end product testing. Consequently, the use of testing to determine
adherence to GHP and validation and verification of HACCP is essential. Chapter 5, Corrective
Action to Reestablish Control, discusses the elements of GHP and HACCP, while Chap. 3, Verification of
Process Control, discusses methods to evaluate the efficacy and integrity of these essential programs,
which differs from the statistical tools and assumptions that help interpret testing results.
1.2.1 Validation of Control Measures
Validation involves obtaining evidence that control measures, if properly implemented, are capable
of controlling the identified hazards (Codex 2008a). Validation is essential to demonstrate that GHP
and HACCP systems provide the level of safety assurance required and routine sampling plans are
not likely to be sufficient for validation studies. Validation focuses on the collection and evaluation
of scientific, technical and observational information and generally involves microbiological testing.
The scope of validation testing may extend beyond the control measures typically covered by
HACCP, to include areas such as primary production and consumer handling, which can also affect
the safety of the product at the point of consumption.
Processes can be validated using predictive models, microbiological challenge trials or application
of processing criteria (PCs) that have previously been validated or approved to provide adequate
levels of treatment and margins of safety, sometimes termed safe harbors. Not all of these methods
need to be used, and often a combination of approaches is used to establish sufficient evidence to
validate a process. Guidelines for validation have been developed by Codex Alimentarius (2008a).
Chapter 2, Validation of Control Measures, provides a detailed discussion of process validation
approaches and factors that should be considered. Specific considerations for microbiological studies
and approaches, and considerations in planning and undertaking relevant testing and analysis are
also considered. Practical advice for microbiological challenge studies to produce reliable results
is also presented.
8 1 Utility of Microbiological Testing for Safety and Quality
1.2.2 Verification of Process Control
Verification of control measures involves “the application of methods, procedures, tests and other
evaluations in addition to monitoring, to determine whether a control measure is, or has been, operating
as intended” where monitoring is defined as “the act of conducting a planned series of observations or
measurements of control parameters to assess whether a control measure is under control” (Codex
Alimentarius 1997b). Verification can use of a variety of measurements, including:
• Sensory assessments
• Chemical measurements, e.g., acetic acid and preservative levels, water content
• Physical measurements, e.g., pH, aW and temperature
• Time measurements
• Microbial tests, including tests for toxic metabolites
The development of microbiological criteria relevant to process verification testing, sampling strategy
and choice of the sampling plan, and the analysis and interpretation of the data generated for
decision-making is discussed in Chap. 3, Verification of Process Control. That chapter addresses
consideration of both within-batch and between-batch variability in verification testing. Baseline data
on the performance of the food system are used to characterize the quality and safety of product arising
from the process when it is functioning as intended. Comparing these baseline data with data
from periodic testing can then be used to provide:
1. Assurance that conditions that enable a food process to produce safe products are being
maintained.
2. A basis for analyzing performance trends so that corrective actions can be taken before loss of
control.
3. Insights into the cause for loss of control (e.g., periodicity of contamination).
4. A warning that conditions have changed sufficiently such that the original HACCP plan may need
to be reviewed.
Once established, process control testing typically involves routine testing of a small number of samples.
The microbiological limits for a process control testing program ideally include both an action level and
an upper limit. The action level allows corrective actions to be taken proactively before the upper limit is
reached. To detect such trends towards unacceptable loss of control as soon as possible, and to differentiate
them from extreme results that arise simply from normal variation within the acceptable range, comparison
of the data over time is needed and is usually done through some form of process control analysis,
such as control charting. The specific testing requirements depend on the process control analysis
approach employed, and are discussed and exemplified in Chap. 3.
1.2.3 Verification of Environmental Control
Assessment and control of microbial loads in food processing environments is important because
there is ample evidence that postprocessing contamination can affect product quality and safety.
Environmental testing is undertaken to ensure that GHP measures are effective in minimizing product
contamination from the processing environment. Microbiological testing is used to:
1. Assess the risk of product contamination.
2. Establish a baseline that characterizes when the processing environment is appropriately
controlled.
3. Assess whether control is being maintained.
4. Investigate sources of contamination to be able to implement corrective actions.
1.2 GHP and HACCP 9
Routine environmental sampling is most likely to be applied in food processing plants in which
recontamination of product from the environment could occur after a kill step. For ready-to-eat (RTE)
products for which there is no effective CCP, monitoring farm environments may also be useful.
Environmental sampling is unlikely to be useful at other steps along the food chain. Factors that
contribute to and influence postprocessing contamination as well as strategies and actions to control
pathogens in food processing environments are described in detail in ICMSF (2002b) and summarized
in Chap. 4, Verification of Environmental Control.
1.2.4 Corrective Action to Reestablish Control
Despite the application of food safety management systems, control is sometimes lost with potential
implications for product quality and safety. Evidence of loss of control may be obtained from an onsite
inspection, monitoring GHPs, monitoring or verifying activities, analysis of samples, consumer
complaints or epidemiological information implicating the food operation.
As defined by the Codex Alimentarius Commission (1997b), corrective action is “any action to be
taken when the results of monitoring at the CCP indicate a loss of control.” Control may not only rely
on the HACCP control points, but also on the combined effect of prerequisite programs, other actions
and the HACCP plan; thus evaluation of effective control is not always straightforward.
Unlike HACCP systems in which corrective actions in response to loss of control must be documented
as part of the HACCP plan, there is less clear description of specific actions to respond to
loss of controls relevant to GHP. Chapter 5, Corrective Action to Reestablish Control, describes how
visual inspection and microbiological testing are commonly employed to evaluate prerequisite programs,
and how they can indicate loss of control and reveal the need for more frequent or more effective
cleaning, for more frequent and thorough maintenance of processing equipment, for retraining of
staff in hygiene principles and practices or other actions. Specific testing can also be used to identify
contamination sources.
For control defined in the HACCP plan, the need for corrective actions for CCPs can be revealed
by routine monitoring or from epidemiological or customer complaint data. In these situations, testing
can reveal if the document control criteria were incorrect or have become inadequate. The use of
appropriate testing according to a relevant sampling plan can help to reveal the microbiological consequences
of loss of control and the disposition of the product, e.g., no increased risk, reprocessing
required or product must be discarded.
Chapter 5 considers these topics in greater detail, providing practical advice for assessing points/
processing requiring control, establishing base-line values so that unacceptable deviation can be
recognized, and identifying appropriate use of testing to reestablish control of the operation.
1.2.5 Microbiological Testing in Customer-Supplier Relations
The commercial food chain involves many interacting businesses and supplier-customer relationships,
each implying contracts that define expectations of customers and the commitments of suppliers. For
perishable and semi-perishable foods or ingredients these may include microbiological aspects of the
product, potentially concerning safety, quality and shelf life expectations. For shelf-stable and frozen
foods, microbial shelf life is not relevant, but because of persistence of some pathogens, microbiological
criteria may be relevant especially if resistant pathogens or microbial toxins could be present through
inappropriate handling earlier in the product’s life.
Microbiological criteria and testing in customer-supplier relationships can relate to raw materials,
ingredients, semi-processed and finished products. They can also consider the potential for microbial
10 1 Utility of Microbiological Testing for Safety and Quality
growth in the product. Criteria related to microbial quality and safety can include microbial limits,
product formulation specification, packaging, storage and transport conditions, and time/temperature
conditions that prevent, or minimize to an acceptable degree, the growth of pathogens or spoilage
microorganisms. Evaluation may include microbiological testing, physical-chemical measurements
(e.g., pH, aW, residual chlorine assessment etc.) or even visual assessment (e.g., mold affected fruits,
grains or nuts in a lot do not exceed some defined, acceptable limit).
Criteria may also relate to processing operations, such as those that might be considered in evaluating
a supplier HACCP program. Considerations in defining microbiological or related criteria can
include the point in the production chain, the intended further processing or end-use of the product,
technological feasibility etc. Microbiological testing considerations specifically relevant to customersupplier
relationships are discussed in further detail in Chap. 6, Microbiological Testing in Customer-
Supplier Relations.
1.2.6 End Product Testing to Evaluate Integrity
The relative importance of end product testing must be determined on a product by product basis. For
some products, end product testing is the only point where regulatory limits apply. End product testing
may be used for lot acceptance when there is insufficient process or testing information available
from which to evaluate product safety or utility. Similarly, for products in which no effective CCP is
currently available and there is no other means of assessing product integrity, end product testing may
offer the only alternative. The suggested criteria for lot acceptance in Part II of this book (Chaps.
8–26) are based on baseline data, experience, industry practice, relative risk when ICMSF cases are
considered or existing microbiological criteria that have been developed internationally as a result of
the risk analysis process established by the Codex Alimentarius Commission (see Sect. 7.4). Different
sampling plans may be appropriate in certain situations. Reducing the number of samples may be
entirely appropriate for on-going surveillance activity; whereas increasing the number of samples
may be prudent when investigating significant process deviations or outbreaks. For example, in the
event of a loss of control, sampling frequency should be increased until confidence is achieved that
the process is again under control. Such investigational samples should be analyzed individually
rather than as composites, because this will help in identifying the source of the problem.
1.3 Limitations in Microbiological Testing of Foods
This book aims to provide practical guidance on relevant microbiological testing of foods to help
ensure their safety and quality. Readers should be aware, however, of the limits of confidence one can
have in the results of such testing both from a statistical perspective, and also due to the limitations
in methods for detection and enumeration of microorganisms in foods.
While methodological considerations are discussed briefly in Sect. 7.5, Limitations of
Microbiological Tests, it must be emphasized that estimates for the performance of sampling plans
presented in this book (see Table 7.2) do not take into account any errors that might occur from the
microbiological methods used to determine either the presence or concentration of microorganisms
in foods.
The process of sampling itself can never be completely reliable. The degree to which the microbiological
status of the samples taken can be expected to represent the whole lot or batch of food being
assessed is discussed in Appendix A, Statistical Aspects of Sampling.
References 11
1.4 Conclusions
Microbiological testing is applied to food safety and quality management for a number of reasons
including development of process controls, monitoring and verification of process control, investigation
of the causes of loss of control, and in some situations to directly assess product quality and
safety. Assessment of microbiological quality and safety of foods is often laborious and time consuming,
and a comprehensive microbiological testing program for many products involves more than
routine lot acceptance testing. Currently all microbiological testing methods for end product are
destructive. Accordingly, the goal of a comprehensive program is to infer the quality and safety of
batches of product using process data augmented by relevant microbiological assessment of samples
taken not only from the lot, but also relevant ingredient, in-process, environmental and shelf life. This
process has limitations, both due to the confidence that one can have that the samples are representative
of the lot, and also because methods of isolation, identification and enumeration of microorganisms
from foods are imperfect. These limitations must be understood when designing microbiological
testing program for food safety and quality assurance.
The Commission trusts that this book provides practical guidance to those responsible for the
assurance of microbial quality and safety of foods to fulfill this important role. Specific recommendations
for product categories are provided in subsequent chapters.