Foodborne pathogens

Hazards, risk analysis, and control

Clive McClure W. Blackburn and Peter J. McClure W. Blackburn and Peter J. McClure W. Blackburn and Peter J. McClure W. Blackburn and Peter J. de W. Blackburn and Peter J. McClure W. Blackburn and Peter J. McClure W. Blackburn and Peter J. McClure W. Blackburn and Peter J. McClure W. Blackburn and Peter J. de W. Blackburn and Peter J. de

1.1 Introduction

Clive Blackburn and Peter McClure, Unilever Cloworth, UK

1.1 Trends in foodborne disease

Foodborne disease continues to be common and serious threat to public health all over the world and is a major cause of morbidity.common and serious threat to public health all over the world and is a major cause of morbidity.

Both industrialised and developing countries suffer large numbers of illnesses and the incidence, on a global basis, appears to be increasing.industrialised and developing countries suffer large numbers of illnesses and the incidence, on a global basis, appears to be increasing.

Most foodborne illnesses are mild, are associated with acute gastrointestinal symptoms such as diarrhoea and vomiting Sometime foodborne disease is much more serious and is diarrhoea and vomiting Sometime foodborne disease is much more serious and is life-threatening particularly in children in developing countries, in children in developing countries, and infection can also be followed by chronic sequelae or disability.sequelae or disability.

In many countries where information on foodborne diseases is collected, the total number of cases has been increasing over the past 20-30 years (Kaferstein et al., 1997). For example,in the UK, figures have risen from just under 10, 000 cases recorders in 1977, to more than 90, 000 cases in 1998. Most European countries have reported a doubling of example in Salmonellosis cases between 1985 and 1992 (Anon, 1995b). In the UK, for instance, there were 12846 infections (23 per 100,000) in 1981 compared with 33600 (58 per 100,000) in 1994 (Anon, 1995a).

In North America, there has been a notable increase in infection caused by Salmonella Enteritidis since the late 1980s (St Louis et al., 1988; Levy et al., 1996). Some of the increases recorded are undoubtedly due to improved system for information collection and reporting, better diagnoses and greater awareness of improved system for information collection and reporting, better diagnoses and greater of awareness of food safety, but these changes do not explain the general increases observed.

In recent years, the increased awareness of food safety, changes in regulatory and educational measures and changes in practice in food production have led to decreases in of particular foodborne diseases in some regiond.

For example, in the year 2000, Salmonellosis in the UK was at its lowest level since 1985, with a 54% decrease in the number of reported cases compared with the previous year. A decrease in the number of cases of Salmonellosis has also been observed recently in the US (Olsen et al.,2001) these particular decreases in Salmonellosis have been attributed to vaccination programmes and other changes that have been implemented in these regions. programmes for poultry and other changes that have been implemented in these regions.

Decreases in the number of cases of listeriosis have observed in the UK and US in recent year. However, for other pathogens such as campylobacters, numbers of associated cases continues to rise at a steady rate in many countries.

1.5 Control of foodboren

complexity of the global food market means that the control of foodborne disease is joint responsibility and requires action at tion at all level from the individual to international groups, and all parts of the supply chain from the farm to the fast-food restaurant.

The tools used and approaches taken to ensure control require different emphasis, depending on a number of factors such as where food materials have come from, how they have been processed and handled and how they are stored.

The risk of foodborne illness can be reduced by using existing technologies, such as pasteurisation and refrigeration, and by adopting some dborne illness can be reduced by using existing technologies, such as pasteurisation and refrigeration, and by adopting some simple precautions such as avoiding cross-contamination by separation of raw and cooked foods and aration of raw and cooked foods and employing good hygienic practices.

Although the onus is on us is on prevention of foodborne disease, valuable lessons can be learned by reviewing food poisoning statistics and incidents.

This turn can provide a focus for effective control measures to help reduce food poisoning (Bryan, 1988).

Ranking the factors that contribute to outbreaks of foodborne diseases can indicate trends and also differences in the different foodborne pathogens reflecting their association with raw material and physiological properties.

For many foodborne diseases, multiple choices for prevention are available, and the best answer may be to apply several steps simultaneously, for example, measures both to eliminate organisms during the food process and to reduce the likelihood of the organisms being present in the first place.

A better understanding of how pathogens persist in animal reservoirs (such as farm herds) challenge of foodborne disease lay in preventing contamination of human food with sewage of animal manure.

In the future, prevention of foodborne diseases will increasingly depend on control contamination of feed and water consumed by the animals themselves (Tauxe, 1997).

1.6 Rationale for this book

Ultimately, the control of foodborne pathogens requires the understanding of a number of factors including the knowledge of the possible hazards, factors including the knowledge of the possible hazards, their likely occurrence in different products, their physiological properties, the risks they pose to the consumer and the availability and effectiveness of the different preventative/intervention measures.

This aim of this book is to help provide this understanding, while there are good reference texts for the microbiologist on foodborne pathogens, there are less that relate current research to to practical strategies for hazard identification, risk assessment, and control.

This text takes the nonspecialist, particularly those whose role involves the safety of food processing operations.

Part 1 look at general techniques in assessing and managing microbiological hazards.

After a review of analytical methods and their application, there are chapters on modelling pathogen behaviour and carrying out behaviour and carrying out risk assessment as the essential foundation for effective food safety management. as the essential foundation for effective food safety management.

The following chapters then look at good management practices at key stages in the supply chain, starting with farm production and ending with the consumer.

In between there are chapters on hygienic plant design and sanitation, and safe process design and operation.

These provide the foundation for what makes for effective HACCP systems implementation.

This discussion of pathogen control than provides a context for Part 2 which looks at what this means in practice for major provides a context for Part 2 which looks at what this means in practice for major provides a context for Part 2 which looks at what this means in practice for major pathogens such as Pathogenic E. colo, Salmonella, Listeria, colo, Salmonella, Listeria, colo, Salmonella, Listeria, and Campylobacter. Each chapter discusses pathogen characteristics, detection methods, and control procedures. Part 3 then look at then look at then look at and as well as emerging potential hazards such as Mycobacterium paratuberculosis and the increasingly important of as emerging potential hazards such as Mycobacterium paratuberculosis and the increasingly important of as emerging potential hazards such as Mycobacterium paratuberculosis and the increasingly important of as emerging potential hazards such as Mycobacterium paratuberculosis and the increasingly important of non-bacterial hazards such as toxigenic fungi, viruses, parasites, chronic disease.

2 Detecting pathogens in food

Dr. Roy Betts, Cambden, and Chorleywood Food Research Association, UK, and Dr. Clive Blackburn, Unilever R&D Colworth, UK

2.1 Introduction

The detection enumeration of microorganisms either in food or on food contact surfaces from an integral part of any Quality control or Quality assurance plan.

Microbiological tests done on food can be divided in two types:in two types:in two types:

(a) ) Quantitative or enumerative, in which a group of microorganisms in the sample is counted and the result expresses as the number of the ample is counted and the result expresses as the number of the ample is counted and the result expresses as the number of the ample is counted and the result expresses as the number of the organisms present per unit weight of sample; OR (b) Qualitative or presence/absence, in which the requirement is simply to detect whether a particular organism is present or absent in a known weight of sample.

The basis methods used for the testing of microorganisms in foods is very well established, and relies on the incorporation of a food sample into a nutrient medium in which microorganisms can replicate thus resulting in a visual indication of growth.

Such methods are simple, adaptable, convenient and generally inexpensive.

However, they have two drawbacks:

Firstly, the tests rely on the growth of organisms in media, media, which can take many days and y days and result in a long test elapse time; and

Secondly, the methods are manually oriented and are thus labour intensive.

Over recent years, there has been considerable research into rapid and automated microbiological methods.

The aim of this work has been to reduce the test elapse time by using methods other than growth detect and/or count microorganisms and decrease the level of manual input into tests by automating methods as much as possible.

These rapid and automated methods have gained some acceptance and application within the food industry.

Microbiology methods are fundamental to Quality Control (QC), but with the inexorable move towards a Quality Assurance (QA) approach to food safety, they have been the brunt of much denigrating.

However, microbiological testing, even with all its limitations, is now being seen as an essential tool as part of this assurance, albeit with a shift in application and emphasis.

This chapter considers the application of microbiological methods in the identification of hazards, the assessment of risk and hazard control, as well as providing a comprehensive overview of the principles behind both conventional and rapid and automated methods.

A comparison of Quality Assurance and Quality Control her is showing the different, source: From Kilsby (2001).

Factor quality control (QC) approach is Reactive, Reliance for delivering Safety Measurement, Focus on Legal and Commercial issues.

Factor quality assurance (QA) approach is Preventative, Reliance for delivering Safety Central Standards and processes, Focus on Consumer issues.

2.2 A comparison of Quality Control and Quality Assurance

QC and QA are two different approaches to deliver Safety, both systems share tools, but the emphasis is very different.

Both approaches are they need totally different organizations, structures, skills, resource and ways of working (Kilsby, 2001).

QC is a reactive approach influenced by the pressures in the external world. In a QC organisation, the emphasis is on measurement, which needs to be robust and statistically relevant, and focus is on legal and commercial issues.organisation, the emphasis is on measurement, which needs to be robust and statistically relevant, and focus is on legal and commercial issues.

In contrast, QA is a preventive approach driven by the company's internal standards. The emphasis is on operational procedures, which must be robust and regularly reviewed, and the focus is on the consumer.

2.3 Ues of microbiology methods in Quality Control system Ues of microbiology methods in Quality Control system

In a QC system, measurement is relied upon to deliver quality and safety. This means the microbiological methods must be robust and the result that are produced must be statistically relevant.

This, in turn, places great importance on the use of sampling plans, which are covered briefly later.

Raw material and finished products cts cts have to be tested on a regular basis, often according to the risk they pose. For raw material, the onus is on the buyer to analyse samples samples samples samples and reliance place on is positive release rather than supplier assurance ier assurance ssurance for compliance with standards.

Microbiology methods can differ widely in their comparative advantages and disadvantages. These relative benefits and limitations may influence the choice of microbiological method for a particular task.

Factors that may influence the choice of microbiological method

* Performance factor should be considered these issues: should be considered these issues: should be considered these issues: should be considered these issues: Sensitivity, specificity, accuracy, precision, reproducibility, repeatabilityrepeatability

hese issues: should be considered these issues: Total test time (presumptive/confirmed results), 'hands-on' time, time constraints should be considered t* Time factor

* Ease of use factor should be considered these issues: Complexity, automation, robustness, training requirement, sample throughout, result interpretation

* Standardisation factor should be considered these issues: should be considered these issues: should be considered these issues: should be considered these issues: Validation, accreditation, international acceptance

* Cost factor should be considered these issues: should be considered these issues: should be considered these issues: should be considered these issues: Cost/test, capital outlay/equipment running cost, labour costslabour costs

For example, for products with a short shelf-life, rapidity of test result may be an important factor.However, when maximising the volume of material sampled is maximising the volume of material sampled is ample throughput and low cost/test may be higher on the priority list.

In recent years a plethora of rapid test kits has become available that, to a greater or lesser extent, have helped to expedite, simplify, miniaturise and automate miniaturise and automate methodology.

The drive for standardisation, validation and international acceptance of standardisation, validation and international acceptance of methods, with regard to [Good Laboratory Practices] and accreditation, means that this is often a constraint on method selection.

The are several problems associated with relying on testing for product safety assurance (van Schothorst and Jongeneel, 1994).

In order to apply any statistical interpretation to the result, the contaminant should be distributed homogeneously through the batch. AS microbiological hazards are usually heterogeneously distributed this means that there is often a major discrepancy between the microbiological status of the batch and microbial test results (Anon, 1986).

Even if the microbial distribution is homogeneous, it still may be prohibitive to test a sufficient number of sample units for all the relevant hazards to obtain meaningful information.

Microbiological testing testing testing testing detects only the effects and neither identifies nor controls the cause.cause.

2.4 Sampling

although this chapter deals with the this chapter deals with the this chapter deals with the this chapter deals with the methodologies employed to test food, it is important for the r the microbiologist to consider sampling.

No matter how good a method is if the sample has not been taken correctly and is not representative of the batch of food that is has been taken from, then the test result is meaningless.

It is useful to devise a sample plan in which result are interpreted from a are interpreted from a rather than a single result.

It is now common for microbiologists to use two or three class sampling plans, in which the number of individual samples to be se two or three class sampling plans, in which the number of individual samples to be tested from one batch are specified, together with the microbiological limits the apply.

These type of type of sampling plan are fully described in (Anon, 1986).

Once a sampling plan has been devised then a representative portion must be taken for analysis. In order to do this, the microbiologist must understand the food product and its microbiology in some detail.

Many chilled products will not be homogeneous mixtures mixtures but will be made up of layer or sections: A good example would be a prepared sandwich. It must be decided if the microbiological result is needed for the whole sandwich (i.e. bread and filling), or just the brad, or just the filling, indeed in some cases one part of a mixed filling may need to be tested, when this has been decided then the sample for analyses can be taken, using the appropriate aseptic technique and sterile sampling implements bread and filling), or just the brad, or just the filling, indeed in some cases one part of a mixed filling may need to be tested, when this has been decided then the sample for analyses can be taken, using the appropriate aseptic technique and sterile sampling implements (Kyriakides et al., 1996)..The sampling procedure having been developed, the microbiologist will have confidence that sample taken are representative of the food being tested and test method can be used with confidence.

2.5 Use of microbiology methods in a Quality Assurance system

Owing to the difficulty of assuring microbiological safety through testing alone there is now widespread adoption of the is now widespread adoption of the Quality Assurance approach using the Hazard Analysis Critical Control Point (HACCP) system.

Successful implementation of fully Validated HACCP study means that the supposed reliance on microbiological testing, with all its sampling limitations, is relinquished and this should enable a significant reduction in the volume of testing.

Some in the food industry went so far as to Surmise that microbiological testing would become Obsolete (Struikk, 1996).

In reality. however, , microbiology testing has continued albeit with a shift in application and emphasis.

Microbiological methods are needed with a HACCP-based programme for for risk assessment, the control of raw material, the control of the process-line and the line-environment, and for validation and verification of the HACCP program (de Boer and Beumer, 1999).de Boer and Beumer, 1999).

It has been pointed out that although in spite of meticulous adherence to HACCP-based good practices occasional human, instrumental or operational hiatuses can and will occur (Stuijk, 1996).practices occasional human, instrumental or operational hiatuses can and will occur (Stuijk, 1996).

Microbiological methods are still required for trouble-shooting and forensic investigation in order to identify the cause of the contamination and rectify it.

2.5.1 Hazard analysis

The HACCP comprises seven principles, which are further broken down into series of series of stages.

The first principle is to conduct a hazard analysis and the use microbiological test may be required by the HACCP team to gather relevant data.

This may involve determining the incidence of pathogens or indicator organisms in raw material, the efficacy of equipment cleaning procedures, the presence of pathogens (e.g. Listeria) in the Listeria) in the environment, and microbial load in food and on on equipment (Stier, 1993).

The use of molecular characterisation techniques characterisation techniques has further increased the microbiologist's armoury and epidemiological and epidemiological tracking of strains can provide a more in-depth knowledge of the food process.

This may enable the determination of sites of cross-contamination, or sites where stains appear and disappear, thus pinpointing the positions contributing to the final flora of the product, permitting more precise identification of critical control points (CCPs) (Dodd, 1994).

2.5.2 MONITORING CCPs

The HACCP process requires the establishment of system to monitor all identified CCPs.

Most microbiology methods are too slow for monitoring of CCPs, but a notable exception to this is the application of ATP bioluminescence for checking the cleaning of equipment.

Results from these methods can be obtained in only a few minutes, which allows sufficient time for equipment to be recleaned before production begins if they are found to be contaminated.

Although care in the application of these methods is required to prevent being lulled into false sense of security of security (Stier, 1993), the methodology can have a beneficial impact in demonstrating to staff responsible for cleaning the importance of their role.

In the context of HACCP, microbiological specification and criteria play a role in the monitoring of CCPs in food processing and distribution (Hall, 1994) and both conventional and rapid methods have a role to play in the checking of raw materials and monitoring of supplies.

Receipt of raw material is often identified as a CCP, and intake testing may be identified as one of the preventative measures for control.

However, if this is done it is the done it is the context of verifying the supplier's own microbiology assurance procedures.

2.5.3 Validation of HACCP

Validation of the technical accuracy of the hazard analysis and effectiveness of the preventative measures is important before the HACCP study is finalised and implemented.finalised and implemented.

Examples, where microbiology method may be used for validation, include pre-operation checks of cleaning and sanitising, , screening of sensitive raw materials, challenge testing, and monitoring of critical sites for microbiological build-up during processing (Hall, 1994).

For safe product design, a defined reduction defined reduction (e.g. 5 or 6 log10) of target microorganisms may be required, delivered either in one CCP or over a series of process steps.

Quantitative data may be required to demonstrate that the process can deliver the defined level of microbial kill or that the end-product meet the the specification for safety and/or stability.

Microbial method, particularly molecular characterisation characterisation ones, can be useful in answering questions that may arise as part of the HACCP validation exercise.

For example, if a hazardous organism appears in a in a product at a point in the production-line beyond the CCP designed to control it, does this mean failure of the CCP, or does it indicate post-process contamination (Dodd, 1994).

2.5.4 HACCP verification and review

Part of the HACCP process involves establishing procedures for verification to confirm that the HACCP system is working effectively.

Once a HACCP plan is operational, finished product testing can be one means by which its successful implementation is verified.

In addition, microbiological data can provide valuable sources of information for trend analysis and statistical process control.

In theory, a well-functioning HACCP plan should only require occasional testing as part of the verification process.

However, sometimes local legislation, customer, requirements or the company's own standard demand a higher level of a higher level of testing (Stier, 1993).

HACCP living system and as such new hazard may need to be considered and risk assessed.

In addition, change or proposed change to a process may require that microbiological data is generated to ensure that sufficient control is maintained.

2.5.5 Microbiological specifications and criteria

Regardless of whether HACCP is used, microbiological specifications and criteria are still applied to are still applied to foods.

They can serve as a determinant of the acceptability of an ingredient, finished product or process with regard to microbiological Safety and/or microbiological Quality.

In practice, microbiological specifications typically are used both as an internal tool by the manufacturer to judge acceptability against pre-determined standards and as an external measure against customer or government standards (Hall, 1994).

Increasing international trade and the potential for disputes places further emphasis on the need for agreed and reliable agreed and reliable methodologies.

This checking of conformance to specifications may mean that raw material and finished products are held pending the result of microbiological test.

In these cases, faster techniques can help to determine the fate of products more quickly.

2.5.6 Risk assessment

One important area within the food industry where methodology is raising its profile is quantitative risk assessment.

Risk assessment is very much tied in with microbiological data and microbiological examinations of samples of ingredients and end-[roducts may be necessary (de Boer and Beumer, 19999).roducts may be necessary (de Boer and Beumer, 19999).

Risk assessment methods can identify gaps in our knowledge that are crucial to providing better estimates of risk and this may in fact lead to an increase in the level of microbiological testing.

Assessing the risk posed by a 'new' or 'emerging' organism may also highlight deficiencies in current methodology requiring the need for method development.

2.6.1 Conventional microbiological techniques

As outlined in the introduction, conventional microbiological techniques are based on the established of incorporating food samples into nutrient media and incubating for a period of time at allow the microorganisms to grow.

The detection or counting method is then a simple visual assessment of growth. These methods are thus technically simple and relatively inexpensive, requiring no complex instrumentation. The methods are however very adaptable, allowing the enumeration of different groups of microorganisms.

Before testing, the food sample must be converted into a liquid form in order to allow mixing with the growth medium. This Mixture pulsifier) to breaks the sample apart releasing the organisms into diluent. If the organisms sample are stressed incorrectly then they injured or killed, breaks the sample apart releasing the organisms into diluent. If the organisms sample are stressed incorrectly then they injured or killed, or stomacher (e.g. stomacher or homogenised (e.g. homogenisedthus affecting the final result obtained from the microbiological test. The initial phase of the test procedure (Davis and Jones, 1997).

2.6.1 Conventional quantitative procedures

The enumeration of organisms in samples is generally done by using plate count, or most probable number (MPN) methods.

The former are the most widely used, while the latter tend to be used for certain tend to be used for certain organisms (e.g. Escherichia ) or group (e.g. coliforms).coliforms).

2.6.2 Conve ualitative procedures

Qualitative procedures are used when a count of the number of organisms in a sample is not required and only their presence or absence needs to be determined.

Generally, such methods are used to test for potentially pathogenic microorganisms such as Salmonella app., Listeria app., and Campylobacter app.

2.7.1 Rapid and automated methods

The general interest in alternative microbiological methods has been stimulated i part by the increased output of i part by the increased output of food production sites.

This has resulted in the following:

1 - Greater numbers of samples being stored prior to positive release - a redu tion i analysis time would reduce storage and warehousing costs.

2 - A greater sample throughput being required in laboratories - the only way that this can be achieved is by increased laboratory size and staff levels, or by using more rapid and automated methods

3 - A requirement for a longer shelf-life in the chilled foods sector - a reduction in analysis time could expedite product release thus increasing the shelf-life of the product.foods sector - a reduction in analysis time could expedite product release thus increasing the shelf-life of the product.

4 - The increased application HACCP procedures - rapid methods can be used in HACCP verification procedures.

There are a number of different techniques referred to as rapid methods and most have little in common with each other or with the conventional procedures that they replace.

The methods can generally be divided into quantitative and qualitative tests, the former giving a measurement of the number of organisms in a sample, the latter indicating only presence or absence.

Laboratories considering the use of rapid methods for routine testing must carefully consider their own requirement before purchasing such a system.

Every new method will be unique, giving a slightly different result, in a different timescale with varying levels of timescale with varying levels of automation and sample throughput.

In addition, some methods may work poorly with of food or may not be able to detect the specific organisms or group that is required.

All of these points must be considered before a method is adopted by a laboratory.

It is also of importance to ensure that staff using new methods are aware of the principles of operation of the techniques and thus have the ability to troubles-shoot in the method clearly shows erroneous results.

2.7.1 Electrical methods

The enumeration of microorganisms in solution can be achieved by one of two electrical methods, one measuring particle number and size, the other monitoring metabolic activity.

Particle counting

The counting and sizing of particles can be done with the 'Coulter' principle, using instruments such as the Coulter Counter (Coulter Electrics, Luton).

Metabolic activity

Stewart (1899) first reported the use of electrical measurement to monitor microbial growth. In order to use electrical system to enumerate organisms in food, the sample must initially be homogenised.

2.7.2 Adenosine triphosphate (ATP) bioluminescence

The non-biological synthesis of ATP in the extracellular environment has been demonstrated (Ponnamperuma et al., 1963), but it is universally accepted that such sources of ATP are very rare (Huermnekens and Whiteley, 1960).

ATP is a high-energy compound found in all living cells and it is an essential component in the initial biochemical steps of substrate utilisation and in the synthesis of cell material.

Microscopy methods

Microscopy is well-established and sample technique for enumeration of microorganisms.

One of the first its use was for rapidly counting bacteria in films of milk stained with the dye methylene blue (Breed and Brew, 1916).

One of the main advantages of microscope methods is the speed with which individual analyses can be done, however, this must be balanced against the high manual workload and potential for operation fatigue caused by constant microscopic counting.

Microbial ecologists first stains made use of such compounds to visualise and count microorganisms in natural water (Francisco et al.,1973; Jones and Simon, 1975).

Hobbies et al, (1977) first described the use of Nuclepore polycarbonate membrane filters to capture microorganisms before fluorescent staining, with enumeration was considered in depth by (Pettipher et al., 1980), the method developed by the latter author being known as the direct epifluorescent filter technique (DEFT).

Flow cytometry

Flow cytometry is a technique based on the rapid measurement of cells as they flow in a liquid stream past a sensing point (Carter and Meyer, 1990).

The cells under investigation are inoculated into the centre of a stream of fluid (known as the sheath fluid). The flow cytometry can provide a rapid and sensitive method for the rapid enumeration of microorganisms and its suitable staining system for separation of microorganisms from food debris the system fitted with a number of light detection allow the analysis of sample and for many parameters at once.

Solid phase cytometry

A relatively new cytometric technique has been developed by Chemuneex (Maisons-Alfort, France) based on solid phase cytometr.

Labelling techniques used to detect particular organisms of food containing particulate material.

2.7.4 Immunological methods

Antibodies antigens

Immunological methods are based on the specifi binding reaction occurs between an antidody and antigen to which it isdirected

Labels

The labels that can be used with antibodies are of many types and include radiolabels, fluorescent agents, Luminescent chemicals and enzymes; and addition agglutination reactions can be used to detect the binding of antibody to antigen.

Immunoassay conclusions

In conclusion, immunological methods have been extensively researched and developed.

The are now a range of system that allows the rapid detection of the specific organisms to which they are directed.

2.7.5 Nucleic acid hybridisation

Nucleic acids

The specifi characteristic of any organisms depends on particular sequence of the nucleic acids contained in its genome.

The nucleic acids themselves are made up of a chain units each consisting of a sugar (deoxyribose or ribose, depending on whether the nucleic acid is DNA or RNA), a phosphorus-containing group and one of four organic purine or pyrimidine bases, DNA is constructed from two of these chains arranged in double helix and held together by bonds between the organic bases.

The bases specifically bind adenine to thymine and guanine to cytosine.

It is the sequence of bases that make different organisms unique.

The development of nucleic acid probes

Probes for organisms in food

Probes - the future

Nucleic acid amplification techniques

Polymerase chain reaction (PCR)

Reverse transcriptase PCR

NASBA

Commercial PCR-bases kits

Separation and concentration of microorganisms from foods

Future trends

The food industry has the responsibility to produce safe and wholesome food and providing this assurance is ultimately the microbiological goal.

A microbiology test that could analyse a batch of food non-destructively, online and with the required accuracy, sensitivity and specificity is the 'Holy Grail' and would provide this assurance. However, our current technical capability fall well short of this ideal situation

In summary, methods have an important place in our armoury against the threats posed by microorganisms in food. Owing to the diversity of applications and user requirement and the shift from Quality Control to Quality Assurance, Methodology still plays a key role in assuring food safety and new methods still have the potential to bring benefits.

Modelling the growth, survival and death of bacterial pathogens in foods

Dr David Legan and Dr Mark Vandeven, Kraft Food North America; and Dr Cynthia Stewart and Martin Cole, Food Science Australia

3.1 Introduction

Simple mathematical models have been used in food microbiology since the early 1920s when much work was done to support the safe operation of canning processes.

To calculate the process time needed to produce 'sterility' in canned foods' (Bigelow et al., 1920) came up with a lethal rate curve to relate the reciprocal of time needed to destroy all spores present in the food to process temperature.gvivi

Ball 1923 introduced the term 'z' to describe the slope of this curve with the value of the slope equal to the temperature change needed to effect a 1 log change in the time.

He also presented formulae and graphical methods to calculate the total lethality of thermal process including come-up and cooling times.

At about the same time, the classic work of Esty and Meyer (1922) on thermal resistance of Clostridium botulinum spores and gave rise to the so-called 'botulinum cook', or minimum process lethality required to destroy 1,000000000000 spores of C. in foods with pH greater than 4.5.

The familiar term 'D' to define the time to cause 1 log reduction in microbial population at a given temperature was not introduced until (Stumbo et al., 19950), that concept having previously been represented by (zeta), giving rise to some confusion between zed & zeta.

According to Lambert and Hohnston (2000), similar models were used even earlier for assessment of chemical disinfection.

The fermentation industries developed more sophisticated models for growth rates, product yield, mass transfer requirements, etc., in the interests of designing more efficient processes (Pirt, 1975).

However, the widespread acceptance of mathematical modelling in food microbiology has gained ground slowly only since the early 1980s.

Application in food microbiology include models that predict the growth rate of bacterial pathogens in response to product or environmental factors such as water activity (w a), temperature or pH (Buchanan and Philips, 1990; Gibson et al., 1988; McClure et al., 1997; Sutherland and Bayliss, 1994). These can help food processors to produce safe products with less reliance on Laboratory testing Growth models can be used to design safe product formulations, to set appropriate storage conditions and to explore the maximum interval between cleaning and sanitising for process equipment.

Models that can predict the rate of the death of pathogens or spoilage organisms can be used to design safe and effective processes.

The potential of models is to help improve product safety was identified by the UK and government in the 1980s and both funded sophisticated programmes to develop models for growth, survival, and death of foodborne pathogens.

The success of those programmes resulted in suites of the models that are available for consultations.

The UK government models reside in a software package called Food MicroModels available form the Food MicroModels subsidiary of Leatherhead Food Research Association

The approach to developing those models was described by (McClure et al., 1994a).

The US government mode its models freely available and they can be downloaded from the US Department of Agriculture website.

While the sciences and applications of modelling in microbiology are becoming ever more sophisticated (Baranyi and Roberts, 2000(, of modelling,

This chapter is designed to be a practical guide to modelling, supported by references to primary sources of modelling information.

Our aim is to give readers an appreciation of the principle involved in creating useful models and help them to identify soundly based models.

3.2 Approaches to modelling

3.2.1 Principles

By 'model' we men an euation that decribes or predicts the growth, survival or death of microoganisms in foods.

In food microbiology, these models are empircical.

In othe words the simply relate the microbial growth, survival or death responses to the levels of the controlling factors throughout the experimental design space.

They tell us nothing about the physiological mechanisms or biological, chemical or physical principles that drive the responses.

For this reason they are sometimes known as 'black-box' models.

Empirical models should not used outside the range of the factors used to create them because there is no underlying principle on which to base extrapolation. Hance, we must carefully consider the range over which they be used before beginning experimentation.

The ranges may be decided from our knowledge of the conditions found in relevant product categories and/or knowledge of the likely microbial responses, e.g. minimum a w or pH growth of organisms of intreset. Microbial responses to single factors are well document (ICMSF, 1996) but responses to factors acting together are still much lee weel characterised.

A simplifying common approach is to experiment in liquid laboratory media that are homoeneous and less complex than the foods that we are ultimately interested in.